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Biotechnology and Bioengineering

Volume 37, Issue 7 , Pages 614 - 626

Published Online: 18 Feb 2004

Copyright 1991 John Wiley & Sons, Inc.


Article

A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing

Victor Goetz, Magali Remaud, David J. Graves *

Department of Chemical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6393

*Correspondence to David J. Graves, Department of Chemical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6393



__________________________________________________________

_________________________________________________________
Weapons-Grade Anthrax:


http://www.asph.org/document.cfm?page=847

Summary of 2004 Environmental Health Conference

Introduction


The fifth annual Environmental and Occupational Health

conference, "Environmental Health Risk: Assessment, Management

and Communications", was held from July 11-13, 2004 in

Minneapolis, MN. The generous support provided by the Health

Resources Services Administration, the Centers for Disease Control

and Prevention, and the National Institute for Environmental Health

Sciences made it possible for this year's effort to be our most

successful yet. We would also like to acknowledge NSF

International for their support of the conference. The efforts of the

Association of Schools of Public Health Environmental and

Occupational Health Council resulted in a valuable and memorable

experience for all the participants.

The presenters were:

John Cicmanec, DVM, MS, U.S. EPA
Overview of Environmental Health Issues related to agriculture

John L. Cicmanec, DVM, MS, U.S. Environmental Protection


Agency: Weapons-Grade Anthrax:

The Infectious Dose-50 in

Rhesus Monkeys Derived from a
Biologically-based Model for Use

in Human Risk Assessment€�

One of the significant discoveries following the bioterrorist attacks

of October 2001 was that a modified form of Bacillus anthracis

(Ames strain) was the causative agent. Physical alteration of the

inoculum had occurred; the electrostatic charge had been altered

and the resulting spores were 1 to 3 microns in diameter. Eight

separate inhalation studies have been identified in which non-human

primates were used for inhalation exposure to B. anthracis to

determine the Infectious Dose50 ( Druett 1953, Henderson,1949,

Estep 2003,etc.) Depending on the spore particle size and strain

used, these values ranged from 4000 to 682,000 spores. Although

some studies used spores that were one micron in diameter,

conventional spore preparations will aggregate so that many of the

inhaled particles will often range from four to twelve microns. The

primary advantage that is gained through the use of spores with an

altered electrostatic charge is that particles do not clump and

essentially all of the inoculum can be deposited directly in the lungs.

In order to adjust for the amount of the conventional inoculum in the

non-human primate studies that was deposited in nasal passages,

pharynx, and tracheobronchial regions, a methodology that has been

developed for chemical particulate inhalation exposure (using the

MMAD and sigma g) has been used as a model. Conveniently,

cadmium chloride and radio-labeled polystyrene microspheres share

the same target cell, alveolar macrophages, as anthrax spores.

Through the use of ranking the dose response of these two

surrogates and zones of deposition in the respiratory tract, similar

dosage adjustments can be made for anthrax spores of various

particle sizes. Use of this model enables us to predict that the ID50

for the modified form of anthrax is at least 15 to 500 times lower

than for conventional spores. (This presentation does not represent

US EPA policy)

http://www3.interscience.wiley.com/cgi-bin/abstract/107622942/



ABSTRACT

Biotechnology and Bioengineering
Volume 37, Issue 7 , Pages 614 - 626

Published Online: 18 Feb 2004

Copyright 1991 John Wiley & Sons, Inc.


Abstract | References | Full Text: PDF (1554k) |

A novel magnetic silica support for use in chromatographic and

enzymatic bioprocessing

Victor Goetz, Magali Remaud, David J. Graves *

Department of Chemical Engineering, University of Pennsylvania,

Philadelphia, Pennsylvania 19104-6393


*Correspondence to David J. Graves, Department of Chemical

Engineering, University of Pennsylvania, Philadelphia, Pennsylvania

19104-6393

Keywords
chromatography • immobilized enzyme • magnetically stabilized

fluidized bed • silica

Abstract
A limited number of support matrices have so far been developed for

use in magnetically stabilized fluidized bed (MSFB) applications. We

have developed a versatile magnetic silica support which can be

derivatized readily for both adsorption chromatography and enzyme

immobilization by well-known techniques. A magnetic pellicular bead

is prepared by electrostatically depositing alternating layers of

colloidal silica and cationic polymer onto macroscopic nickel core

particles. The polymer is then burned out and the silica partially

sintered to yield a porous shell with 5-80 m2/g of surface area. This

magnetic composite was tested as a support for immobilizing

invertase. Sucrose was continuously converted to its component

monosaccharides with nearly constant activity over the first 8 days

and retention of 50% of initial activity after 25 days.
Received: 24 July 1990; Accepted: 13 September 1990

Digital Object Identifier (DOI)

10.1002/bit.260370704 About DOI

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1: Biotechnol Prog. 1990 Nov-Dec;6(6):452-7.

Plant cell culture using a novel bioreactor: the magnetically stabilized fluidized bed.

Bramble JL, Graves DJ, Brodelius P.

Department of Chemical Engineering, University of Pennsylvania, Philadelphia 19104.

A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared to the results obtained in shake flask culture.

PMID: 1366835 [PubMed - indexed for MEDLINE]






1: Biotechnol Prog. 2003 Nov-Dec;19(6):1721-7. Related Articles,Links

Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption.

Tong XD, Sun Y.

Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is promising for use in liquid MSFB for protein adsorption.

Publication Types:





1: Biotechnol Prog. 2003 Nov-Dec;19(6):1721-7. Related Articles,Links

Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption.

Tong XD, Sun Y.

Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is promising for use in liquid MSFB for protein adsorption.

Publication Types:




Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed


Handan Yavuz1 , Ridvan Say2 , Mge Anda1 , Necmi Bayraktar3 and Adil Denizli1
1Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey
2Department of Chemistry, Anadolu University, Ankara, Turkey
3Faculty of Medicine, Urology Department, Hacettepe University, Ankara, Turkey

BioMagnetic Research and Technology 2004, 2:5 doi:10.1186/1477-044X-2-5


Published 26 August 2004

Abstract


Background

Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders.

Methods

Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed.

Results

Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity.

Conclusions

Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1366835&dopt=Abstract

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BioMagnetic Research and Technology
Volume 2

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Review
.
Magnetic techniques for the isolation and purification of proteins and peptides


Ivo Safarik1, 2 and Mirka Safarikova1
1Laboratory of Biochemistry and Microbiology, Institute of Landscape Ecology, Academy of Sciences, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic
2Department of General Biology, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

BioMagnetic Research and Technology 2004, 2:7 doi:10.1186/1477-044X-2-7

The electronic version of this article is the complete one and can be found online at: http://www.biomagres.com/content/2/1/7


Received 10 November 2004
Accepted 26 November 2004
Published 26 November 2004

2004 Safarik and Safarikova; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract




Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques.


Outline Introduction

Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References



Isolation, separation and purification of various types of proteins and peptides, as well as of other specific molecules, is used in almost all branches of biosciences and biotechnologies. Separation science and technology is thus very important area necessary for further developments in bio-oriented research and technology. New separation techniques, capable of treating dilute solutions or solutions containing only minute amounts of target molecules in the presence of vast amounts of accompanying compounds in both small and large-scale processes, even in the presence of particulate matter, are necessary.

In the area of biosciences and biotechnology the isolation of proteins and peptides is usually performed using variety of chromatography, electrophoretic, ultrafiltration, precipitation and other procedures, affinity chromatography being one of the most important techniques. Affinity ligand techniques represent currently the most powerful tool available to the downstream processing both in term of their selectivity and recovery. The strength of column affinity chromatography has been shown in thousands of successful applications, especially in the laboratory scale. However, the disadvantage of all standard column liquid chromatography procedures is the impossibility of the standard column systems to cope with the samples containing particulate material so they are not suitable for work in early stages of the isolation/purification process where suspended solid and fouling components are present in the sample. In this case magnetic affinity, ion-exchange, hydrophobic or adsorption batch separation processes, applications of magnetically stabilized fluidized beds or magnetically modified two-phase systems have shown their usefulness.

The basic principle of batch magnetic separation is very simple. Magnetic carriers bearing an immobilized affinity or hydrophobic ligand or ion-exchange groups, or magnetic biopolymer particles having affinity to the isolated structure, are mixed with a sample containing target compound(s). Samples may be crude cell lysates, whole blood, plasma, ascites fluid, milk, whey, urine, cultivation media, wastes from food and fermentation industry and many others. Following an incubation period when the target compound(s) bind to the magnetic particles the whole magnetic complex is easily and rapidly removed from the sample using an appropriate magnetic separator. After washing out the contaminants, the isolated target compound(s) can be eluted and used for further work.

Magnetic separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube or another vessel. There is no need for expensive liquid chromatography systems, centrifuges, filters or other equipment. The separation process can be performed directly in crude samples containing suspended solid material. In some cases (e.g., isolation of intracellular proteins) it is even possible to integrate the disintegration and separation steps and thus shorten the total separation time [1]. Due to the magnetic properties of magnetic adsorbents (and diamagnetic properties of majority of the contaminating molecules and particles present in the treated sample), they can be relatively easily and selectively removed from the sample. In fact, magnetic separation is the only feasible method for recovery of small magnetic particles (diameter ca 0.1 – 1 μm) in the presence of biological debris and other fouling material of similar size. Moreover, the power and efficiency of magnetic separation procedures is especially useful at large-scale operations. The magnetic separation techniques are also the basis of various automated procedures, especially magnetic-particle based immunoassay systems for the determination of a variety of analytes, among them proteins and peptides. Several automated systems for the separation of proteins or nucleic acids have become available recently.

Magnetic separation is usually very gentle to the target proteins or peptides. Even large protein complexes that tend to be broken up by traditional column chromatography techniques may remain intact when using the very gentle magnetic separation procedure [2]. Both the reduced shearing forces and the higher protein concentration throughout the isolation process positively influence the separation process.

Separation of target proteins using standard chromatography techniques often leads to the large volume of diluted protein solution. In this case appropriate magnetic particles can be used for their concentration instead of ultrafiltration, precipitation etc. [3].

The purpose of this review is to summarize various methodologies and strategies which can be employed for the isolation and purification of target proteins and peptides with the help of magnetic materials. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. All these information will help the scientists to select the optimal magnetic material and the purification procedure.


Outline Necessary materials and equipment

Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References

Figures

Figure 1
Examples of batch magnetic separators applicable for magnetic separation of proteins and peptides





The basic equipment for laboratory experiments is very simple. Magnetic carriers with immobilized affinity or hydrophobic ligands, magnetic particles prepared from a biopolymer exhibiting affinity for the target compound(s) or magnetic ion-exchangers are usually used to perform the isolation procedure. Magnetic separators of different types can be used for magnetic separations, but many times cheap strong permanent magnets are equally efficient, especially in preliminary experiments.

Magnetic carriers and adsorbents can be either prepared in the laboratory, or commercially available ones can be used. Such carriers are usually available in the form of magnetic particles prepared from various synthetic polymers, biopolymers or porous glass, or magnetic particles based on the inorganic magnetic materials such as surface modified magnetite can be used. Many of the particles behave like superparamagnetic ones responding to an external magnetic field, but not interacting themselves in the absence of magnetic field. This is important due to the fact that magnetic particles can be easily resuspended and remain in suspension for a long time. In most cases, the diameter of the particles differs from ca 50 nm to approx. 10 μm. However, also larger magnetic affinity particles, with the diameters up to millimetre range, have been successfully used [4]. Magnetic particles having the diameter larger than ca 1 μm can be easily separated using simple magnetic separators, while separation of smaller particles (magnetic colloids with the particle size ranging between tens and hundreds of nanometers) may require the usage of high gradient magnetic separators.

Commercially available magnetic particles can be obtained from a variety of companies. In most cases polystyrene is used as a polymer matrix, but carriers based on cellulose, agarose, silica, porous glass or silanized magnetic particles are also available. Examples of magnetic particles used (or usable) for proteins and peptides separation can be found elsewhere [5-7].

Particles with immobilised affinity ligands are available for magnetic affinity adsorption. Streptavidin, antibodies, protein A and Protein G are used most often in the course of protein and peptides isolation. Magnetic particles with above mentioned immobilised ligands can also serve as generic solid phases to which native or modified affinity ligands can be immobilised (e.g., antibodies in the case of immobilised protein A, protein G or secondary antibodies, biotinylated molecules in the case of immobilised streptavidin).

Also some other affinity ligands (e.g., nitrilotriacetic acid, glutathione, trypsin, trypsin inhibitor, gelatine etc.) are already immobilised to commercially available carriers. To immobilise other ligands of interest to both commercial and laboratory made magnetic particles standard procedures used in affinity chromatography can be employed. Usually functional groups available on the surface of magnetic particles such as -COOH, -OH or -NH2 are used for immobilisation, in some cases magnetic particles are available already in the activated form (e.g., tosylactivated, epoxyactivated etc).

In the laboratory magnetite (or similar magnetic materials such as maghemite or ferrites) particles can be surface modified by silanization. This process modifies the surface of the inorganic particles so that appropriate functional groups become available, which enable easy immobilisation of affinity ligands [8]. In exceptional cases enzyme activity can be decreased as a result of usage of magnetic particles with exposed iron oxides. In this case encapsulated microspheres, having an outer layer of pure polymer, will be safer.

Biopolymers such as agarose, chitosan, kappa carrageenan and alginate can be easily prepared in a magnetic form. In the simplest way the biopolymer solution is mixed with magnetic particles and after bulk gel formation the magnetic gel formed is mechanically broken into fine particles [9]. Alternatively biopolymer solution containing dispersed magnetite is dropped into a mixed hardening solution [4] or water-in-oil suspension technique is used to prepare spherical particles [10].

Basically the same procedures can be used to prepare magnetic particles from synthetic polymers such as polyacrylamide, poly(vinylalcohol) and many others [11].

In another approach used standard affinity or ion-exchange chromatography material was post-magnetised by interaction of the sorbent with water-based ferrofluid. Magnetic particles accumulated within the pores of chromatography adsorbent thus modifying this material into magnetic form [12,13]. Alternatively magnetic Sepharose or other agarose gels were prepared by simple contact with freshly precipitated or finely powdered magnetite [12,14].

