CA1127571A - Method of magnetic separation of cells and the like, and microspheres for use therein - Google Patents

Method of magnetic separation of cells and the like, and microspheres for use therein

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Publication number
CA1127571A
CA1127571A CA345,986A CA345986A CA1127571A CA 1127571 A CA1127571 A CA 1127571A CA 345986 A CA345986 A CA 345986A CA 1127571 A CA1127571 A CA 1127571A
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Prior art keywords
microspheres
protein
parts
albumin
cells
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French (fr)
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Andrew E. Senyei
Kenneth J. Widder
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KENDREW BIOSYSTEMS Inc
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KENDREW BIOSYSTEMS Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16051—Methods of production or purification of viral material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/30—Magnetic particle immunoreagent carriers the magnetic material being dispersed in the polymer composition before their conversion into particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/84—Polymer coating, e.g. gelatin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/828—Protein A

Abstract

METHOD OF MAGNETIC SEPARATION OF CELLS AND THE LIKE, AND MICROSPHERES FOR USE THEREIN
ABSTRACT
Magnetically-responsive microspheres having Protein A
associate with the outer surfaces thereof are reacted with antibodies selective to the cells, bacteria, or viruses to be separated from a mixed population to attach the antibodies in oriented relation with their Fab arms extending outwardly, and the microspheres are then used in a magnetic separation procedure. The preferred microspheres are prepared from a mixture of albumin, Protein A, and magnetic particles, the microspheres being prepared so that the Protein A is present in the exterior surfaces for antibody binding.

Description

11~7S'~

BAC~GROUND AND ~IOR ART

This invention relates to the fractionation of heterogeneous populations of cells or the like to isolate a rel2tively homogeneous sub-po?ulation of a specific cell type. ~ore specifically, the improvement of this invention -elates to masne~ic sor'.ins of cells, bacteria, or viruses.

A general procedure for magnetic sorting of cells, bacteri2, and viruses is disclosed in ~nited States patent 3,970,518, issued July 20, 1976. In tha' procedure, uncoated particles of a magnetic material, such as iron oxide, are contacted with a high concentration liquid dispersion of the selective ant-ibody, and a~ter sufficient antibody has adhered to the magnetic particles, the coated par,icles are contacted with the mixed population to be fractionated, the select cell or the like binding to the magnetic particles, and the bound cells are then separated magneticallv from the remainder of the population. As a further ste?, the select cells may be separated from the magnetic material, bv the use of a cleaving agent solution and magne.ic removal of the magnetic s2rticles.

~ hile there are literature reports describing the use of magnetic microspheres in cell sorting, there is no literature verification that uncoated magnetic particles can be made to erfectively bind with antibodies. In the published procedures, the particles of magnetic material are contained in microspheres formed from polymers, ~hich can be chemicallv cou?led to anti-i~ !
bodies. See, for example: Molday et al, Nature, 268, 437 (1977), ~ronick et al, Science, 200, 1074 (1978); and Antoineet al,~ ImmunochemistrY, 15, 443 (1978). These references -
- 2 -_. . _ _ ~ _ _ , , _.,, ~

~ 27573~

describe magneticaily-responsive microspheres formea from acrvlate ?olymers, such as hydroxyethyl methacrvlate, or polyacrylamide-asarose microspheres. Such microspheres can be chemically cou~led to antibodies with glutaralaehyde or other di-aldehyde. As described by the cited ~olday (1977) .. ..
and Kornick rer-erences~ one procedure involves the chemical attachment of diaminoheptane spacer groups to the microspheres, which are then chemically linked to the antibodies by glutaraldehyde reaction. Although effective bonding of the antibodies can be obtained, such procedures are difricult since aggregation of microspheres can readily occur and the pre?ara-tive procedure is time consuming. For example, the reaction to attach spacer sroups m~y require from five to twelve hours of chemical reaction time, and subsequent dialysis to remove the excess reagent. The coupling of the antibodies mav then require another twelve to twenty-four hours followed bv dialysis to remove excess coupling agent. Further, such an.ibody reagents may not be used efficiently, since an excess of the antibodies will usually need to be ?resent during the chemical coupling.

