Transcript
Page 1: CELL FRACTIONATION WITH AFFINITY LIGANDS CONJUGATED … · CELL FRACTIONATION WITH AFFINITY LIGANDS CONJUGATED TO AGAROSE-POLYACROLEIN MICROSPHERE BEADS S. MARGEL*, M. OFARI AN ZDM

J. Cell Sci. 62, 149-159 (1983) 149Pnnted in Great Britain © The Company of Biologists Limited 1983

CELL FRACTIONATION WITH AFFINITY LIGANDSCONJUGATED TO AGAROSE-POLYACROLEINMICROSPHERE BEADS

S. MARGEL*, M. OFARIM AND Z. ESHHARfThe Departments of Plastics Research and f Chemical Immunology, The WeizmannInstitute of Science, Rehovot, Israel

SUMMARY

A new effective insoluble support useful for cell fractionation based on agarose-polyacroleinmicrosphere beads (APAMB) of diameters 150-250 ^m has been developed. The synthesizedpolyacrolein (PA) microspheres, of average diameter 0-2[*m, are provided with reactive aldehydegroups through which various ligands containing primary amino groups are bound covalently in asingle step at physiological pH. Antibodies coupled to the microspheres are very effective forlabelling of cell surface receptors on human red blood cells and mouse lymphoid cells.

A PAM B were obtained by encapsulating the PA microspheres with agarose. Antibodies and lectinsbound to the APAMB served to construct affinity columns for the separation of red blood cells andmurine lymphocyte subpopulations. Anti-human red blood cell antibodies coupled to anti-immunoglobulin APAMB are effective in separating human from turkey red blood cells, whereaseither anti-Thy 1-2 anti-immunoglobulin antibodies or soybean agglutinin coupled to APAMB haveproved useful for the separation of T and B cells from heterogeneous population of spleen cells. Theseparation procedure is simple, rapid and effective. The viability of the fractionated cells is unaffectedby the procedure and the recovery of the cells is high: between 80 % and 100 %.

INTRODUCTION

The understanding of many biological phenomena, such as the molecular mechan-ism involved in cell recognition and interaction, cell specialization and malignancy,requires the development of a reliable method for fractionating various cell sub-populations. Many fractionation methods based on various properties of cells havebeen developed. The main methods include cell electrophoresis (Boehmer, Shortman& Nossal, 1974), fluorescent sorting (Hulett, Bonner, Sweet & Herzenberg, 1973),gravity sedimentation (Faguet, 1974; Reisner& Sharon, 1980), glass, nylon and cottoncolumns for removal of adherent cells (Julius, Simpson & Herzenberg, 1973), and af-finity chromatography with various types of beads, e.g. Sepharose (Nicola, Burgess,Metcalf & Battye, 1978), agarose-polyacrylamide beads (Antoine, Ternynck,Rodrigot & Avrameas, 1978) and magnetic microspheres (Jovin & Jovin, 1980;Molday, Yen & Rembaum, 1977).

In this paper the synthesis of a new effective insoluble support for cell fractionation,consisting of agarose-polyacrolein microsphere beads (APAMB) is described. Thepolyacrolein microspheres situated on the surface of the APAMB were used forcovalent binding of appropriate amino ligands in a single step, at physiological pH.

• To whom reprint requests should be directed.

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150 S. Margel, M. Ofarim and Z. Eshhar

APAMB with bound ligands were used for specific labelling and, thereby, separationof the desired cell population. The separation procedure is simple, rapid and effective.The viability of the fractionated cells is unaffected by the procedure and the recoveryis high.

MATERIALS AND METHODS

ReagentsThe following reagents were purchased from commercial sources: goat anti-rabbit immuno-