Magnetoliposomes (magnetic derivatives of standard liposomes), either in the original form or after immobilization of specific proteins, have the potential for the separation of antiphospholipid antibodies [15], IgG antibodies [16] and other proteins of interest [17].

Recently also non-spherical magnetic structures, such as magnetic nanorods have been tested as possible adsorbent material for specific separation of target proteins [18].

Magnetic separators are necessary to separate the magnetic particles from the system. In the simplest approach, a small permanent magnet can be used, but various magnetic separators employing strong rare-earth magnets can be obtained at reasonable prices. Commercial laboratory scale batch magnetic separators are usually made from magnets embedded in disinfectant-proof material. The racks are constructed for separations in Eppendorf micro-tubes, standard test tubes or centrifugation cuvettes, some of them have a removable magnetic plate to facilitate easy washing of separated magnetic particles. Other types of separators enable separations from the wells of microtitration plates and the flat magnetic separators are useful for separation from larger volumes of suspensions (up to approx. 500 – 1000 ml). Examples of typical batch magnetic separators are shown in Fig. 1.

Flow-through magnetic separators are usually more expensive, and high gradient magnetic separators (HGMS) are the typical examples. Laboratory scale HGMS is composed from a column packed with fine magnetic grade stainless steel wool or small steel balls which is placed between the poles of an appropriate magnet. The suspension is pumped through the column, and magnetic particles are retained within the matrix. After removal the column from the magnetic field, the particles are retrieved by flow and usually by gentle vibration of the column.

For work in dense suspensions, open gradient magnetic separators may be useful. A very simple experimental set-up for the separation of magnetic affinity adsorbents from litre volumes of suspensions was described [19].

Currently many projects require the analysis of a high number of individual proteins or variants. Therefore, methods are required that allows multiparallel processing of different proteins. There are several multiple systems for high throughput nucleic acid and proteins preparation commercially available. The most often used approach for proteins isolation is based on the isolation and assay of 6xHis-tagged recombinant proteins using magnetic beads with Ni-nitriloacetic acid ligand [20]. The commercially available platforms can be obtained from several companies such as Qiagen, USA (BioRobot and BioSprint series), Tecan, Japan (Te-MagS) or Thermo Electron Corporation, USA (KingFisher).


Outline Basic principles of magnetic separations of proteins and peptides

Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References



Magnetic separations of proteins and peptides are usually convenient and rapid. Nevertheless, several hints may be helpful to obtain good results.

Proteins and peptides in the free form can be directly isolated from different sources. Membrane bound proteins have to be usually solubilized using appropriate detergents. When nuclei are broken during sample preparation, DNA released into the lysate make the sample very viscous. This DNA may be sheared by repeated passage up and down through a 21 gauge hypodermic syringe needle before isolation of a target protein. Alternatively, DNase can be added to enzymatically digest the DNA.

Magnetic beads in many cases exhibit low non-specific binding of non-target molecules present in different samples. Certain samples may still require preclearing to remove molecules which have high non-specific binding activity. If preclearing is needed, the sample can be mixed with magnetic beads not coated with the affinity ligand. In the case of immunomagnetic separation, magnetic beads coated with secondary antibody or with irrelevant antibodies have been used. The non-specific binding can also be minimised by adding a non-ionic detergent both in the sample and in the washing buffers after isolation of the target.

In general, magnetic affinity separations can be performed in two different modes. In the direct method, an appropriate affinity ligand is directly coupled to the magnetic particles or biopolymer exhibiting the affinity towards target compound(s) is used in the course of preparation of magnetic affinity particles. These particles are added to the sample and target compounds then bind to them. In the indirect method the free affinity ligand (in most cases an appropriate antibody) is added to the solution or suspension to enable the interaction with the target compound. The resulting complex is then captured by appropriate magnetic particles. In case antibodies are used as free affinity ligands, magnetic particles with immobilised secondary antibodies, protein A or protein G are used for capturing of the complex. Alternatively the free affinity ligands can be biotinylated and magnetic particles with immobilised streptavidin or avidin are used to capture the complexes formed. In both methods, magnetic particles with isolated target compound(s) are magnetically separated and then a series of washing steps is performed to remove majority of contaminating compounds and particles. The target compounds are then usually eluted, but for specific applications (especially in molecular biology, bioanalytical chemistry or environmental chemistry) they can be used still attached to the particles, such as in the case of polymerase chain reaction, magnetic ELISA etc.

The two methods perform equally well, but, in general, the direct technique is more controllable. The indirect procedure may perform better if affinity ligands have poor affinity for the target compound.

In most cases, magnetic batch adsorption is used to perform the separation step. This approach represents the simplest procedure available, enabling to perform the whole separation in one test-tube or flask. If larger magnetic particles (with diameters above ca 1 μm) are used, simple magnetic separators can be employed. In case magnetic colloids (diameters ranging between tens and hundreds of nanometres) are used as affinity adsorbents, high-gradient magnetic separators have usually to be used to remove the magnetic particles from the system.

Alternatively magnetically stabilised fluidised beds (MSFB), which enable a continuous separation process, can be used. The use of MSFB is an alternative to conventional column operation, such as packed-bed or fluidised bed, especially for large-scale purification of biological products. Magnetic stabilisation enables the expansion of a packed bed without mixing of solid particles. High column efficiency, low pressure drop and elimination of clogging can be reached [21,22].

Also non-magnetic chromatographic adsorbents can be stabilized in magnetically stabilized fluidized beds if sufficient amount of magnetically susceptible particles is also present. The minimum amount of magnetic particles necessary to stabilize the bed is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations [23].

Biocompatible two phase systems, composed for example from dextran and polyethylene glycol, are often used for isolation of biologically active compounds, subcellular organelles and cells. One of the disadvantages of this system is the slow separation of the phases when large amounts of proteins and cellular components are present. The separation of the phases can be accelerated by the addition of fine magnetic particles or ferrofluids to the system followed by the application of a magnetic field. This method seems to be useful when the two phases have very similar densities, the volumetric ratio between the phases is very high or low, or the systems are viscous. Magnetically enhanced phase separation usually increases the speed of phase separation by a factor of about 10 in well-behaved systems, but it may increase by a factor of many thousands in difficult systems. The addition of ferrofluids and/or iron oxide particles was shown to have usually no influence on enzyme partioning or enzyme activity [24,25].

Proteins and peptides isolated using magnetic techniques have to be usually eluted from the magnetic separation materials. In most cases bound proteins and peptides can be submitted to standard elution methods such as the change of pH, change of ionic strength, use of polarity reducing agents (e.g., dioxane or ethyleneglycol) or the use of deforming eluents containing chaotropic salts. Affinity elution (e.g., elution of glycoproteins from lectin coated magnetic beads by the addition of free sugar) may be both a very efficient and gentle procedure.


Outline Examples of magnetic separations of proteins and peptides

Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References

Tables

Table 1
Examples of proteinases and peptidases purified by magnetic techniques



Table 2
Purification of lysozyme by magnetic techniques



Table 3
Examples of polysaccharide and disaccharide hydrolases purified by magnetic techniques



Table 4
Examples of other enzymes purified by magnetic techniques



Table 5
Examples of antibodies purified by magnetic techniques



Table 6
Examples of DNA/RNA/oligonucleotide/aptamer binding proteins purified by magnetic techniques



Table 7
Purification of albumin and haemoglobin by magnetic techniques



Table 8
Examples of other proteins purified by magnetic techniques



Table 9
Examples of peptides purified by magnetic techniques





Magnetic affinity and ion-exchange separations have been successfully used in various areas, such as molecular biology, biochemistry, immunochemistry, enzymology, analytical chemistry, environmental chemistry etc [26-29]. Tables 1, 2, 3, 4, 5, 6, 7, 8, 9 show some selected applications of these techniques for proteins and peptides isolation.

In the case of proteins and peptides purifications, no simple strategy for magnetic affinity separations exists. Various affinity ligands have been immobilised on magnetic particles, or magnetic particles have been prepared from biopolymers exhibiting the affinity for target enzymes or lectins. Immunomagnetic particles, i.e. magnetic particles with immobilised specific antibodies against the target structures, have been used for the isolation of various antigens, both molecules and cells [5] and can thus be used for the separation of specific proteins.

Magnetic separation procedures can be employed in several ways. Preparative isolation of the target protein or peptide is usually necessary if further detailed study is intended. In other cases, however, the magnetic separation can be directly followed (after elution with an appropriate buffer) with SDS electrophoresis. Magnetically separated proteins and peptides can also be used for further mass spectroscopy characterization [30,31]. The basic principles of magnetic separations can be used in the course of protein or peptide determination using various types of solid phase immunoassays. Usually immunomagnetic particles directly capture the target analyte, or magnetic particles with immobilised streptavidin are used to capture the complex of biotinylated primary antibody and the analyte. The separated analyte is then determined (usually without elution) using an appropriate method. A combination of magnetic separation with affinity capillary electrophoresis is also possible [32].

Enzyme isolation is usually performed using immobilised inhibitors, cofactors, dyes or other suitable ligands, or magnetic beads prepared from affinity biopolymers can be used (see Tables 1, 2, 3, 4).

Genetic engineering enables the construction of gene fusions resulting in fusion proteins having the combined properties of the original gene products. To date, a large number of different gene fusion systems, involving fusion partners that range in size from one amino acid to whole proteins, capable of selective interaction with a ligand immobilized onto magnetic particles or chromatography matrices, have been described. In such systems, different types of interactions, such as enzyme-substrate, receptor-target protein, polyhistidines-metal ion, and antibody-antigen, have been utilized. The conditions for purification differ from system to system and the environment tolerated by the target protein is an important factor for deciding which affinity fusion partner to choose. In addition, other factors, including protein localization, costs for the affinity matrix and buffers, and the possibilities of removing the fusion partner by site-specific cleavage, should also be considered [33,34]. As an example, isolation of recombinant oligohistidine-tagged proteins is based on the application of metal chelate magnetic adsorbents [35,36]. This method has been used successfully for the purification of proteins expressed in bacterial, mammalian, and insect systems.

Antibodies from ascites, serum and tissue culture supernatants can be efficiently isolated using magnetic particles with immobilized Protein A, Protein G or anti-immunoglobulin antibodies. Protein A, isolated from Staphylococcus aureus, binds the Fc region of IgG of most mammalian species with high affinity, leaving antigen specific sites free. Protein G, isolated from Streptococcus sp., reacts with a larger number of IgG isotypes. It has a higher binding affinity to immunoglobulins than Protein A, however, it also interacts with the Fab regions of IgG, although the affinity is ten times lower than for the Fc region [37]. Antiphospholipid antibodies were successfully isolated using magnetoliposomes [15].

Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers binding proteins can be immobilised to magnetic particles and used for isolation of target proteins.

DNA/RNA binding proteins (e.g., promoters, gene regulatory proteins and transcription factors) are often short-lived and in low abundance. A rapid and sensitive method, based on the immobilization of biotinylated DNA/RNA fragments containing the specific binding sequence to the magnetic streptavidin particles, can be used. The bound DNA/RNA binding proteins are usually eluted with high salt buffer or change of pH [38].

Other types of proteins were isolated using specific affinity-based procedures. For example, plasminogen immobilized on magnetic particles was used to separate scrapie and bovine spongiform encephalopathy associated prion protein PrPSc from its conformer which is a cellular protein called PrPC. In fact, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes [39,40].

Magnetic separation was also successfully used for the recovery of proteins expressed in the form of inclusion bodies, involving at first chemical extraction from the host cells, then adsorptive capture of the target protein onto small magnetic adsorbents, followed by rapid collection of the product-loaded supports with the aid of high gradient magnetic fields [41].

A new approach for analytical ion-exchange separation of native proteins and proteins enzymatic digest products has been described recently [31]. Magnetite particles were covered with a gold layer and then stabilized with ionic agents. These charged stabilizers present at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins and peptides from aqueous solutions. The species trapped by the isolated particles were then characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) after a simple washing.

Magnetic solid phase extraction (MSPE) enables to preconcentrate target analytes from larger volumes of solutions or suspensions using relatively small amount of magnetic specific adsorbent. Up to now this procedure was used for preconcentration of low-molecular weight xenobiotics [42,43] but using suitable magnetic adsorbents the MSPE could be used to preconcetrate target proteins and peptides as well.

Sometimes the removal of certain proteins will reveal functions involving the depleted proteins or will help in the course of subsequent protein isolation. As an example, Dynabeads have been used to remove involved proteins from Xenopus egg extracts for analyses of the cell mitosis mechanisms [44,45]. Rapid removal of contaminating proteolytic enzymes from the crude samples could increase yields of sensitive proteins due to the limitation of their proteolysis [46].

A combination of mechanical cell disintegration and magnetic batch affinity adsorption was used to simplify the isolation of intracellular proteins. Magnetic glass beads were used because of their hardness and rigidity [1].

An example of quite different protein purification strategy can also be mentioned. Proteins associated with the endocytic vesicles of Dictyostelium discoideum were separated after magnetic isolation of the vesicles that was accomplished by feeding the amoebae with dextran-stabilized iron oxide particles. The cells were broken, the labelled vesicles were magnetically separated and then disrupted to release proteins which were resolved by SDS-PAGE. After „in-gel“ digestion with endoproteinase Lys-C or Asp-N the generated peptides were used for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles [47]. Analogous procedure was used for the separation and study of peroxisomes proteins when at first peroxisomes were separated using magnetic beads with immobilized specific antibodies and then the protein content of the separated peroxisomes was analysed [48].


Outline Conclusions

Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References



Standard liquid column chromatography is currently the most often used technique for the isolation and purification of target proteins and peptides. Magnetic separation techniques are relatively new and still under development. Magnetic affinity particles are currently used mainly in molecular biology (especially for nucleic acids separation), cell biology and microbiology (separation of target cells) and as parts of the procedures for the determination of selected analytes using magnetic ELISA and related techniques (especially determination of clinical markers and environmental contaminants). Up to now separations in small scale prevail and thus the full potential of these techniques has not been fully exploited.