Another disadvant2ge of magnetic particle or micros?here separation methods as described in the art is that the anti-bodies are attached to the microspheres in a random manner.
A_ Antigen-binding occurs through the Fab regions of the antibodies which are in the outer portions of the arms. hith random attachment of the antibodies, one or both of the Fab arms may be unavailable for antigen-bindina. Thus, an e~cess or antibody must be used to assure that the coated microspheres effectively bind to the an~igens associated with the cells or other bodies being sorted.

1~27S7~

SIJ-lM';~R" OF INV._I~TIG~

The ~-esent invention utili7es s.aphylococc21 Pro,ein to o-~ercome ,he limitations OL prior art masnetic sorting pro-cedures, as desc~ibed above. It is known that staphvlococcal Protein ~. selectively binds to an.ibocies through the Fc region o the antibodies, ~hich is located in tne tail portions of the antibodies remote from the Fab arms. See Forsgren et al, J. Immunol., _ , 19 (1967). Heretorore; however, this pro?ertv o' Protein A has not been utilized to form magnetic micros?heres.
Protein h has been coupled to Sepnarose beads (cross-linked agarose sels) to provide a column material with immunoglobulin-binding properties. The column may be used 'or affinity chroma-tography, for-example, of the IgG fraction o~ serum. Such chromatograpnic column materials are commercially available.

Protein A has also been used in procedures for cell separation by densitv gradient ce~trifucation. See, for example, Ghe.ie et al, Scand. J. Immunol., 4, 471 (1975). In a typical procedure, sheep erythrocvtes are coated with Protein A by CrC13 cou?linc, anc the coated eryth~ocytes are then con-tacted with mouse lymphocy.es which have been ?reviouslv reacted with antibodies to ?repare the cell surfaces for binding to Protein A, tnèreby resulting in rosetting of the l~mphocytes around the erythrocytes. The resulting rosetted cells are recovered by density gradient centrifugation.

In accordance with the present invention, as distinguishec from prior art procedures, magnetically-responsive microspheres are prepared having Protein A associated with the surfaces therec~~, and the resultins microspheres are first reacted with the select antibocies before the microspheres are used for cell se?ara~ion.

1~27S7~

With the microspheres used in the method of this invention the antibodies are thereby arranged in oriented attachment on their outer surfaces with the Fab arms of the antibodies extendino outwardly. The effectiveness of the microspneres for anti~en bindins and use in magnetic sorting p-ocedures is thereby maximized. This greatly increases the efficiency with which the select antibodies may be used. Eurther, it eliminates the need for chemic~ cou?ling of the antibodies.

In a preferred emDodiment, the microspheres are prepared by mixing Protein A with a polymer matrix material which does not mask the antibody-binding sites of the Protein A. The resultinq microspheres having the Protein A in the outer sur~aces thereof do not require chemical couplins of the Protein A to preformed microspheres. Albumin appears to be a particularly suitable matrix material for preparing such microspheres. When the microspheres are formed from an aaueous admixture of albumin, Protein A, and magnetic particles, the Pro~ein A is effectively available in the outer sur_aces of the microspheres, in effect, forming sur'ace layers on the micros?heres with the Protein A in high concentration. The explanation for this result is not fully understood, but ap?ears to relate to the wetting agent or surface tension properties of Protein A when dispersed in ar a~ueous solution in admixture with albumin.

DETAILED DESCRIPTION

In its broad method aspect, the present invention relates to a metnod for the separation of a select po?ulation of cells, bacteria, or viruses from a mixed pc?ul2tion thereof, in which the microspheres containing magnetic particles are coated with a layer of an'ibodies which selectively bind to the select population. Tne coated microspheres are cont2c~ted 1~2757~