It can be expected that further development will be focused at least on two areas. The first one will be focused on the laboratory scale application of magnetic affinity separation techniques in biochemistry and related areas (rapid isolation of a variety of both low- and high-molecular weight substances of various origin directly from crude samples thus reducing the number of purification steps) and in biochemical analysis (application of immunomagnetic particles for separation of target proteins from the mixture followed by their detection using ELISA and related principles). Such a type of analysis will enable to construct portable assay systems enabling e.g. near-patient analysis of various protein disease markers. New methodologies, such as the application of chip and microfluidics technologies, may result in the development of magnetic separation processes capable of magnetic separation and detection of extremely small amount of target biologically active compounds [49].

In the second area, larger-scale (industrial) systems are believed to be developed and used for the isolation of biologically active compounds directly from crude culture media, wastes from food industry etc., integrating three classical steps (clarification, concentration and initial purification) into a single unit operation [50]. It is not expected that extremely large amounts of low cost products will be isolated using magnetic techniques, but the attention should be focused onto the isolation of minor, but highly valuable components present in raw materials. Of course, prices of magnetic carriers have to be lowered and special types of low-cost, biotechnology applicable magnetic carriers and adsorbents prepared by simple and cheap procedures have to become available. The existence of inexpensive and effective magnetic separators enabling large-scale operations is necessary, as well.

In the near future quite new separation strategies can appear. A novel magnetic separation method, which utilizes the magneto-Archimedes levitation, has been described recently and applied to separation of biological materials. By using the feature that the stable levitation position under a magnetic field depends on the density and magnetic susceptibility of materials, it was possible to separate biological materials such as haemoglobin, fibrinogen, cholesterol, and so on. So far, the difference of magnetic properties was not utilized for the separation of biological materials. Magneto-Archimedes separation may be another way for biological materials separation [51].

It can be expected that magnetic separations will be used regularly both in biochemical laboratories and biotechnology industry in the near future.


Acknowledgements




The research is a part of ILE Research Intention No. AV0Z6087904. The work was supported by the Ministry of Education of the Czech Republic (Project No. ME 583) and Grant Agency of the Czech Academy of Sciences (Project No. IBS6087204).


Outline References

Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References


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10. Safarikova M, Roy I, Gupta MN, Safarik I: Magnetic alginate microparticles for purification of α-amylases.
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15. Rocha FM, de Pinho SC, Zollner RL, Santana MHA: Preparation and characterization of affinity magnetoliposomes useful for the detection of antiphospholipid antibodies.
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16. Zollner TCA, Zollner RD, de Cuyper M, Santana MHA: Adsorption of isotype "E" antibodies on affinity magnetoliposomes.
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http://www.devicelink.com/ivdt/archive/05/03/001.html

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Originally Published IVD Technology March 2005

Assay Development

Paramagnetic microparticles for optimized biological separations

Polymer-based particles with highly irregular surfaces enhance the

existing advantages of the technology through their large surface

area and high density.

Debra A. Sesholtz, Mitchell T. Gore, Robert L. Frescatore, and Kim

M. Stever


Magnetically responsive particles have been utilized for a variety of

laboratory applications, including nucleic acid separation, protein

purification, cell isolation, therapeutics, and diagnostics. Their use

can greatly simplify biological separations and reduce assay times.

Often, they eliminate the need for column chromatography or

centrifugation.

In addition, magnetic separation procedures are usually scalable,

both up and down, and often allow the establishment of systems

coupling fully automated, high-throughput separation with molecule

detection. As with most biological separation procedures, the issues

of binding capacity and specificity are critical. Higher binding

capacities characteristic of magnetic microparticles are usually

associated with increased sensitivity and lower cost per assay. And

magnetic carriers have recently been finding employment in clinical

and biomedical applications in the area of targeted drug delivery.

This article reviews applications for magnetic-particle-based

technology, with a focus on the enhanced characteristics of

irregular, high-surface-area magnetic particles.

Magnetic Particle Anatomy

Magnetic microparticles are composed primarily of ferrimagnetic

materials. In cell separations, the magnetic labels most commonly

used are ferrimagnetic magnetite (Fe3O4) and maghemite

(g-Fe2O3). Although the particles under discussion are often

referred to as magnetic—and for ease they will be here—they are,

strictly speaking, superparamagnetic. This term designates particles

that will be responsive to a magnetic field but that will not

themselves become magnetized. Such particles disperse easily once

they are removed from the magnetic field.

Some magnetic particles are polymer based. In these, the

magnetically responsive material is added to a polymer matrix by

one of two processes: either it is entrapped during the

polymerization process, or it is attached to the particle after

polymerization. The particles are then usually overgrown in either

case, in order to encapsulate the magnetic material and provide

functional groups on the surface.


Figure 1. A BioMag superparamagnetic particle is a crystalline

aggregate of magnetite encapsulated by aminosilane and, as shown

by this transmission electron micrograph, has a highly irregular

shape. Its surface area relative to mass is more than 100 m2/g (click

to enlarge).
Magnetic particles can also be composed of crystalline aggregates

that are encapsulated by any of a variety of polymers such as

polystyrene, dextran, or silanes. With this type of particle, as with

polymer-based particles, it is important that the magnetic material

be completely encapsulated. This is to prevent leaching of the iron

complexes, which can cause problems in biological systems.

Practical Advantages

The introduction of magnetic particles in biological applications such

as immunology, cell separation, molecular biology, and diagnostics

has made processes for separating cells, DNA, and proteins faster

and easier.

In cell separation applications, a particular type of cell can be

isolated from a complex mixture by numerous means, among them

fluorescence-assisted cell sorting via flow cytometry;

density-gradient-based methods; and magnetic- particle-based

methods. Cell isolations based on magnetically responsive particle

technology offer many advantages over other procedures. The main

one is that they are relatively simple to perform. Target cells can be

isolated from fairly crude mixtures, including blood, tissue

homogenates, stool, soil, microbial culture media, water, and food,

among others. Also, the procedures are gentle, can be scaled up or

down as necessary, and can replace centrifugal, filtration, or

chromatographic separation. These attractive properties allow the

development of automated cell isolation systems.

Magnetic particles are used also in the study of biological processes

using whole cells, where the goal may be simply to detect the

presence of pathogens such as Escherichia coli, Salmonella, and

Staphylococcus in clinical, food, or environmental samples.1-3

Another objective may be to detect the expression of a eukaryotic

cell surface marker.4,5 Researchers may also find it useful to isolate

a particular cell type in order to study it in a controlled manner. In

converse fashion, they may deplete a cell type by means of

magnetic-particle-based separation in order, by its absence, to study

its influence on biological processes.

For the most part, cell separations are performed by using an

antibody– magnetic-particle complex; however, antigens can also be

attached for the capture of antigen-specific cells such as

antibody-producing cells from hybridoma cultures and

antigen-specific B-cells, and also for the biopanning of antibody

phage-display libraries.6,7 Lectins as well can be attached to the

surfaces of magnetic particles and used as capture molecules for

polysaccharides and glycoproteins expressed on the surfaces of

eukaryotic and prokaryotic cells. In the reverse approach, magnetic

particles with attached oligosaccharides can be used to isolate

lectin-expressing cells.7

Cell separation has been used in clinical applications for the

identification and isolation of circulating tumor cells in peripheral

blood.8 Another use has been the depletion of T-cells from donor

bone marrow in order to combat graft-versus-host disease in

patients receiving bone marrow transplants.9

Magnetic particles are also employed extensively to purify nucleic

acids, proteins, and cellular organelles. While these types of

separations can be affinity based, like cell separations, they can also

rely largely on the principles of conventional chromatographic

separations. Magnetic-particle-based separations of nucleic acids

have been performed by means of ion-exchange functionalized

particles, silica-coated particles, nonspecific absorption, and

sequence-specific oligonucleotide capture. Several manufacturers

provide products suitable for protein purification via magnetic

particles functionalized with capture molecules. Particles

functionalized with nickel or glutathione molecules can be used to

purify recombinant proteins containing 6X His or GST affinity tags;

protein A or protein G for immunoglobulin purification; and particles

containing secondary or primary antibodies for magnetic

immunoprecipitation.

A Mobile Surface Area

Magnetic separations are somewhat analogous to chromatographic

separations in that an increase in particle surface area usually

results in larger binding capacity. The major difference, of course, is

that, in the former, the solid phase is mobile and responsive to

magnetic fields.

One manufacturer, Polysciences Inc. (Warrington, PA), has

developed superparamagnetic particles that have an irregular,

bumpy surface, which dramatically increases the surface area of the

particle. Trade named BioMag, these particles are made of a

crystalline magnetite formulation encapsulated with aminosilane

(see Figure 1). Their irregular surface provides 20 to 30 times more

surface area than similar-sized spherical particles. An irregular

magnetic particle 1 m in breadth has a surface area in excess of

100 m2/g, as opposed to 4–8 m2/g for a 1-m-diameter spherical

particle.

The greater surface area of the irregular particles translates into

greater binding capacity. When compared with a similar-sized

spherical particle in experiments, BioMag oligo (dT) particles bound

significantly larger amounts of polyadenylated messenger RNA

(mRNA) (see Figure 2). In another experiment, BioMag streptavidin

particles and two other magnetic streptavidin particles that were

spherical were used to bind radiolabeled biotinylated

oligonucleotides. The irregular particles bound approximately twice

as much as one type of spherical particle and more than eight times

the amount bound by the other spherical particle (see Figure 3).

Particle size can influence cell separation results, as well.

Experiments were conducted to compare equal masses of 1-m

irregular magnetic particles with 1.8-m irregular magnetic particles

for depletion of CD45 positive white blood cells. Use of the smaller

particles resulted in levels of depletion approximately 15% greater

at the highest volume of particles tested (see Figure 4). Again, the

higher capture rates of the smaller (BioMagPlus) particles are

attributable to their higher ratio of surface area to mass.

The size of magnetic particles used in biological separations can

vary dramatically. As might be expected, the size of the particle in

large part determines how it will behave in solution and what type of

magnetic separation device will have to be used. Magnetic particles

can be broadly grouped as large particles (0.75 to about 5 m) and

small particles (less than 0.75 m, and mainly 10–200 nm).

All three sizes are used in cell and antigen capture applications. In

the case of the colloidal labels, cell labeling kinetics are quite rapid;

little or no mixing is required.7 The effects of Brownian motion on

larger particles is minimal, making mixing of these particles

necessary for efficient antigen capture.

Size is a factor to consider not only in regard to mixing kinetics; the

effect of magnetic-particle size on magnetic responsiveness also is

important. While there are some exceptions, in general, the smaller

the magnetic particle, the stronger the magnetic field needed for

effective separation. This can be confounded, however, by

differences in the amount of magnetite or maghemite contained in

the particles. For example, when larger particles containing small

amounts of magnetically responsive material are used, longer

separation times or more-powerful magnetic fields are required.

Shape, Density, and Hydrodynamics



Figure 2. The isolation of polyadenylated mRNA by irregularly

shaped magnetic oligo (dT)20 (BioMag) and by spherical

(Competitor D) magnetic oligo (dT)20 particles was compared

experimentally over a range of particle amounts. The irregular

particles isolated significantly more mRNA at all levels, this greater

binding capacity reflecting the greater surface area (click to

enlarge).
The relationship of the hydrodynamic motion of magnetic particles

to the capture of bacteria has been recently described.1,2 In these

studies, the motion of the particles in an aqueous suspension was

considered to be controlled by gravity, buoyancy, and friction. The

authors used the mass of the particle, the density of the solution, the

density of the particle, gravitational acceleration, the viscosity of

the solution, and the radius of the particle to predict mathematically

the total volume of solution traversed by the particles.

An important distinction must be noted: whereas the hydrodynamic

radius of spheres is the same as the radius of the sphere, the

hydrodynamic radius of nonspherical particles is determined

primarily by their shape.1 The quantitative expression developed by

the researchers predicts that particles with larger mass, higher

density, and a nonspherical shape favor bacterial capture.


Figure 3. Irregularly shaped streptavidin-conjugated BioMag

particles and two different spherical polymer

streptavidin-conjugated magnetic particles were compared for their

ability to bind radiolabeled biotinylated oligonucleotides. The

maximum binding for the irregular particles was just under 200

pmol, compared with approximately 100 and 15 pmol for the other

particles. These results reflect, in part, differences in surface area

among the three types of particles (click to enlarge).
Streptavidin-conjugated irregular particles with a surface area of

1000 cm2/mg and a density of 2.5 g/ml were compared with an equal

mass of spherical magnetic streptavidin-conjugated polymer

particles having a surface area of 80 cm2/g and density of 1.3 g/ml,

using biotinylated antibodies to E. coli O157. In this comparison,

capture of E. coli was two to three times greater when particles of

higher density were used, and capture required less mixing time with

these higher-density particles.1 Additionally, when the capture

efficiencies of high-density smaller particles (1-m particles) and

high-density larger particles (18-m particles) with the same

antibody content were compared, the larger particles exhibited more

than 40 times the capture of E. coli O157 after 60 minutes of

incubation.2 This agrees with the mathematical model the

researchers developed, which predicted that the larger particles

would have a total sedimentation volume more than 100,000 times

that of the smaller particles.

In other words, the larger and more dense the particle, the more

volume the particle will be exposed to during the mixing and capture

phase of the assay. Therefore, according to these studies, when

antibody concentration is held constant, the rate-limiting step

controlling bacterial capture is the frequency of the

bacteria-particle collision, which is favored when particles of greater

density and size are used.

Applications

Many reviews have been published on the use of magnetic-particle

technology in genomics and proteomics, drug discovery, biomedicine,

and clinical applications.10-13 Biological and biomedical applications

of magnetic-particle technology have been employed widely for

DNA, RNA, protein, and cell separations in genomic, proteomic, and

immunological research. The ability to automate magnetic-particle

separations and obtain high-purity products while reducing reagent

costs, eliminating laborious and time-intensive steps, and minimizing

the mechanical stress to which products are exposed makes the

technology attractive for high-throughput applications such as

sample preparation. And in drug discovery, the screening of large

numbers of compounds to identify potential drug candidates is

another application for which automated high-throughput

magnetic-particle technology is well suited.


Figure 4. Results of the experimental depletion of CD45 positive

white blood cells by irregularly shaped BioMag (1.8-m) and

BioMagPlus (1-m) amine particles conjugated to anti-CD45

antibodies. For both particles, the greater the volume of particles,

the greater the depletion percentage. BioMag Plus, with a smaller

average diameter, presents an increased surface area, which is

reflected in greater capture rates (click to enlarge).
Magnetic-particle-based technology has also been used for clinical

screening and diagnostic applications. Their ability to covalently

couple with proteins, enzymes, antibodies, and other ligands makes

magnetic particles suitable for direct use in bioassays or as affinity

ligands for the capture of target molecules and cells.10 The ability of

these particles to bind protein molecules without significantly

changing the biochemical activity of the proteins is the basis for

magnetic-particle-based immunoassays. Magnetic particles coated

with leukocyte-specific antibodies have been used to detect and

remove tumor cells from whole blood samples, bone marrow, and

prepared mononuclear cells, and have enabled subsequent

diagnosis.11

In addition to the analysis of clinical samples, magnetic particles

have been used for the detection of pathogenic organisms in food

and environmental samples. Particles are coated with antibodies

targeted to surface-specific proteins on the microorganism, then

testing to identify the organism is conducted.
The relative simplicity and speed of procedures based on

magnetic-particle technology well suits the design of rapid diagnostic

tests. Because target molecules can be isolated from fairly crude

mixtures, sample preparation is minimized. Also,

magnetic-particle-based technology allows target molecules to be

concentrated in order to increase signal intensities, thus enhancing

detection.

In addition to magnetic-particle technology lending itself well to

automation for high-throughput sample preparation for biological

and biomedical research, the use of magnetic particles in drug

targeting and delivery for clinical applications is also being actively

investigated. Magnetic particles bound with established drugs can be

employed for site-specific drug targeting in the human body using a

magnetic field.13 Site-specific targeting can increase the amount of

drug delivered directly to the target area, while at the same time

minimizing systemic distribution or exposure to normal cells.

More-localized targeting of a drug may also make possible reduction

in the dosage.12 Many types of targeted drug delivery systems are

being investigated, including magnetoliposomes, magnetic

nanoparticles, and biodegradable magnetic particles.13

For magnetic particles to be effective for drug targeting, they must

be smaller than, or comparable in size to, a cell, virus, protein, or

gene so that they can get near the target of interest.12 In

performing targeted drug delivery, biocompatible ferrofluids or

colloidal suspensions of magnetic particles in a liquid carrier are

injected into the body. A strong magnetic field gradient is produced

over the target site outside the body, drawing the particles to the

target region.

The first reports of magnetic-particle targeting of sarcoma tumors

in rats with a cytotoxic drug (doxorubicin) were encouraging.12 This

has led the way to continued investigation and, more recently,

preliminary human trials. In addition to treating tumors and

cancerous cells, magnetically controlled drug targeting may be used

to deliver other types of drugs, such as antiinflammatories, steroids,

antibiotics, and any others that can be bound reversibly to

ferrofluids.

The future holds promise of increased use of magnetic-particle

technology and delivery systems. One potential application is the

transfer of genes into cultured cells or bacteria for the production of

pharmacological biological products.12 Magnetic particles might also

serve as detection probes to replace current radioactive,

fluorescent, or chemiluminescent modes of detection.10 And

specifically targeted magnetic particles could be used to initiate a

biochemical reaction within a cell.12

Conclusion

The various uses of magnetic particles for biological separations

speak to the many benefits they confer. Biomagnetic separations are

simple, robust, scalable, and amenable to automation. Furthermore,

many options exist with regard to magnetic-particle characteristics,

allowing selection of a type of particle that suits an application well.

Success in biomagnetic separation procedures is highly dependent on

the specificity and avidity of the capture molecule, the abundance of

target, and other factors. Particle morphology, size, and density are

important for the significant influence they can have on separation

effectiveness. For example, particle diameter being constant,

particles having irregular shape will present more surface area to

the separation and demonstrate better capture of target analytes.

Additionally, recent studies have shown that factors that influence

the hydrodynamics of mixing can strongly affect the efficiency of

pathogen capture, with particles of greater density and larger

diameter exhibiting higher levels of pathogen capture.

The wide use of magnetic particles in biological separations has led

to interest and research in their potential for clinical applications.

The ability to couple specific drugs to magnetic microparticles small

enough to enter the body has led to investigation into the use of

magnetic particles as targeted drug-delivery systems, as well as

studies of their utility for gene transfer and in vivo tracking. The

specificity and sensitivity of magnetic-particle technology promises

to stimulate the development of a range of future applications.

References

1. SI Tu et al., “The Capture of Escherichia coli O157:H7 for Light

Addressable Potentiometric Sensor (LAPS) Using Two Different

Types of Magnetic Beads,” Journal of Rapid Methods and

Automation in Microbiology 10 (2002): 185–196.

2. SI Tu et al., “Factors Affecting the Bacterial Capture Efficiency

of Immuno Beads: A Comparison between Beads with Different Size

and Density,” Journal of Rapid Methods and Automation in

Microbiology 11 (2003): 35–46.

3. MJ Payne, S Campbell, and RG Kroll, “Lectin-Magnetic

Separation Can Enhance Methods for the Detection of

Staphylococcus aureus, Salmonella enteritidis, and Listeria

monocytogenes,” Food Microbiology 10 (1993): 75–83.

4. A Thiel, A Scheffold, and A Radbruch, “Immunomagnetic Cell

Sorting—Pushing the Limits,” Immunotechnology 4 (1998): 89–96.

5. JT Kemshead and J Ugelstad, “Magnetic Separation Techniques:

Their Application to Medicine,” Molecular and Cellular

Biochemistry 67, no. 1 (1985): 11–18.

6. C Sawyer, J Embleton, and C Dean, “Methodology for Selection

of Human Antibodies to Membrane Proteins from a Phage-Display

Library,” Journal of Immunological Methods 204 (1997): 193–203.

7. I Safarik and M Safarikova, “Use of Magnetic Techniques for the

Isolation of Cells,” Journal of Chromatography B722 (1999): 33–53.

8. U Bilkenroth et al., “Detection and Enrichment of Disseminated

Renal Carcinoma Cells from Peripheral Blood by Immunomagnetic

Cell Separation,” International Journal of Cancer 92 (2001):

577–582.

9. F Vartdal et al., “Depletion of T Lymphocytes from Human Bone

Marrow. Use of Magnetic Monosized Polymer Microspheres Coated

with T Lymphocyte Specific Monoclonal Antibodies,”

Transplantation 43 (1987): 366–371.

10. ZM Saiyed, SD Telang, and CN Ramchand, “Application of

Magnetic Techniques in the Field of Drug Discovery and

Biomedicine,” Biomagnetic Research and Technology 1, no. 2

[online] (2003); available from Internet:

http://www.biomagres.com/content/1/1/2.

11. MB Meza, Designing Microsphere-Based Tests and Assays (The

Latex Course) (Fishers, IN: Bangs Laboratories, 2002).

12. QA Pankhurst, J Connolly, SK Jones, and J Dobson,

“Applications of Magnetic Nanoparticles in Biomedicine,” Journal of

Physics D: Applied Physics 36 (2003): R167–R181.

13. AS Lbbe, C Alexiou, and C Bergemann, “Clinical Applications

of Magnetic Drug Targeting,” Journal of Surgical Research 95

(2001): 200–206.

Debra A. Sesholtz is laboratory products business manager at

Polysciences Inc. (Warrington, PA). Mitchell T. Gore, PhD, formerly

of Polysciences, is now regional manager, mid-Atlantic, at

Integrated DNA Technologies Inc. (Coralville, IA). Robert L.

Frescatore is a senior chemist, and Kim M. Stever is an application

specialist at Polysciences. The authors can be reached at

sesholtz@polysciences.com, mgore@idtdna.com,

rfrescatore@polysciences.com, and kstever@polysciences. com,

respectively.

Copyright 2005 IVD Technology

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192MAGNETIC SOLID-PHASE EXTRACTION OF TARGET ANALYTES FROM LARGE VOLUMES OF URINEM. Safarikova & I. SafarikLaboratory of Biochemistry and Microbiology, Institute of Landscape Ecology, Na Sadkach 7, 370 05 Ceske Budejovice, Czech RepublicINTRODUCTION: Analysis of urine is often used to detect the presence or determine theconcentration of various biologically activecompounds or xenobiotics. Urine has become thepreferred specimen since many compounds can bedetected for longer time periods in urine than inblood. Furthermore, urine collection does notrequire phlebotomy, and urine is typically an ample and stable sample. In most cases the concentrationof target analytes is low and thus the analytes haveto be preconcentrated before the application of anappropriate analytical procedure. There is a wide range of preconcentrationprocedures available that can be used individuallyor sequentially according to the complexity of thesamples, the nature of the matrix, the analytes, and the instrumental techniques available. The use of an extraction technique is common in the pretreatment of most types of sample. Both liquid-liquid andsolid-phase extraction procedures are widely usedfor the urine analytes analysis.A great increase in the use of solid-phase extraction (SPE) as a preconcentration step has occurredrecently. Solid-phase extraction can effectivelyhandle small samples using only small volumes oforganic solvents and very simple equipment(usually a small column and a syringe). In some cases, however, it is needed to extract very lowamounts of the target analytes from larger volumes of samples. In this case standard SPE requiresadditional equipment such as solid-phase extraction vacuum manifolds.Recently a new extraction procedure, calledmagnetic solid-phase extraction (MSPE), has alsobeen developed [1]. This procedure based on theadsorption of the target analyte on relatively smallamount of magnetic specific adsorbent enables tohandle liter volumes of samples. Using MSPE with subsequent elution very low concentrations in ppb(g/L) range of some compounds can be detected.There are two main advantages of this procedurewhen compared with standard column SPE. Atfirst, it is the possibility to perform the extractionsteps in a very simple way, without the need ofexpensive equipment, even using larger amount ofsample (up to 1000 mL of liquid can bemanipulated without problems). At second, MSPEcan be performed not only in solutions, but also insuspensions. This is a general advantage ofmagnetic separation techniques due to the fact thatmajority of accompanying impurities arediamagnetic and do not interfere with magneticparticles during the magnetic separation step. The use of MSPE in urine analysis was tested using silanized magnetite particles with immobilizedreactive copper phthalocyanine dye (C.I. ReactiveBlue 21; see Fig. 1) as an affinity ligand. Thisligand interacts specifically with polyaromatichydrocarbons having three or more conjugated rings [2] and with diamino- and triaminotriphenylmethane dyes [3]. This adsorbent can thus be used for thepreconcentration of various compounds with proven or suspected carcinogenic properties [2]. Fig. 1: Possible structure of the C.I. Reactive Blue 21. R – reactive linker arms of the followingstructure: SO2NHC6H4SO2CH2CH2OSO3HFor experiments crystal violet (Basic Violet 3, CI42555; also known as gentian violet orhexamethylpararosaniline chloride; chemicalstructure is shown in Fig. 2) was used as a modelanalyte. This dye belongs into the letter group ofcompounds interacting with copper phthalocyaninederivative. Because of its potential cancerousEuropean Cells and Materials Vol. 3. Suppl. 2, 2002 (pages 192-195) ISSN 1473-2262
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193property FDA put this dye on the Food and DrugAdministration’s (FDA’s) priority list for drugs that need analytical methods development.Fig. 2: Chemical structure of crystal violet Concern about the health risks and carcinogenicityassociated with the use and contact with variousdyes and other xenobiotics requires a routinelaboratory method to be developed to monitor thepresence of target dyes and dye metabolites in body fluids. In this paper we demonstrate the use ofMSPE for the preconcentration of a model dye(crystal violet) from human urine. Due to the factthat this dye exhibits high extinction coefficient(112,000 M-1cm-1at 590 nm) subsequent spectrophotometric detection can easily beperformed. METHODS: In model experiments fine magnetiteparticles with immobilized reactive copperphthalocyanine dye (blue magnetite) were used asan affinity adsorbent. Blue magnetite was preparedin a similar way as described recently [3]. Iron(II,III) oxide (10 g; Aldrich, USA) wassuspended in 5 % nitric acid and boiled in aclosed vessel at 100 C for 60 minutes. Afterthorough washing with distilled water, 40 mL of10 % water solution (pH 4.0) of 3-aminopropyl-triethoxysilane (Sigma, USA) were added to thesedimented magnetite. The suspension was mixed in a water bath at 80 C for 4 h. Then the silanizedmagnetite was thoroughly washed with water,suspended in 200 mL of water and mixed with 4 gof Ostazin turquoise V-G (C.I. Reactive Blue 21;produced by Spolek pro chemickou a hutni vyrobu, Usti nad Labem, Czech Republic) and 12 g ofsodium chloride. The suspension was warmed to 70 C and 15 min later 10 g of anhydrous sodiumcarbonate were added. The suspension was stirred at 70 C for 4 h and then the mixture was left overnight at ambient temperature without mixing. The blue magnetite particles were thoroughlywashed with water and the remaining free dye wasremoved using an extraction with methanol in aSoxhlet apparatus. The extracted particles werethen repeatedly washed with methanol – 2 % acetic acid (50:1, v/v). The washed blue magnetiteparticles were stored in water at 4 C. The dryweight of 1 mL of the settled blue magnetite was322 mg. The copper phthalocyanine content of theblue magnetite was ca 80 mol per g of the dryadsorbent (determined from elemental analysis forcopper using a PU 7450 ICP spectrometer (Pye-Unicam, England) after mineralization of bluemagnetite with concentrated nitric acid).Various volumes of human urine (usually 100 mLor more; obtained from healthy male donors) werespiked with various amounts of crystal violet andsubsequently 400 L of water suspension ofmagnetic adsorbent (corresponding to 100 L ofsedimented adsorbent) was added. The suspensionwas stirred for 3 – 4 hours at room temperature.After that magnetic particles were captured to thebottom of the beaker or flask using an appropriatemagnet or flat magnetic separator and urine waspoured off taking care not to lose the adsorbent.The adsorbent was washed several times with water and transferred into a test tube (test tube magneticseparators MPC-1 or MPC-6 from Dynal, Norway were used for magnetic separation). After washingand removing all water 1.5 mL of methanol – 2 %acetic acid (50:1, v/v) solution was added to theadsorbent. The elution proceeded for ca 20 minutes. The eluate was used for the spectrophotometric measurement when the position of the crystal violet peak was verified using a crystal violet standard. RESULTS: Crystal violet was used as a model dye in order to test a new preconcentration procedureMSPE (magnetic solid-phase extraction) for thedetection and determination of low concentration of target analytes in urine. The procedure wasdeveloped for standard spectrophotometers havingthe possibility to record spectra in visible region oflight and for use of standard semi-microcuvettes (working volume ca 1.5 ml). In previous experiments magnetic carrier withimmobilized copper phthalocyanine dye (bluemagnetite) efficiently adsorbed chromatic form ofcrystal violet from water solutions [3]. Nevertheless this dye can also be separated from other liquidsamples, such as urine. It was shown in preliminary experiments that majority of the tested dye (added
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194in amounts 0.25 - 1 g into 100 mL of urine) was adsorbed on blue magnetite in approximately 120min; further prolongation of the incubation time did not improve the dye adsorption and subsequentrecovery (data not shown). This incubation period(ca 2 hours) was used in all further experiments. Fig. 3 shows an example of eluates spectra obtained after MSPE of crystal violet from male urine. It can be seen that very low amounts of the dye can bedetected spectrophotometrically. After MSPEclearly visible peak of crystal violet can be found at the concentration 0.5 g/100 mL urine (i.e., 5 ppb) and even at the half concentration (2.5 ppb). Itcorresponds to the concentration of crystal violet 6 - 12 nmol/L. The samples showing a peakcorresponding to that one of crystal violet (with themaximum at ca 585 - 590 nm) can be considered as presumptive positive and the presence of crystalviolet can be verified e.g. using a HPLC procedure. Fig. 3: Spectra of a standard and eluates (1.5 mL) after magnetic solid-phase extraction of crystalviolet from 100 mL of male urine. A – controlsample (unspiked urine); B – urine spiked with0.25 g of crystal violet; C – urine spiked with 0.5 g of crystal violet; D – urine spiked with 1.0 g of crystal violet; E – crystal violet (1.0 g per 1.5 mL of the elution solution).The recovery of crystal violet was ca 60 – 75 %depending mainly on the batch of magnetic affinityadsorbent used for the MSPE. The reproducibilityof the technique was determined from five replicatemeasurements for one concentration (1.0 g per100 mL of urine) of crystal violet. The typicalrelative standard deviation of the adsorption andelution efficiencies (measured as the absorbance ofthe eluate at the peak maximum) was 9.3 %. DISCUSSION & CONCLUSIONS: Isolation and preconcentration of compounds present in solutions or suspensions in very low concentrations belongsto the basic operations in analytical, bioanalyticaland environmental chemistry. The aim of this work was to develop a simple and ready to use procedure for the preconcentration and subsequent detectionor determination of low concentrations of targetanalytes in urine. Crystal violet was chosen as amodel compound because it belongs (together withe.g. malachite green) to a group of compounds that are linked to an increased risk of cancer [4]. Thisdye is commonly used for many purposes (e.g. such as an antimicrobial or antifungal agent)nevertheless its use has already been controlled insome individual member states within the European Community.The results confirmed the applicability of MSPEprocedure for a treatment of biological samples,specifically urine. Using MSPE low concentrations (5 ppb) of the native dye (in a chromatic form) can be detected spectrophotometrically. Of course,different situation can be expected if the dyes (orother xenobiotic compounds) enter the human body and their metabolic modification takes place. Rapid metabolic conversion of crystal violet to thecolorless leuco form can be expected followed by its slow release into urine. The leuco form cannot besimply separated using the described procedure. An appropriate oxidizing process may lead to theconversion of leuco form into the chromatic form of crystal violet, which could then be separated andsubsequently detected using the describedprocedure. The optimized oxidation /preconcentration procedure for the detection ofmetabolically modified crystal violet will be studied soon. It can be supposed that MSPE can also be used for the preconcentration of other xenobiotics from urine provided that suitable affinity ligands (e.g.,antibodies) will be available. MSPE could thus beefficiently used as a simple screening andpreconcentration procedure that can be followed by a more precise chromatography analysis in casesuspected urine samples are found. Currently wetest other types of magnetic adsorbents (bothspecific and non-specific ones) which could be used for magnetic solid-phase extraction of variousxenobiotics from human urine.
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195It can also be expected that MSPE could be usedfor the isolation and preconcentration of importantminority biologically active compounds directly from urine and other body fluids such as blood and milk. REFERENCES: 1 M. Safarikova and I. Safarik(1999) J Magn Magn Mater 194: 108-112.2 H. Hayatsu (1992) J. Chromatogr 597: 37-56. 3I. Safarik, M. Safarikova and N. Vrchotova(1995) Collect Czech Chem Commun 60: 34-42. 4J.J. MacDonald and C.E. Cerniglia (1984) Drug Metab Dispos 12: 330-336.ACKNOWLEDGEMENTS: The research is apart of ILE Research Intention No. AV0Z6087904. The experimental work was supported by theNATO Collaborative Linkage Grant No.LST.CLG.977500, Ministry of Education of theCzech Republic (Project No. OC 523.80) and Grant Agency of the Czech Academy of Sciences (Project No. S6087204).