wi~h ~he mixed po?ulation, and the boun~ selec, populâtion is magnetically sep2rated from the rest of the mixed ?o?ulation.
The method improvement is characterized by modifying the surfaces of the microspheres prior to coating them with antibodies to provide staphylococcal Protein A distributed thereover in ad'neren~ relation to the microspheres. The micros?heres zre then contacted wi~h antibodies wnich bind the Protein A and which also bind selectively to the select population. By this means the antibodies are arranged in orlented attachment on the surfaces of the microspheres with their Fab arms extending ouLwardly. Thereafter, the rest of the steps of the magnetic separation are carried out, as is known in the art. Preferably, the microspheres are formed of a polymer matrix material in aæmixture with the magnetic particles and Protein A, such as an albumin matri}: material in an amount of 100 parts per 5 to 40 parts of Protein A. Alternatively, however, the Protein A
may be chemically-bonded to the exterior surfaces of the microspheres to provide a Protein A coating thereon.

Where chemical coupling procedures are used, the micro-s?heres may be formed from any matrix material which can be chemically cou~led to Protein A, including albumin or other amino acid poiymer, and synthetic polymers, such as acrylate polymers. 'For example, the microspheres mav be former~ from methyl methacrylate, hydroxyethyl methacrylate, methacrylic acid, ethylene glycol dimethacrylate, agarose polvmers, polv-acrylamiàe polymers, or mixtures of such polymers. Protein A
may be directly coupled to solid support surfaces containing magne.icallv responsive materials by several procedures. See, for example, ~lolday et al, J. Cell Biology, 64, 75 (1975).-~

l~Z757~

Microspheres can be derivatized with either aminocaproic acidor diaminoheptane which provide ex~ended functional groups Cor col-pling proteins ~o insolubilized matrixes. Alternatively, with solid surfaces alreacy containing functionally available grou?s (i.e. amino groups on albumin micros?heres) a direct slutaraldehyde cou?ling of Protein A may be accomplished.

An alternate preferred procedure is to incorporate the Protein A in the microspheres by admixing it with the matrix material prior to the for~ation of the microspheres, a d carryinc out the preparation so that the Protein A is available in the outer surfaces of the microspheres. Suitable procedures for preparing such microspheres will therefore be described, but it should be understood that the present invention in its broad method aspect is not limited to the use of such preferred microspheres.

For use in the present invention, the Protein A can be pre?ared from Staphylococcus aureus by procedures described in the literature. See, for example, Forsgren et al, J. Im~u~., 97, 822 (196~); and Kronvall et al, Immunochemistry, 7, 12~
- (1970). Staphylococcal Protein A is also available from commer-cial sources, such as Pharmacia Fine Chemicals, ~iscataway, ~ew Jersey.

~- The preferred matrix material fo~ forming the micros?heres - by admixture with Protein A is an amino acid polymer, such as albumin. Animal or human albumin may be used, for exam?le, h~man serum albumin. Other water-soluble proteins ca~ be used -- such as hemoslobin, o synthetic amino acid polymers including : polv-L-lysine and poly-L-glutamlc acld.
.:::::
::::::

~ . _............ ., "

~Z75~

When the Protein A is ?remixed with the ma~rix pol~mer, and the micros?heres formed therefrom, sufficient Protein A
should be incluced so tnat the outer surfaces of the micros?heres -v~ill bind antibodies throush the seiective action of the Protein A.
In general, the microspheres may contain from 2 to 40 par~s by weight of Protein A per 100 ?arts of the matrix polymer such as albumin. Preferred ?roportions are from about 10 to 35 parts of the Protein A per 100 parts of the matrix polymer.

A sufficient amount of finely-divided particles or a magnetic material should also be included so that the micro-s?heres are magnetically-responsive. ~or example, the magnetic particles may be ferri- or ferro-magnetic compounds, such 25 magnetic iron oxides. Other useable magnetic materials in particulate form are disclosed in U.S. patent 3,970,518. A
preferred magnetic material is magnetite (Fe3O~). Depending on the size of the microspheres, the magnetic particles may range in size from 100 to 20,000 .~ngstroms. The microspheres may contain from 10 to 150 ?arts by weight of the magnetic material per 100 parts of the matrix polymer. The microspheres may range in size from 0.2 to 100 microns in diameter. Pre-ferably, however, the microspheres have an average size in the range from about 0.5 to 2.0 microns. With microspheres in this size range, i~ is preferred that the masnetic particles have diameters of no. over 300 Angstroms, such as an averase size of about 100 Ansstroms.