_________________________________________________________


particles are not attracted to each other

Since there is usually no magnetic re-manence the particles are not

attracted to each otherand therefore they can be easily suspended

into a ho-mogeneous mixture in the absence of any externalmagnetic

Reld.
--------------------------------------------------------------------------------
Page 1
theories and technologies that will be needed to bringthese ideas to

fruition. The tasks are challenging, evenformidable, and require the

commitment of scientistsand engineers as well as the provision of

vast fundsfor research, development and capital investment

bygovernments and industry.Further ReadingCarberry JJ (1976)

Chemical and Catalytic Reaction Engin-eering. New York:

McGraw-Hill.Choudry VR and Doraiswamy R (1971) Industrial

Engine-ering and Chemical Product Research and Development10:

218.Durand JP, Boscher Y and Dorbon M (1990) Journal

ofChromatography 404: 49.Emmett PH (1957) Advances in

Catalysis, vol. 9, p. 645.New York: Academic Press.Hall WK,

MacIver DS and Weber HP (1979) Industrial andEngineering

Chemistry 52: 425.Guichon G and Gonnard F (1979) Analytical

Chemistry51: 379.Mile B, Adlard ER, Roberts GM and Sewell PA

(1988)Catalysis Today 2: 685.Mile B, Ryan TA, Tribeck TD,

Zammitt MA and HughesGA (1994) Topics in Catalysis 1: 153.Mile

B, Howard JA, Tomietto M, Joly HA and Sayari(1996) Journal of

Materials Science 31: 3080.Petroff N, Hoscheitt A and Durand D

(1993) HydrocarbonProcessing (International Edition) 72:

103.Rideal EK and Taylor HS (1919) Catalysis: Theory andPractice.

London: Macmillan.Rooney JJ (1985) Journal of Molecular

Catalysis 31:147.Somorjai GA (1984) Chemical Society Review 13:

321.Zelter MS (1993) Hydrocarbon Processing

(InternationalEdition) 72: 103.CATALYST STUDIES:

CHROMATOGRAPHYIsolation: Magnetic TechniquesI. S[ afar\

eHk and M. S[ afar\ eHkovaH , Institute of LandscapeEcology,

Academy of Sciences,C[ eske& Bude\ jovice, Czech

RepublicCopyright^2000 Academic PressThe Rrst experiments with

magnetic separation ofcells date from the 1950s when lymphocytes

weremagnetically separated after in vitro phagocytosis ofiron

granules. The real boom in the application ofmagnetic labels for cell

isolation came in the 1980sand since then an enormous amount of

work has beendone in both the development and application of

thistechnique.Magnetic separation of cells has several advantagesin

comparison with other techniques. In general, themagnetic

separation procedure is gentle, facilitatingthe rapid handling of

delicate cells in an unfriendlyenvironment. It permits the cells of

interest to beisolated directly from crude samples such as blood,bone

marrow, tissue homogenates, stools, food, culti-vation media, soil

and water. The cells isolatedby magnetic separation are usually

pure, viable andunaltered. Magnetic separation is a simple, fast

andefRcient procedure and the whole separation processcan be

performed in the same tube. Large differencesbetween magnetic

permeabilities of the magnetic andnonmagnetic materials can be

exploited in developinghighly selective separation methods. The

separationprocedure can easily be scaled up if large quantities

ofliving cells are required.Principles of Magnetic Separationof

CellsTwo types of magnetic separation can be distin-guished when

working with cells. In the Rrst type,cells to be separated

demonstrate sufRcient intrinsicmagnetic moment so that magnetic

separations canbe performed without any modiRcation. There

areonly two types of such cells in nature: magnetotacticbacteria

containing small magnetic particles withintheir cells and red blood

cells (erythrocytes) contain-ing high concentrations of paramagnetic

haemoglo-bin. In the second type, cells of interest have to betagged

by a magnetic label to achieve the requiredcontrast in magnetic

susceptibility between the label-led and unlabelled cells. The

attachment of magneticlabels is usually attained by the use of

afRnity ligandsof various types, which can interact with

targetstructures on the cell surface. Usually

antibodies2260III/CATALYST STUDIES: CHROMATOGRAPHY/

Isolation: Magnetic Techniques
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Page 2
against speciRc cells surface epitopes are used, butother speciRc

ligands can also be employed. Thenewly formed complexes have

magnetic propertiesand can be manipulated using an appropriate

mag-netic separator.The magnetic separation process for the

puriRca-tion of target cells usually consists of the followingthree

fundamentals steps:1. The suspension containing cells of interest

ismixed with magnetic labels. Incubation time isusually not longer

than 30}60 min. Then the mag-netic complex formed is separated

using a mag-netic separator and the supernatant is discarded orused

for another application.2. The magnetic complex is washed several

times toremove unwanted contaminants. The selected cellswith

attached magnetic labels can be used directly,e.g. for cultivation

experiments. Alternatively,cells can be disrupted and the cell

content analysedusing various methods.3. If necessary, a variety of

detachment procedurescan be used to remove the magnetic labels

fromthe separated cells. After detachment, the mag-netic label is

removed from the suspension ina separator and free cells are ready

for furtherapplications and analyses.Necessary EquipmentMagnetic

labels and magnetic separators are neces-sary to be able to perform

efRcient cell separation.Typical examples are given below.Magnetic

LabelsWith the exception of erythrocytes and

magnetotacticbacteria, the cells to be isolated have to be

magneti-cally labelled in order to be amenable to

magnetictreatment. Magnetic labelling can be performed

withmagnetic and superparamagnetic particles (c. 1 mand more in

diameter), magnetic colloids (c.50}200 nm), magnetoliposomes or

with molecularmagnetic labels. In most cases the magnetic

propertiesof the labels are caused by the presence of smallparticles

of magnetite (Fe3O4) or maghemite ( -Fe2O3); in some cases,

chromium dioxide particles orferrite particles have been

used.Magnetic and superparamagnetic particles Mag-netic and

superparamagnetic particles (typical dia-meter c. 1}5 m) attached to

the target cells can easilybe removed from a suspensionwith a simple

magneticseparator. Since there is usually no magnetic re-manence

the particles are not attracted to each otherand therefore they can

be easily suspended into a ho-mogeneous mixture in the absence of

any externalmagnetic Reld. Magnetic particles typically

compriseRne grains of iron oxides dispersed throughout theinterior

of a polymer particle (in many cases ofa monosized type), the

surface chemistry of which canbe modiRed to provide a range of

different linkingmethods. Alternatively, silanized particles of

mag-netic iron oxides or magnetic porous glass can be usedfor the

same purpose.A number of particulate magnetic labels can

bepurchased commercially. Up to now, in most applica-tions,

monosized polymer particles marketed asDynabeads (Dynal, Oslo,

Norway) in various formshave been used. Dynabeads are prepared

from mono-sized macroporous polystyrene particles which

aremagnetized by an in situ formation of ferromagneticmaterial

inside the pores. Other commercially avail-able magnetic particles

can be successfully used forcell separation. A selection of these

products can befound in Table 1.Colloidal magnetic labels Colloidal

magnetic labels(typical size c. 50}200 nm) are prepared by a

varietyof methods which result in Socks composed of poly-mer

(typically dextran, starch or protein) and mag-netite and/or other

iron oxide crystals. A standardprocedure for the synthesis of

superparamagneticdextran nanospheres is performed by

precipitationof iron oxide in the presence of the polysaccharide.To

such materials, ligands (usually antibodies, lec-tins, streptavidin or

biotin) are coupled so they canbe used for cell separation. Using high

gradientmagnetic columns, the labelled cells are easily separ-ated.

Magnetic particles isolated from magnetotacticbacteria (50}100 nm)

composed of magnetitecovered by a stable lipid membrane can also

be usedsuccessfully.Magnetoliposomes Magnetoliposomes are

mag-netic derivatives of ordinary liposomes prepared

byincorporation of colloidal magnetic particles into thelipid vesicles.

When magnetoliposomes are associatedwith antibodies they can

label and/or selectively con-centrate the target cells.Molecular

magnetic labels Molecular magneticlabels are usually lanthanides

(especially erbium), fer-ritin and magnetoferritin. Erbium in the

form of er-bium chloride (ErCl3) has been used for

magneticlabelling of a variety of cells. Different Er3#bindingsites,

such as carboxyl groups in glycoproteins or theCa2#receptor sites on

the cell wall are responsible forthe binding of erbium

ions.III/CATALYST STUDIES: CHROMATOGRAPHY/ Isolation:

Magnetic Techniques2261
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Page 3
Table 1 Selected examples of commercially available magnetic

particles and colloidal magnetic labels used or usable for

magneticseparation of cellsNameDiameter ( m)

Polymercomposition/surfacemodificationEnd groups andactivation

possibilityImmobilizedcompoundsManufacturer/supplierBioMag&1S

ilanized iron oxides }COOH}NH2Secondary Abs,anti-CD

Abs,anti-fluorescein Ab,protein A, protein G,streptavidin,

biotinPerseptiveBiosystems,Framingham, MA, USADynabeads

M-280Dynabeads M-450Dynabeads

M-5002.84.55PolystyreneTosyl-activatedSecondary Abs,anti-CD

Abs, Absagainst Escherichiacoli O157, Salmonella,Listeria,

Crypto-sporidium, streptavidin,oligo(dT)Dynal, Oslo,

NorwayEstapor&1Polystyrene}COOH}NH2Prolabo,Fontenay-sous-

Bois,FranceFerrofluids0.1350.175Modified

hydrophilicprotein}COOH}NH2Secondary Abs,protein

A,streptavidinImmunicon, HuntingdonValley, PA, USAM 100M

104M 1081}10Cellulose}OHScigen,

Sittingbourne,UKMACSMicrobeads0.05Dextran}OHSecondary

Abs,anti-CD Abs,streptavidin, biotinMiltenyi Biotec,Bergisch

Gladbach,GermanyMagneticmicroparticles1}2Polystyrene,cellulose,

polyacrolein}COOH}NH2Secondary Abs,protein A,protein

G,streptavidinPolysciences,Warrington, PA,

USAMagneticnanoparticles0.09}0.6Starch, dextran,chitosan}OH,

}COOHStreptavidin,biotin, protein AMicro caps,

Rostock,GermanyMagNIM0.050.250.5}COOH}NH2Secondary

Abs,Ab against E. coliO157, streptavidin,protein ACardinal

Associates,Santa Fe, NM,

USAMagneticparticles&1Polystyrene}COOH}NH2Secondary

Abs,Protein A,streptavidin,Bangs Laboratories,Fishers, IN,

USAMPG5Porous glass}NH2,

hydrazide,glycerylStreptavidin,avidinCPG, Lincoln Park,

NJ,USAXM200Microsphere3.5Polystyrene}COOHSecondary

Abs,protein AAdvancedBiotechnologies,Epsom, UKFerritin is a

naturally occurring, soluble iron stor-age protein in mammals. For

magnetic modiRcationof cells cationized horse spleen ferritin

(ferritincoupled with N,N-dimethyl-1,3-propanediamine)ex-hibiting a

net positive charge at pH 7.5 is usuallyused. Under these conditions

the cationized ferritinreadily forms ionic bonds with the anionic sites

on thecell membrane. Magnetic derivatives of ferritin

calledmagnetoferritin, prepared by controlled reconstitu-tion

conditions, have also been used for cell labelling.Magnetic

SeparatorsA variety of magnetic separators is available on

themarket, starting with very simple concentrators

for2262III/CATALYST STUDIES: CHROMATOGRAPHY/

Isolation: Magnetic Techniques
--------------------------------------------------------------------------------
Page 4
Figure 1 (See Colour Plate 63). Example of a magnetic separ-ator

(Dynal MPC-M) for work with microcentrifuge tubes of

theEppendorf type, with a removable magnet plate to facilitate

easywashing of magnetic particles. Courtesy of Dynal, Oslo,

Norway.Figure 2 (See Colour Plate 64). Examples of

laboratory-scalehigh gradient magnetic separators. Left:

MiniMACS separationunit; right: MidiMACS separation unit, both

with inserted columns.Courtesy of Miltenyi Biotec, Bergisch

Gladbach, Germany.Figure 3 (See Colour Plate 65).

Computer-controlled magneticcell sorter CliniMACS. Courtesy of

Miltenyi Biotec, Bergisch Glad-bach, Germany.one test tube and

ending with complicated fully auto-mated devices. In many cases,

especially when work-ing with larger labels, very cheap

home-mademagnetic separators can be used successfully.Batch

magnetic separators Batch magnetic separ-ators are usually made

from strong rare-earth per-manent magnets embedded in

disinfectant-proofmaterial. The racks are designed to hold various

sizesand numbers of tubes. Some of the separators havea removable

magnet plate to facilitate easy washingof magnetic particles (Figure

1). Test tube magneticseparators enable separation of magnetic

particlesfrom volumes ranging between about 5 L and50 mL. It is

also possible to separate cells from thewells of standard

microtitration plates. Magneticcomplexes from larger volumes of

suspensions (up toapproximately 500}1000 mL) can be separated

usingSat magnetic separators. More sophisticated mag-netic

separators are available, e.g. those based on thequadrupole and

hexapole magnetic conRguration.Flow-through magnetic separators

Flow-throughmagnetic separators are characterized by the Sow

ofthe liquid and suspended cells through the separationsystem.

These systems are usually more expensive andmore complicated in

comparison with batch separ-ators, but for preliminary experiments

simple devicescan also be used.Laboratory-scale high gradient

magnetic separtors(HGMS; Figure 2) are composed of small

columnsloosely packed with Rne magnetic-grade stainlesssteel wool

which are placed between the poles ofstrong permanent magnets or

electromagnets. Mag-netically labelled cells are pumped through the

col-umn, labelled cells are retained on the steel wool, theReld is

removed and cells are retrieved by Sow andusually by gentle

vibration of the column. Automa-tion of the separation process has

led to the develop-ment of computer-controlled magnetic cell

sorters,such as CliniMACS (Figure 3) and AutoMACS,

bothproduced by Miltenyi Biotec, Germany.III/CATALYST

STUDIES: CHROMATOGRAPHY/ Isolation: Magnetic

Techniques2263
--------------------------------------------------------------------------------
Page 5
Figure 4 (See Colour Plate 66). The Isolex 300i Cell

SelectionSystem. Courtesy of Nexell Therapeutics, USA.A

continuous magnetic sorter based on an elec-trophoresis

counter-Sow chamber has been de-veloped. The injected magnetic

particles are deviatedin the inhomogeneous magnetic Reld and

focused intoa stream that is completely separated from thestreams

of the undeviated particles.The Isolex 300i Magnetic Cell Separator,

producedby Nexell Therapeutics, USA, is intended for theisolation of

stem cells from blood or bone marrow.The whole process is done

automatically and thedevice represents a Sexible platform for future

ap-plications in cell separation (Figure 4).Procedures for Performing

MagneticSeparation of CellsMagnetic separation of cells is usually

performed inone of the following formats:1. Direct method. The

afRnity ligand is coupled tothe magnetic particles, which are then

added dir-ectly to the cell sample. During incubation themagnetic

particles will bind the target cells whichcan be then recovered using

a magnet.2. Indirect method. Target cells are Rrst sensitizedwith a

suitable primary afRnity ligand. After incu-bation, excess unbound

afRnity ligand is removedby washing the cells and then magnetic

particleswith an immobilized secondary afRnity ligandwith afRnity

for the Rrst ligand are added. Themagnetic particles will bind the

target cells, whichcan then be recovered using a magnetic

separator.Another differentiation of magnetic separationtechniques

is based on the selection of the magneti-cally labelled cells.1.

Negative selection. Negative selection is a methodby which a

cellular subset is puriRed by removingall other cell types from the

sample. Both the directand indirect method are applied for negative

selec-tion. The advantage is that the puriRcation processdoes not

involve any direct contact of magneticlabels with the cells to be

isolated.2. Positive selection. The target cells are isolatedfrom the

cell suspension. Both the direct and in-direct method can be used.

The separated magneti-cally labelled cell complexes can be further

charac-terized directly, but in many cases it is necessary toremove

larger magnetic particles from the posit-ively selected cells after

their isolation.3. Depletion of cells. Depletion is a method by

whichone or more unwanted cellular subsets is removedfrom a cell

suspension. Both the direct and theindirect procedure can be applied

for this purpose.Immunomagnetic SeparationImmunomagnetic

separation (IMS) is the most oftenused approach for the isolation of

cells. Most often,a monoclonal antibody is used for IMS, but

alsopolyclonal antibodies are used successfully. IMS canbe

performed in all the formats mentioned above.In the direct method

the appropriate antibody iscoupled to the magnetic particles and

colloids, whichare then added directly to the sample. Ideally,

theantibody should be oriented with its Fc part towardsthe magnetic

particle so that the Fab region is point-ing outwards from the

particle. Several proceduresare available for direct binding of

antibodies(Table 2).The indirect method is also used very often.

Thecell suspension is Rrst incubated with primary anti-bodies which

bind to the target cells. Not only puri-Red primary antibodies have

to be used, crudeantibody preparations or serum can also be

used.After incubation, the unbound antibodies are usuallyremoved

by washing. Then magnetic particles withimmobilized secondary

antibody are added to bindthe labelled cells. Target cells } primary

antibodycomplexes } can be also captured by protein A orprotein G

immobilized on magnetic carriers. Alterna-tively, biotinylated or

Suorescein-labelled primaryantibodies and magnetic particles with

immobilizedstreptavidin or anti-Suorescein antibody are used

tocapture the target cells.The indirect method is generally more

efRcient inremoving target cells from a suspension because

freeantibodies will Rnd their target antigen more

easily2264III/CATALYST STUDIES: CHROMATOGRAPHY/

Isolation: Magnetic Techniques
--------------------------------------------------------------------------------
Page 6
Table 2 Selected procedures for binding of antibodies on magnetic

particles and colloidsAdsorption of antibodies on hydrophobic

magnetic particles (especially those made of polystyrene)Covalent

binding of antibodies on activated magnetic particles (e.g.

tosyl-activated), or on magnetic particles carrying

appropriatefunctional groups (e.g. carboxy, amino, hydroxy,

hydrazide) using standard immobilization proceduresImmobilization

of secondary antibodies (i.e. antibodies against primary antibodies)

on magnetic particles followed by binding of

primaryantibodiesImmobilization of biotinylated antibodies on

magnetic carriers with immobilized streptavidinImmobilization of

antibodies on magnetic particles with immobilized protein A and

protein GImmobilization of antibodies tagged with oligo dA on

magnetic particles with immobilized oligo dTImmobilization of

antibodies on magnetic carriers with immobilized boronic acid

derivative via their carbohydrate units on the Fc partTable 3

Selected typical procedures for detachment of cells after

immunomagnetic separationIncubation of rosetted cells overnight in

cell culture medium with subsequent mechanical forces such as firm

pipetting, flushing thesuspension 5}10 times through a narrow-tipped

pipetteTrypsin, chymotrypsin and pronase have general applicability

for proteolytic detachment of isolated cellsDetachment with a

polyclonal antibody that reacts with the Fab fragments of primary

monoclonal antibodies on magnetic beads. Thisprinciple is

commercialized by Dynal, Norway (DETACHaBEAD)Using

synthetic peptides which bind specifically to the antigen-bindingsite

of primary antibodies(Baxter Healthcare, Deerfield, IL,

USA)Antibodies immobilized via carbohydrate units on the Fc part

to the magnetic particles with immobilized }B(OH)3groups are

dissociatedwith sorbitolA complex primary antibody}DNA linker can

be split enzymatically using DNaseCryptosporidium oocysts were

successfully released from the immunomagnetic particles by

decreasing the pH of the suspension(adding HCl)than antibodies

bound to magnetic particles. Theindirect technique is recommended

when the targetcell has a low surface antigen density or a cocktail

ofmonoclonal antibodies is used. The direct method isusually faster

and requires less antibody than theindirect method. Also, the direct

method is advant-ageous when one does not want to cover all

antigensites with antibody.Typically, 95}99% viability and purity of

the pos-itively isolated cells can be achieved with a typicalyield of

60}99%. Depletion efRciency often reaches99.9% and leaves

remaining cells untouched. Sequen-tial depletions are markedly

more efRcient.Magnetic particles usually do not have any nega-tive

effect on the viability of the attached cells. Manytypes of magnetic

particles are usually compatiblewith subsequent analytical

techniques such as Sowcytometry, electron and Suorescence

microscopy,polymerase chain reaction (PCR), Suorescence in

situhybridization (FISH) or cultivation in appropriatenutrient media.

In some cases, however, it is neces-sary to remove larger

immunomagnetic particles fromthe cells after their isolation. The

detachment processcan be performed in several ways (Table

3).Incubation time for cell separation is usually5}60 min while the

binding of primary antibodies tosecondary coated magnetic particles

usually takes 30 minor less. In positive isolation, the purity of cells

gener-ally decreases with time, although the yield

increases.NonspeciRc interactions of nontarget cells with

hy-drophobic magnetic particles can be expected. Theseinteractions

can be partially eliminated using bovineor human serum albumin,

casein and nonionic ten-sides such as Tween 20.Magnetic

Separations using other LabelsAntigens Antigens immobilized on

magnetic par-ticles can be used for the isolation of antibody

ex-pressing or antigen-speciRc cells. This approach hasbeen

successfully used for selection of antigen-speciRchybridoma cells or

human antibody-producing celllines.Lectins Lectins immobilized on

magnetic carrierscan interact with saccharide residues on the cell

surfa-ces. A typical example of this approach is the applica-tion of

immobilized Ulex europaeus I lectin whichbinds to terminalL-fucosyl

residues present on thesurface of human endothelial cells. Magnetic

beadscan be released from the isolated cells using a freecompeting

sugar.Oligosaccharides Oligosaccharides immobilized onmagnetic

particles can be used for the rapid isolationof speciRc

lectin-expressing cells. Target cells boundto the magnetic particles

can be released using a freecompeting saccharide

structure.Bacteriophage

Salmonella-speciRcbacteriophageimmobilized to a magnetic solid

phase has been usedfor the separation and concentration of

Salmonellafrom food materials.III/CATALYST STUDIES:

CHROMATOGRAPHY/ Isolation: Magnetic Techniques2265
--------------------------------------------------------------------------------
Page 7
Erbium ions, ferritin and magnetoferritin havebeen used for

magnetic labelling of both prokaryoticand eukaryotic cells.

Magnetotactic bacteria can beintroduced into granulocytes and

monocytes byphagocytosis which enables their magnetic separ-ation.