The procedure p-ev'ouslv published for preparins alb~min microspheres can be used. ~idder et al, J. Pnarm. Sci., 6~, 79 (1979). The preferred procedure is he one aescribed for the heat-stabilized micros~heres. In general, an aqueous mixture is prepared for use in ~ormins the microcapsules, the mixture ~2757~

containins the zlbumin or other hydrocolloid matrix polymer, Protein ~, ana the magne,ic particles. The solid materials are dispersed in water and thoroughly mixed therewith, for example, usins 20 to 40 parts of total solias per 100 parts of water. Su ficient water should be present to form an aqueous gel with the matrix hydrocolloid. In general, the amount of water may rance from 10 to 60 parts per 100 parts of total solids.
Tne aqueous mix is then emulsified wlth an oil, such as a vege-table oil, the emulsification being carried out with vigorous agitation, for example, usins sonication, to obtain a droplet dispersion OT the aqueous mix in the vegetable oil having the requisite droplet size to form the microspheres. Preferably, the emulsification is carried out at low temperatures, such as temperatures in the range of 20 to 30 C. After the emulsion has been formed, the emulsion is added to a larger body of oil, which is preferably the same oil used to form the emulsion.
In ?ractice, cottonseed oil gives good results. To promote the separation of the water droplets, the emulsion can be added in small increments to the oil bath, such as by dro?wise addition. Preferably, also, the addition is accompanied by ra2id stirring of ~he oil into which the emulsion is beins introduced.

~ or p~rpose of the p-esent inven~ion, the droplets may be heat-hardened to stabilize them and thereby proviae the microspheres. This can be conveniently accomplished by using a heated oil bath, that is, by dispersing the emulsion into hot oil, such as oil at a tem?erature in the range o 70 to 160C. The effect of heat stabilization on albumin micros?heres is described in ~nited S,ates patent 3,37,668, issued February 10, 1976.

~ .f.er the hea.-hardening, .he ?-e?area microspheres are separatec lrom the oil. This ~ay be accomplished by cen.rifugation o _ .

~27571 or ~iltration, and the microspheres ~ashed wi,h a suitable organic solvent, such as diethyl ether, to remove the oil from the exterior sur~aces OL the microspheres. The microspheres are tnen ready for reac,ion with a s?ecific antibody, such as an antibody ~re?ared in rabbits. Such rabbit lmmunoglobulins which bind to Pro,ein A include all subclasses of IgG. However, antibodies pre?arec from other sources can be used, providing they also bind to Protein A. ~sually, the antibodies will be applied .o the micros~heres in aqueous suspension. The concentration of the antibodies may be low, since the Protein A will remove the antibodies from the treating solution even at low concentrations.
As previously described, the binding is through the Fc region of the antibodies, thereby providing for an oriented attachment of the antibodies ~ith the antigen-binding Fab arms extendin~
outwardly fro~ the outer surfaces of the microspheres. The microspheres are then ready for use in magnetic cell separation, as previously described in the literature.

The magnetic sor.ing method of this invention and the preferred microspheres for use therein are further described and illustrated in the followins specific examples. For conciseness of description, the examples use certain abbrevia-tions, which have the followins meaninss:

SpA: Staphylococcal Protein A
FITC: flurocein isothiocyanate CRBC: chicken red blood cell SRBC: sheep red blood cell RBC: red blood cell FCS: fetal cal~ serum H~SS: ~ank's balanced saline solution E3C: carDodiimide: l-cyclohexyl-3-(2-morpholi..yl-(~)-e.hyl-carbodiimiGe me.hotoluene sulphonate) .

E~LE I

Magnetic albumin microspheres containins staphylococczl Protei~ A (Sp~) as part of tne ~atrix were pre?ared by an emulsion polymerization method. A 0.5 ml aqueous suspension con-taining a total of 190 mg dry material was made consisting of 66% human serum albumin, 19~ Fe3O4 (particles 15-20 nm) and 15%
S?A. To this, 60 ml of cottonseed oil was added and the emulsion was homogenized by sonication for one minute. The homogenate was added dropwise to 200 ml of constantly stirred cottonseed oil at 120 to 125C for 10 minutes. The suspension was washed four times in diethyl ether by centrifugation for 15 minutes at 2000 xg and stored at 4C until subsequent use.
A sample of microspheres were coupled with FITC-conjugated rabbit IsG by incubation at 37C ror 20 minutes, and examined for surface fluorescence with a fluorescent microscope. The intensity and app2rent uniform distribution of fluorescence indicated that SpA was oriented on the microsphere surlace in a manner that allowed IgG molecules to interact with the Fc binding sites on the SpA.

E~A~L~ II

Microspheres prepared as described in Example I were used to separ2te CRBC from suspensions con.ai..ing both CR~C
and SRBC. Aliquots of CRBC and SRBC were labeled with Cr in order to assess extent of separation as well as cell integrity. Labeling of CRBC was accomplished by incubating ~, 1 x 10 CRBC suspended in 0.2 ml ~anks balanced salt solution (HBSS) containing 2.5% heat inactivated etal calr serum (PCS) with 100~ Ci ~a251CrO4 (1 mCi/ml) for 90 minutes at 37C.
SRBC were labeled by si~ilar treatment with the exception or overnigh- incubation at 37C. Antiboay was coupled to the -llZ75i7~

01 microspheres by incubating 0.5 mg of the microspheres suspended 02 in 0.2 ml of 0.9% NaCl solution containing 0.1% Tween 80 03 (saline-Tween 80) with either 0.5 mg rabbit anti-chicken RBC tIgG
04 fraction) or 0.5 mg normal rabbit IgG for 45 minutes at 37C.
05 Unbound IgG was removed by centrifugation with excess 06 Saline-Tween 80 at 1500 xg for two minutes at 4C. Microspheres 07 were then resuspended in 0.2 ml saline-Tween 80 by briefly 08 sonicating in an ultrasonic waterbath. To this suspension, a 09 mixture of 1 x 106 CRBC and 1 x 106 SRBC in 0.2 ml of HBSS was added. The cells were then incubated with IgG-coated 11 microspheres for 30 mnutes at 37C with mild agitation. Cells 12 bearing adherent magnetic microspheres were removed ~rom 13 suspension by applying a 4000 gauss (gradient - 1500 gauss/cm) 14 bar magnet to the side of each test tube for one minute. Both supernatant and pellet fractions were counted in a Beckman Model 16 8000 gamma counter for 51Cr. Control labeled cells, incubated in 17 saline-Tween 80, were counteA for 51Cr to assess spontaneous 18 release.
19 Based on 51Cr counts, it was found that when 1 x 106 51Cr CRBC in combination with 1 x 106 SRBC were incubated with 21 0.5 mg microspheres bearing anti-CRBC antibodies, 97.8% of the 22 labeled cells were magnetically removed from suspension.
23 Hemocytometer counts of erythrocytes in the supernatant revealed 24 only 0.26~ residual CRBC among the remaining SRBC. Using this method of cell separation, a population of SRBC which was 97-99%
26 homogeneous was generated with 90.5% recovery of the starting 27 SRBC mass. The non-specific adherence of 51Cr CRBC was tested 28 while using microspheres bearing anti-SRBC and normal rabbit IgG
29 respectively, and found to be <10%.

1~27S71 EY~LE III

Microspheres prepared as described in Example I were used to fractionate Lewis ra, splenocytes. Based on the presence or absence of surface immunoclobulins, it is possible to distinsuish between thymus-derived T lymphocytes and bone-marrow derived B lymphocytes. Normal non-IsG bearing spleno-cytes, considered to be predominantly T lymphocytes, were puri'ied by incubating splenocytes with microspheres con.aining rabbit anti-rat IgG.