Submicron magnetic particles of -Fe2O3adhere to the surface of

Saccharomyces cerevisiae,making the cells magnetic and amenable

to magneticseparation.Magnetotactic bacteria, due to the presence

of fer-romagnetic material in their cells, can be

magneticallyseparated without any labelling. Erythrocytes can

beseparated by the high gradient magnetic separationtechnique

after conversion of diamagnetic eryth-rocytes containing

oxyferrohaemoglobin into para-magnetic red blood cells by the

oxidation of the ironatoms in the cell haemoglobin to the ferric

state(methaemoglobin). Erythrocytes, infected by Plas-modium,

contain paramagnetic hemozoin, that isa component of malarial

pigment. The paramagneticmoment of hemozoin is of sufRcient

magnitude toenable the separation of malaria-infected

(hemozoin-bearing) erythrocytes.Magnetic Separations in

Microbiology,Cell Biology, Medicine andParasitologyIMS and, in

some cases, lectin-magnetic separationsare often used in the

above-mentioned disciplines. Inmicrobiology they are especially used

for the detec-tion of pathogenic microorganisms. IMS enables

thetime necessary for detection of the target pathogen tobe

shortened. Target cells are magnetically separateddirectly from the

sample or the pre-enrichment me-dium. Isolated cells can than be

identiRed by stan-dard, speciRc microbiological procedures. IMS is

notonly faster but also usually gives a higher number ofpositive

samples. Also sublethally injured and stressedmicrobial cells can be

very efRciently isolated usingIMS. The most important microbial

pathogens can bedetected using commercially available speciRc

im-munomagnetic particles; they are used for the detec-tion of

Salmonella, Listeria and Escherichia coli O157.New

immunomagnetic particles for the detection ofother microbial

pathogens are under development.Removal of cancer cells is one of

the most impor-tant applications of IMS in the area of cell

biologyand medicine. The Rrst experiments were performedin the

1970s and since then an enormous number ofapplications have been

described. Cancer cells areusually removed from bone marrow prior

to itsautologous transplantation and using IMS they aredetected in

blood. Elimination of graft-versus-hostdisease (GvHD) in allogenic

bone marrow transplan-tation requires an effective removal of T

cells fromthe bone marrow of the donor. A direct methodenabled a

103times depletion of T cells.Magnetic particles are being

increasingly used forisolation of human cell subsets directly from

bloodand other cell sources. B lymphocytes, endothelialcells,

granulocytes, haematopoietic progenitor cells,Langerhans cells,

leukocytes, monocytes, naturalkiller cells, reticulocytes, T

lymphocytes, spermato-zoa and many others may serve as examples.

Cellsfrom other animal and plant species have been suc-cessfully

separated, too.Not only whole cells, but also cell organelles can

beisolated from crude cellular fractions. Dynal (Oslo,Norway) has

developed Dynabeads M-500 Subcellu-lar, which are able to isolate

rapidly more than 99%of target organelles.In the area of

parasitology Cryptosporidium andGiardia are the parasites where

IMS is of interest.Two commercially available kits can be used for

thispurpose. Both products are used in the method

1622:Cryptosporidium in Water by Filtration/IMS/FA(December

1997 Draft) of the US EnvironmentalProtection Agency. In very low

turbidity samples(clean waters), IMS has demonstrated

signiRcantlybetter results than the standard procedures. Whenwater

samples were turbid, the recovery efRciency ofIMS

diminished.Future DevelopmentsMagnetic separation of cells is a

simple, rapid, speci-Rc and relatively inexpensive procedure, which

en-ables the target cells to be isolated directly from crudesamples

containing a large amount of nontarget cellsor cell fragments. Many

ready-to-use products areavailable and the basic equipment for

standard workis relatively inexpensive. The separation process

canbe relatively easily scaled up and thus large amount ofcells can

be isolated. New processes for detachmentof larger magnetic

particles from isolated cells enableuse of free cells for in vivo

applications. Moderninstrumentation is available on the market,

enablingall the process to run automatically. Such devicesrepresent

a Sexible platform for future applications incell separation.IMS play

a dominant role at present but otherspeciRc afRnity ligands such as

lectins, carbohydratesor antigens will probably be used more often in

thenear future. There are also many possibilities tocombine the

process of cell magnetic separationwith other techniques, such as

PCR, enabling theelimination of compounds possibly inhibiting

DNApolymerase. New applications can be expected, espe-cially in

microbiology (isolation and detection of2266III/CATALYST

STUDIES: CHROMATOGRAPHY/ Isolation: Magnetic Techniques
--------------------------------------------------------------------------------
Page 8
microbial pathogens) and parasitology (isolation anddetection of

protozoan parasites). No doubt manynew processes and applications

in other Relds of bio-sciences and biotechnologies will be developed

in thenear future.See Colour Plates 63, 64, 65, 66.See also:

II/Centrifugation: Analytical Centrifugation;Large-Scale

Centrifugation. III /Cells and Cell Or-ganelles: Field Flow

Fractionation.Further ReadingCell Separation and Protein

PuriTcation (1996) Oslo,Norway: Dynal. 165 pp.HaKfeli U, SchuKtt

W, Teller J and Zborowski M (eds) (1997)ScientiTc and Clinical

Applications of Magnetic Car-riers. New York: Plenum Press.Olsvik

", Popovic T, Skjerve E et al. (1994) Magneticseparation techniques

in diagnostic microbiology. Clini-cal Microbiology Reviews 7:

43d54.Recktenwald D and Radbruch A (eds) (1998) Cell Separ-ation

Methods and Applications. New York: MarcelDekker. 352

pp.S[afar\mHk I and S[afar\mHkovaH M (1999) Use of magnetic

tech-niques for the isolation of cells. Journal of Chromatog-raphy B

722: 33d53.S[afar\mHk I, S[afar\mHkovaH M and Forsythe SJ

(1995) The applica-tion of magnetic separations in applied

microbiology.Journal of Applied Bacteriology 78: 575d585.Ugelstad

J, Olsvik ", Schmid R et al. (1993) ImmunoafRn-ity separation of

cells using monosized magnetic poly-mer beads. In: Ngo TT (eds)

Molecular Interactionsin Bioseparations, pp. 229d244. New York:

PlenumPress.Ugelstad J, Prestvik WS, Stenstad P et al. (1998)

Selectivecell separation with monosized magnetizable

polymerbeads. In: AndraK W and Nowak H (eds) Magnetism

inMedicine: A Handbook, pp. 471d488. Berlin: Wiley-VCH

Verlag.UhleHn M, Hornes E and Olsvik " (eds) (1994) Advances

inBiomagnetic Separations. Natick: Eaton Publishing.CELLS AND

CELL ORGANELLES:FIELD FLOW FRACTIONATIONP. J. P.

Cardot, S. Battu,T. Chianea and S. Rasouli,Universite& de Limoges,

Limoges, FranceCopyright^2000 Academic

PressIntroductionAnalysis and sorting of living cells and puriRcation

ofcell organelles are important procedures in the lifesciences. There

is a wide range of techniques andmethodologies available, which can

be divided intothree main groups. The techniques in the Rrst

groupsare based on physical criteria such as species size,density and

shape, and include centrifugation, elutri-ation and Reld Sow

fractionation. Those in the sec-ond group are linked to cell surface

characteristics,while Sow cytometry techniques make up the

thirdgroup. At a fundamental level, Reld Sow fractiona-tion (FFF)

exploits the physical characteristics of thecells or cell organelles.

However, cells or cell or-ganelles exhibit some speciRc

characteristics that canbe described by a multipolydispersity matrix.

Thedifferent physical characteristics of these biologicalmaterials

require different FFF techniques and modesof operation. Special

care must be taken if biologicalintegrity and viability are to be

preserved.Speci\c Cell CharacteristicsCellular materials range in

size from 1 m to 50 m.Cell populations are classiRed by a set of

morphologi-cal, functional and biophysical characteristics.

Thebiophysical characteristics are of particular interestin FFF.

Usually, separations in FFF are inSuenced(but not directed) by

surface properties of the samplecomponents (to avoid

particle}particle or particle}separator interactions). These

properties can bemodulated by the use of appropriate

carrier-phasemodiRers (surfactants). In terms of FFF

separations,mass, size and density appear to be the major Rrstorder

parameters. However, size is generally deRnedby the radius or the

diameter of a sphere whosevolume is identical to that of the cell.

Size can there-fore be deduced accurately if the cell of interest

isperfectly spherical. However, this is not usually thecase, and the

sphericity index, I, is then used:I"4.84(V2/3)SIn this equation V is

the cell volume and S is itssurface area can be difRcult to determine.

In terms ofcell population, these dimensions are averages andshould

be associated with a variance. These generalIII/ CELLS AND

CELL ORGANELLES: FIELD FLOW FRACTIONATION2267

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--------------------------------------------------------------------------------

CHAPTER 4.
PRODUCT HARVESTING AND FORMULATION
OF MICROBIAL INSECTICIDES

Contents - Previous - Next

4.1 Product Harvesting

Harvesting microorganisms from submerged fermentation is often

difficult due to the low concentration of the products, their

thermolabile nature and in some cases their poor stability.

Stabilizing adjuvants may have to be incorporated immediately

post-harvest to prevent spore death and/or germination. Rapid

drying or the addition of specific biocidal chemicals may be required

to prevent growth of microbial contamination in the broth or

centrifuge slurry (Soper and Ward, 1981).

Spore-forming Bacillus thuringiensis are usually concentrated prior

to drying by centrifugation or filtration. Centrifugation using a

continuous centrifuge concentrates the product from 2-3 %

suspended solids to 15-20 %. Centrifugation may result in some loss

of suspended solid as well as loss of dissolved materials. Such losses

may not be acceptable and concentration using this technique can

often be omitted. Following concentration, one of the technique

mixes the crystal/spores slurry with lactose, adjuvants such as

wetting agents, spreader-stickers or dispersing agents, and the

whole product is spray-dried at 175oC (Dulmage, 1981). The dry

product is blended and/or mixed with additional formulation

adjuvants before packaging and/or use. The lactose added may act

as a cryoprotectant or it may help to prevent clumping Dulmage and

Rhodes, (1971). Dulmage at al. (1970) (see chapter 2) developed an

alternative drying technique for laboratory preparations where

spray drying facilities are not available; this technique of recovery

of B. thuringiensis is based on the lactose-acetone processing (see

2.1.1). Many patents exist such as a foam flotation process for

separating B.t. sporulation products.

Fungal blastospores obtained in submerged culture are much less

stable than conidia and are consequently difficult to process after

harvesting. Laboratory cultures are frequently freeze-dried with or

without protectants, but even dried product may have short viability.

Verticillium lecanii freeze-dried blastospores, for example, have a

half-life at 5, 20 and 30oC of 11, 4 and 2 days, respectively. Blachere

et al.(1973) harvested Beauveria brongniartii by centrifugation

before mixing with silica powder, osmotically active materials (such

as sucrose and sodium glutamate), anti-oxidizing agents (sodium

ascorbate) and a mixture of liquid paraffin-polyoxyethylene glycerin

oleate. The resultant paste was then dried at 4oC in ventilated

drying closet. Blastospores dried in this fashion were viable for 8

months at 4oC (Blachere et al., 1973).

Belova (1978) dried Beauveria bassiana product in five different

ways: vacuum, freeze, spray-drying, drying by mixing with an inert

filler, and in a fluidized bed with an inlet temperature of 40oC and

an outlet of 30oC. The virulence of the fluidized bed-dried material

is enhanced and the process accelerated by precipitation using a

calcium carbonate, surfactant, silica gel mixture. In addition, a

sulphite liquor is mixed in. Drying in a vacuum desiccator produced

samples of high viability and virulence, some remaining active for 1

year at 4oC storage. Spray-dried B. bassiana spores together with

the culture media led to a complete loss of viability. However, using

a 2 % molasses mixture as a protective medium permitted retention

of viability and efficacy, although to a lesser extent than for the

freeze-dried product.

Globa (1980) recovered Beauveria bassiana from fermenter broth

by precipitation with calcium carbonate. Fargues et al. (1979)

spray-dried B. brongniartii conidiospores coated with bentonite clay,

yielding 50-70 % viable spores. However, blastospores were too

sensitive for this technique and these were lyophilized with

powdered milk and glycerin. The spray-dried conidia showed no loss

of viability after 18 months storage at 5oC: lyophilized blastospores

were still viable after 8 months.

4.2 Formulation

Angus and Luthy (1971), Couch and Ignoffo (1981) mentioned that

the development of microbial insecticide formulation closely

paralleled that of chemical insecticides. Pesticide formulation is the

process of transforming a pesticide chemical into a product which

can be applied by practical methods to permit its effective, safe and

economic use. Important specific differences, of course, do exist

because microbial insecticides do not directly depend on the effect

of a poisonous chemical but exploit the activity of living (or

self-replicating) entities. An exception is the enterotoxinosis caused

by Bacillus thuringiensis where a pre-formed toxic glycoprotein is

essential for infection to occur.

The aim of the formulator then is to avoid practices that might

inhibit or harm the pathogen and wherever possible to enhance the

possibility of infection. Thus, not only must one avoid agents in any

way antimicrobial but also, with B. thuringiensis, any compounds

capable of denaturing the glycoprotein comprising the toxic crystal.

Any attempts to utilize a particular species of micro-organism in an

insecticide formulation should be based on an intimate knowledge of

the host-parasite relationships. Generally, however it is the

multi-plication of a microorganism in the host tissues that leads to

disease and death.

4.2.1 Definitions
A microbial pesticide formulation is a physical mixture of living

entities with inert ingredients which provides effective and economic

control of pests.

Formulation of a pathogen product with an extensive shelf-life (>18

month) is critical to industrialization.

In commercial development of a basic formulation of an

entomopathogen, technology concerns maintaining pathogen

viability and virulence during the production process and developing

a product form which preserves or enhances these properties. To do

this, knowledge of the biology of pathogen and target insect is

essential. Effect of temperature, humidity and media (inert carrier)

on the entomopathogen can turn out to be the most important.

4.2.2 Additives
Spreaders

Spreaders or wetting agents are added to the water diluent to

ensure "wetting" of the surface to be sprayed. Many materials have

been used including dried milk, powdered casein, gelatin, saponins,

soaps etc. In so far as microbial insecticides are concerned, it is

essential that the compound used should encourage premature

growth or germination and that it should not inhibit successful

establishment of the pathogen. Some factors are likely to be rather

subtle, e.g. although detergents such as sodium dodecyl sulphate do

not inactive the crystal toxin of B.t, they open up the structure of

the crystal and make it more sensitive to destruction by other

means.

Table Additives that have been added to preparations of microbial

insecticides.

Diluent (dust) pathogen Adhesives and Stickers pathogen

Talc B.t., Tung oil B.t.
Celite B.t., Molasses B.t.
Starch B.t., Powdered skin milk B.t., V
Synthetic silicates B.t., F Methocel B.t., V
Bentonite B.t., Corn syrup B.t., V
Lactose B.t., V Latex D B.t.
Microcell B.t., Casein B.t.
Attagel B.t., Neosil A B.t., F
Pyrax B.t., Folicote B.t.

Wetting agents and spreaders Emulsifiers

Alkyl fenols B.t., Tween 80 B.t., V, F
Sandovit B.t., 9 D 207 B.t., V
Novmol B.t., Pinolene 1882 B.t.
Petro AG B.t., Span 80 B.t., V
Colloidal X77 B.t., V Triton N60 B.t., F
Triton X100 V Triton GR7M B.t., F
Triton 155 B.t., Atlox 848 B.t.
Tween 20 F Atlox 849 B.t.
Tween 80 F Atlox 3404/849 F
Triton X45 V Atplus 448 F
Triton X114 B.t., Atplus 300 F, V

Liquid Vehicles Botanicals

Water Citrus pulp
Preformed oil in water emulsion Corn cob
Preformed water in oil emulsion Corn meal
Edible oil, Wheat bran
Corn oil, Grape pomace
Crude sorbitol Apple pomace
Aromatic spray oils, Rice hulls
Emulsified cottonseed oil, Cracked corn

Suspending agents for B.t.

Bentone 38
CAB-O-SIL
SOLOID

4.3 Oil Suspension Formulation

Uniform suspension is prepared by preliminary wetting of the dry

microbial insecticide with an emulsifer water mixture before final

dispersion in oil carrier. Edible oil is suitable for microbial pesticide

formulation. The storage of fungal spores under edible oil preserve

its viability for sufficient time. Advantage of edible oil is its

nonphytotoxicity and its residua might be acceptable.

4.4 Dusts or Wettable Powder

In the case of one B.t., Formulation, the pathogen is cultured on a

semi-solid medium so that it is preferable to process it as a dust or

wettable powder rather than attempt to separate the spores and

crystals from the medium solids. When grinding and mixing material

containing the pathogens to obtain a sufficiently fine powder, care

should be taken to avoid increase in temperature or physical damage

that would harm the pathogen.

The choice of the filler for use in a dust formulation is subject to the

general provision that it is nontoxic to the pathogen being used.

The decision as to the most desirable form (spray or dust) varies

with the crop being protected, the target insect, climatic conditions

and, of course, the particular pathogen being used.

4.5 Suspension Concentrates (sc)

Are also known as flowable emulsions, colloidal suspensions or

dispersions. Biological agents are not soluble and dispersing medium

is edible oil, which is not aggressive to biological agents.

A flowable must have a satisfactory viscosity for handling purposes;

it should disperse spontaneously or with slight stirring when poured

into water before application.

4.5.1 Processing
Simplest method to produce flowables is by adding thickeners and

thixotropic agents to biological agent in powder form. The use of

thixotropic agents enables the production of flowables which at the

application point do not exceed 2,000 MPa`s. Commercially

available flowables have viscosities ranging from 200 to 2,000

MPa`s. This results then in an excellent self dispersibility. Flowables

should disperse spontaneously when poured into water, but roping

my be tolerated if the flowable disperses rapidly upon agitation.

The coarse and wide range of particle sizes produced by this

technique may cause sedimentation and ultimately claying or

cauling, due to the absence of interfacial forces preventing close

packing.

In commercial flowable production, "wet milling" techniques are

preferred as they provide the most economical and direct means of

producing the desired average particle size of 1-5 microns in

diameter. Possible mills include attritors, sand mills, and ball (roller)

mills for batch or continuous process. The use of an attritor or

sand-mill requires the preparation of a premixture, which usually

consists of all the ingredients (such as siloxyl) at their required

concentrations.

4.5.2 Function of the Surfactants
Surfactants play an important role as basic component in flowable

formulations. They are responsible for wetting of the pesticide

particles before and during the milling process. They help to liquefy

the unmilled premixture in the milling chamber. They help to

stabilize the micronised particles in the dispersing medium. The role

of the surfactant in every step of the production process can be

described as follows:

4.5.3 Wetting
When a dry pesticide is mixed with water during flowable production

process, the surfaces of individual particles must be wet and the air

between the particles must be displaced to make efficient wet

milling possible. As little air entrainment as possible is desired by the

formulator, because the air remaining on the surfaces after milling,

causes flocculating when bubbles of the neighbouring particles

coalesce. This coalescence reduces the dispersion stability.

The most widely used wetting agents are ethoxylated alcohols and

ethoxylated nonylphenols, which should have very low foaming

properties. Foam formulation should always be avoided during the

production process and also during spraying on the field. Foaming

can reduce the uniformity and the effectiveness of the pesticide in

the field.

The concentration on wetting agent varies usually from 0.5 % to 3

% depending on the concentration, the morphology and the surface

properties of the active ingredient.

4.5.4 Milling Aid
During the milling process, surfactants help to rewet and disperse

the newly formed particles. Bad rewetting will result in paste

formulation and the whole milling chamber will be blocked. During

the milling process, the temperature can easily rise up to 60oC. As

known, desorption of the surfactant from the particles take place at

the cloud point. Therefore it is necessary to use wetting and

dispersing agents with a sufficiently high cloud point. Usually,

nonionic surfactants are selected with a cloud point at least 10oC

above the milling temperature and the maximum required storage

temperature.

4.5.4.1 Stabilization
Because of the high loading and the small particle size of the active

ingredient, the dispersed particles have an inherent tendency to

irreversibility flocculate due to the London-Van der Waals forces of

attraction. Adsorption of a dispersing agent on the particles will

generate repulsive forces between the particles or eliminate the

attractive forces between the particles.

Electrostatic repulsion occurs when ionic surfactants are adsorbed

onto particles. The charge imparted by the surfactant causes

particles to repel each other. Electrostatic repulsion is most

important in aqueous flowables.

Steric hindrance results from the adsorption on nonionic surfuctants

having long chains which are soluble in the dispersing medium. When

two particles approach, the solvated chains interact to prevent

irreversible agglomeration. This type of repulsion is important in

both aqueous and oil-based flowables.

4.5.4.2 Milling Conditions
PREMIXTURE (wet grinding mills) Ideally, the premixture should

consist of all the ingredients at the required concentration and yet

be pumpable. The ingredients are:

1) dispersing medium (water, edible oil)
2) active biological agent (conidia, blastospores, spores and crystals)
3) wetting agent (Atlox 4862)
4) thickening or viscosity modifying agents (Atlox 1086 or Bentone

38 [0.1-1.0 % w/v] or Rhodopol 23 [0.05-0.3 w/v] or other type of

Atlox (1096, 4868 B)
5) stabilizer (if necessary)

The active biological agents

B.t. - spores and crystals
Fungi - Metarhizium anisopliae, conidia strong hydrophobic
- Beauveria bassiana, conidia medium hydrophobic
- Verticillium lecanii, conidia or blastospores hydrofility

These particles are under 20 microns but all of them aggregate and

must be milled. Polyethylene glycol preserves aggregation and can

be used during wet milling.

For oil based flowables can used:

Renex 702 / Atlox 1045 A Atlox 4884 / Atlox 1045 A
Atlox 4856 B / Atlox 4885 Atlox 4856 B / Atlox 3386 B
Atplus 300 F

These pairs provide:

The good emulsification of the oil when added to the water.

The required stability of the emulsion in the spray tank.

Furthermore the combination ATPLUS 300F/oil improves the

efficiency and the selectivity of the applied pesticide. As a thickener

"BENTONE 38" (NL Industries USA) can be used. This thickener is

activated by a polar solvent to ensure a good thickening effect. The

polar solvent is generally added whilst preparing the master solution.

Example of formulation of microbial insecticide:

250 - 100 g powder mix of spores and crystal B.t. (or conidia)
30 g Atplus 300F
68 g Bentone 38
to 1 litre of water or oil

4.6 Suggested Evaluation Techique of Flowables

Test of mechanical stability

The mechanical stability is tested by shaking the flowable on shaker

for half an hour. A good flowable will not thicken or gel under the

influence of shaking.

4.6.1 Suspensibility
Suspensibility, or dilution stability is the degree to which a flowable

stays suspended when diluted in water. It is expressed as the volume

percent of setting in a dilution mixture at various time intervals. The

degree of flocculating is noted by the number of inversions of the

test cylinder required to redisperse the sediment.

4.6.2 Storage Stability
Samples are stored in sealed bottles at -10oC, room temperature,

40oC for several months. Afterwards, they are periodically

inspected for:

a) bleeding; the amount of liquid separation on top of the flowable is

expressed as a percent of the total sample depth. The bleeding

should be minimal.
b) thickening; the absence of thickening is checked by probing with

a glass rod or by measuring the viscosity
c) sedimentation; the absence of packing is checked by inserting into

the flowable a glass rod to check the bottom of the container for any

sedimentation. Packing should be absent and any non-uniformity of

the suspension should be easily correctable with mild agitation.

4.6.3 Viscosity
The viscosity is measured at one day, one week and one month

intervals. A minimum variation in viscosity is tolerated. The main

criterion is to maintain pourability.

4.6.4 Bloom
While conducting the suspensibility test, the bloom is observed and

rated qualitatively on a three point scale: good, fair and poor I.O.U..

The scale is ranging from total spontaneity to no spontaneous

dispersion.

4.6.5 Biological Activity
The biological activity is measured at one day, one week and every

month intervals during storage under evaluated temperature to way:

Test of germination of spores, LD50 on target pest in lab.

4.7 Evaluation of Separation Process "Recovery"

Each process for the isolation and purification of bioproduct from

microorganisms consists of a sequence of individual process steps.

The choice of the individual process steps is governed by the

properties of the by-product to be isolated.

The yield of the step: The yield of the step, given in amounts (e.g., g

or kg), or in units (or mega units = millions of units, documents the

scale of the process step and the batch size and permits conclusions

concerning the equipment used.

The percentage yield of a step:

units (or amount) after purification
100 . --------------------------------------------- =% of efficiency
units (or amount) before purification

The common evaluation of enrichment factor and percentage yield

of the step permits a statement concerning the efficiency of the

technology used in the process step. The product of purification

factor times percentage yield of the step is termed the efficiency.

Economic aspects must also be included, especially the

manufacturing costs of the bioproduct. For calculating these, all

types of costs, such as:

- material costs - costs for waste treatment,
- personnel costs - overheads
- energy costs - costs of repair
- depreciations must be added and the result divided by the yield of

the step.

The manufacturing costs are given in

total costs total costs
.------------------ or -------------------------------
kg of product mega units of bioproduct

and they integrate the economic and technical parameters of the

process step.

The evaluation of the total process results from the evaluation of

the individual process steps. Thus, the percentage total yield may be

obtained by multiplying all the percentage step yield, and the

manufacturing costs of the end product by adding all the types of

costs of all process steps and dividing by the overall yield.

The technical and economic classification of a process into process

steps does not only permit an optimization of the process but also an

immediate adaptation to necessary changes in the process even

when they are independent of the process itself.

References:

ANGUS, T.A., and LUTHY, P. 1973: Formulation of microbial

insecticides. In: Burges H.D. and N.W. Hussey: Microbial control of

insects and mites. Acad. Press, London and New York., pp.623-636.

BELOVA, R.N., 1978: Proc. 1st Joint US/USSR Conf. Prod. Selec

Stand. Entomopath. Fungi, Jurmula (Riga) Latvia SSR, 20-21 May,

pp. 102-119.

BLACHERE, H., CLAVEZ, J., FERRON, P., CORRIE, G. and

PERINGER, P. 1973: Ann. Zool. Ecol. Anim. 5, 69-79.

DULMAGE, H.T. and Cooperators, 1981: In: Microbial control of

pests and plant diseases. Ed. H.D. Burges,pp. 191-220. Acad. Press,

London and New York.

DULMAGE, H.T. and RHODES, R.A. 1971: In:Microbial control of

insects and mites. Ed. H.D. Burges and N.W. Hussey, pp. 507-540.

Acad. Press, London and New York.

FARQUES J., ROBERT, P.H., and REISINGER, O. 1979: Ann.

Zool.

Ecol. Anim. 11 (2) 247-257.Ferron P.,1978: Annu. Rev. Entomol. 23:

409-442.

GLOBA, L., 1980: U.S.S.R. Patent SU 73 85 71.

SOPER, R.S. and WARD, M.G. 1981: Beltsville Symposia in

Agricultural Research. Vol.5: Biological Control in Crop Production,

pp. 161-180.

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Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax B’nai B’rith Wassington dc 1997 person of interest person of interest person of interest victor victor victor goetz goetz biologist microbiologist chemist biochemist vaccine bacteris anthracis b.anthracis person of interest person of interest person of interest victor victor victor goetz goetz biologist microbiologist chemist biochemist vaccine bacteris anthracis b.anthracis person of interest person of interest person of interest victor victor victor goetz goetz biologist microbiologist chemist biochemist vaccine bacteris anthracis b.anthracis person of interest person of interest person of interest victor victor victor goetz goetz biologist microbiologist chemist biochemist vaccine bacteris anthracis b.anthracis dpore spores
Berry Berry Berry Berry md MD
Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax freerepublic the great satan anthrax letters anthrax anthrax anthrax investigation anthrax letters anthrax letters anthrax anthrax anthrax investigation anthrax letters Ft Detrick USAMRIID Camel club Assaad antrax antrax 005 ripley zack Don Foster forensic analysis handwriting anthrax investigation fbi anthrax anthrax anthrax investigation spores lost stolen ames ames anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city anthrax city

Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax goetz anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax
Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax victor investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax investigation fbi anthrax anthrax anthrax Steven hatfill anthrax