A cellular suspension of spleen cells was obtained by teasing rat spleen on a metal screen in HBSS with 10% heat inactivated FCS. The cells were washed three times and over-layered on Ficoll-Hypaque (specific cravity 1.072). The sradien, was then centrifuged at 1200 xg for 25 minutes at 25C, to eliminate dead cells and red blood cells. The resultant inter-face band was removed and assessed ^or viabilitv by trypan blue dye exclusion. The number of IgG bearing cells was determined by incubatina the cells at 37C with FITC conjugated rabbit anit-rat IgG and counting the number of fluoroscent labeled cells. Rabbit anti-ra. IsG, normal rabbit IgG, and rabbi~ anti-chicken RBC were coupled to 0.5 mg the SpA microspheres (0.5 mg IgG/0.5 mg microspheres). Spenocytes (2 x 106) suspended in HBSS with 2.5% heat inactivated FCS were added to the 0.5 mg . .
microspheres. In order to minimize the capping phenomenon and maintain viability, reaction mixtures were incubated for 2.5 hours at 4C. Cells with adherent microspheres were separated magnetically as described in Example II, and resultant super-natants were anal~zed for total cell count, viability, anG fluores-cence. The results are summarized in Table A.

l~Z7S7~

Vizbility o~ un~ractionatec cells after centrifugation in Ficoll-~ypacue was 96~. Su?ernatant cell viability after masnetic sepa-a,ion W25 93~, aemonstrating a minimal loss of vizbility. Between ~7 to 51~ of unfractionated splenocytes were IgG-bearing cells as determined by fluorescence micro-sCo?~. However, after magnetie separation of s?lenoeytes in the experimental grou?, only 0.5~ of the supernatant eells had detectable IgG on tneir surfaee, showing a highly enriehed population of non-IgG-bearins lym?hocytes.

Antibody specificity was verified b~ demons'rating negligible depletion of IgG-bearing cells following incubation of splenocytes tith microsphe~es eontaining either normal rabbit IgG or anti-CRBC. Rat thymocytes, normally con'ainins 4 to 6%
IgG bearing lymphocytes, were to~ally depleted of these cells afte- ineubation with mierospheres eoupled with anti-rat IgG.

The sensitivity of the system was tested by serial dilutions of microspheres bearing rabbit an,i-chicken RBC with the addition of 1 x 10 Cr CRBC at each dilution. Incubation of microspheres and cells was carried out for 30 minutes at 37C. Cells with adheren, micros?heres were masneticallv removed and bo,h pellet and supe-natant ~-actions counted for 51Cr.
Percent CRBC bounc to the microspheres was linearly related to the amount of microspheres present until mierosphere saturation ocel~red. No less than 99% bindins of 1 x 10 CRBC was observed when ~104 ~ g of mierospheres were used.

-- 1~ --l~Z757~

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:1 ~ Q
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.

Foo~notes to Table A

(1) 2 x 106 cells/0.2 ml -~BSS + 2.5% PCS, incubated ~ith 0.-5 mg microspheres bearing antibody as indica.ed. The reaction mixture was incubated for 2.5 hours at 4C.

(2) Tne percent of IgG-bearins cells not removed by magnetic ....
microspheres was determined by incubating su?ernatznt cells with 0.1 ml EITC conjugated rabbit anti-rat IgG
for 20 minutes at 3i~C. The amount of contaminant IgG-bearing cells was determined by fluorescence microscopy.
In addition, 95.3% Or the expected non-IgG-bearins splenocytes was found in the supernatant as de~ermined by triplicate counts in a hemocytometer of non-fluorescent cells.
(3) Between 4q to 51~ starting splenocytes are IgG-bearing cells as de.ermined b~ fluorescence microscopy, and 4 to 6% of ra. th~rmocytes are IgG-bearing cells as determined by the same method.

E~A~PL~ IV

~ agnetic albumin microspheres were prepared as described in Example I, except that Protein A was omitted. The amount of albumin was correspondingly increased so that the dry material used to form the microspheres wzs 81~ albumin and 19% Fe3O4.
Protein A can be applied to the microspheres thus formed as described in Exam?le V.

EXA~PLE V

5mg/ml Protein A in HBSS is pre?ared as a starting solution. For the one step acueous carbodiimide coupling 10 mg of ~DC is adced to 20 mg of eitner an acrylate polymer microsphere ~2757~l matrix prederiv2tized with~ aminocaproic acid, or 20 mg of albumin microspheres suspended in 10 mls of the starting Protein A solution and allowed to react for 4 hrs 2t 4C
with vigorous stirring. The coupling reaction is tnen terminated by addition of 0.4 ml of 0.2 ~, glycine solution p~
7.9. ~nbound Protein A and unreacted EDC is removed by washing ax i~ HBSS. The Protein A coated microspheres 2re then coupled to appropriate antisera by incubation o- 2 mg of microspheres with 1 ml of antiserum at 37C for 10 mins with slight agi~ation. The alternative coupling agent is glutaraldenyde 1.25~ solution which is added to 10 mls of H3SS
solution (pH 7.4) containing 50 mg Protein A and 20 mg of either albumin microspheres or 20 mg of diaminoheptane derivatized acrylate microspheres, allowed to react for 2 hrs at 37C with slight asitation. ~nreacted Protein A and eY~cess glutaraldehyae is removed by centrifugation w2shing 4 X with HBSS. Antibody is attached to microspheres as described above in prior eY~am?les.

...

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for separation of a select population cells, bacteria, or viruses from a mixed population thereof, in which microspheres containing magnetic particles are coated with a layer of antibodies which selectively bind to the select population, the coated microspheres are contacted with said mixed population so that said microspheres are bound to the select population, and said bound select population is magneti-cally separated from the rest of said mixed population, wherein the improvement comprises: prior to coating said microspheres to provide staphylococcal Protein A distributed thereover in adherent relation to said microspheres, then contacting said microspheres with antibodies which bind to Protein A and which also bind selectively to said select population, whereby said antibodies are arranged in oriented attachment on the surfaces of said microspheres with their Fab arms extending outwardly, and thereafter carrying out the rest of the step of said method.
2. The method of claim 1 in which said microspheres are formed of a polymer matrix material in admixture with said magnetic particles and said Protein A.
3. The method of claim 2 in which said matrix material is albumin and said microspheres contain from 2 to 40 parts by weight of Protein A per 100 parts of albumin.
4. The method of claim 1 in which said Protein A is chemically-bonded to the exterior surfaces of said microspheres.
5. Microspheres for magnetically sorting of cells, bacteria, or viruses, comprising microspheres formed from an amino acid polymer matrix material in admixture with staphylococcal Protein A, and being of a size from 0.2 to 100 microns diameter, said microspheres containing from 2 to 40 parts by weight of said Protein A per 100 parts of said amino acid polymer and said Protein A being present in the exterior surfaces of said microspheres for antibody binding, said microspheres also containing magnetic particles of a size from 100 to 20,000 Angstroms and in an amount sufficient to make said microspheres magnetically-responsive.
6. The microspheres of claim 5 in which said amino acid polymer is albumin.
7. The microspheres of claim 5 or claim 6 in which said Protein A is present in an amount of from 10 to 30 parts by weight per 100 parts of said amino acid polymer.
8. Microspheres for magnetically sorting of cells, bacteria, or viruses, comprising microspheres formed from an aqueous mixture of albumin, staphylococcal Protein A, and magnetic particles, said microspheres having an average diameter of from 0.5 to 2.0 microns, and containing from 2 to 40 parts of said Protein A per 100 parts of said albumin and said Protein A being present in the exterior surfaces of said microspheres for antibody binding, said magnetic particles being of a size not over 300 Angstroms and being present in an amount sufficient to make said microspheres magnetically-responsive.
9. The microspheres of claim 8 in which said magnetic particles are Fe3O4 and are present in an amount of from 10 to 150 parts by weight per 100 parts of said albumin.
10. The microspheres of claim 8 or claim 9 in which said Protein A is present in an amount of from 10 to 35 parts by weight per 100 parts of said albumin.
CA345,986A 1979-02-28 1980-02-19 Method of magnetic separation of cells and the like, and microspheres for use therein Expired CA1127571A (en)

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