globulin G (GaRIgG), mouse Ig and poly-L-lysine HBr (Miles Yeda, Rehovot, Israel), rabbit anti-human red blood cells (RaHRBC) (Cappel Lab. Inc., Cochranville, PA), bovine serum albumin(BSA, Sigman, St Louis, MO), horse serum (HS) (Bio Lab., Jerusalem, Israel), tetramethylrhodamine isothiocyanate (Research Organics Inc., Cleveland, Ohio), fluorescein isothiocyanate(FITC), polyethylene oxide, M, average 100000 (Polyscience, Warrington, PA), trypan blue(Fluka, Buchs, Switzerland), D-galactose (BDH, Poole, England), peanut oil (Amend, Irvington,NJ), agarose A and Sepharosc 4B (Pharmacia Fine Chemicals, Uppsala, Sweden), acrolein,glutaraldehyde, ethylenediamine, sodium azide, ammonium sulphate and ethanol amine (Aldrich,Milwakee, WIS). Soybean agglutinin was a kind gift from N. Sharon's group, The WeizmannInstitute of Science, Rehovot, Israel. Acrolein was distilled at atmospheric pressure before use. Theamino derivative of tetramethyl rhodamine isothiocyanate was obtained by dissolving thetetramethyl rhodamine isothiocyanate in an excess of ethylenediamine in a weight ratio of 1:20.GaRIgG was purified according to a previously published procedure (Margel, Zisblatt & Rem-baum, 1979). Monoclonal anti-Thy 1-2 antibodies were obtained by the ammonium sulphateprecipitation of the ascitic fluid obtained from the inoculation of (AKR/J X Balb/c)Fi mice withthe HO-13-4 hybrid cells (Rothstein et al. 1979). Goat anti-mouse Ig (GaMIg) antiserum wasobtained by hyperimmunizing goats with mouse (Fab)2 fragments. Purified antibodies were thenobtained by affinity chromatography on normal mouse Ig coupled to Sepharose 4B. Tetramethylrhodamine isothiocyanate conjugated to GaMIg and FITC conjugated to monoclonal anti-Thy 1-2were obtained according to Brandtzaegl (1973).

Synthesis of poly acrolein microspheresPolyacrolein (PA) microspheres were prepared by polymerizing acrolein (10%, w/v) in the

presence of polyethylene oxide (0-5 %, w/v) as surfactant with a cobalt radiation source (1 Mrad).Fluorescent microspheres were obtained by earring out the polymerization in the presence of0'00S % (w/v) of the amino derivative of tetramethyl rhodamine isothiocyanate. The microsphereswere subsequently extensively dialysed against distilled water and then centrifuged four times at2000£ for 20 min. The microspheres, of average diameter 0-2fun, as determined by scanningelectron microscopy (SEM), were redispersed in phosphate-buffered saline, 0-01 M, pH 7-2 (PBS)or in distilled water.

Preparation of cell suspensionsHuman red blood cells (RBC) and turkey RBC were obtained by collecting whole normal blood

into tubes containing citrate as anticoagulant. The RBC were washed by centrifugation four timesat 250gfor 7 min at 4°C. After washing the cell pellet was resuspended in PBS or Hanks' solution.

Mouse splenocytes and thymocytes were obtained from female 8- to 10-week old Balb/c mice.The lymphoid organs were removed and teased into Hanks' solution at 4 °C. After filtration throughnylon gauze the cells were washed four times by successive centrifugation at 350 £ for 10 min at 4 °C.The cell pellet was then resuspended in PBS or Hanks' solution. Cells were counted in a haemo-cytometer and their viability determined using trypan blue.

Labelling of human RBCRhodaminated PA microspheres were shaken for 2 h at 4°C with purified GaRIgG (1 mg micro-

spheres, 0-1 mg GaRIgG in a total volume of 0'lSml of PBS). Unbound antibody was then

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Cell fractionation with immuno-microspheres 151

removed by passing the microsphere suspension through a Sepharose 4B column and monitoringthe separation spectrophotometrically at 280 nm. The free aldehyde groups of the microsphere-antibody conjugate were quenched by shaking the beads for 4h at 4°C with either BSA solution(2%, w/v) or with ethanol amine solution brought to pH7-2 with HC1.

A mixture of human and turkey RBC was shaken for 50min at 4°C with RaHRBC (10* cells ofeach type with 0'8/ig RaHRBC in 0-1 ml PBS). The cells were then separated and washed fourtimes by spinning the cell suspension at 500 £ for lOmin. The cell mixture was then shaken at 4CCfor 1 h with the GaRIgG-conjugated microspheres (106 sensitized HRBC, 106 turkey RBC, 1 mgimmuno-microspheres in 0-2ml PBS). The cells were separated from unreacted immuno-microspheres by centrifugation 3 times at SOO^for lOmin. The cell pellet was resuspended in PBSand examined by fluorescent light microscopy.

Labelling of mouse splenocytes bearing surface immunoglobulins (B cells)Rhodaminated microspheres were shaken for 3h at 4°C with GaMIg (1 mg microspheres,

0-05 mg GaMIg in 0-1 ml PBS). The separation of unbound antibody and the quenching of freealdehyde groups were achieved as described for the labelling of human RBC.

Purified mouse splenocytes (106) were then shaken with the GaMIg-bound microspheres for 1 hat 4°C. The cells were separated from unreacted immuno-microspheres by spinning three times at500^ for 15 min at 4CC. The labelled cells were resuspended in PBS and examined by fluorescentlight microscopy.

The control used for this experiment consisted of GaMIg-conjugated microspheres and mousethymocytes.

Synthesis ofAPAMBA solution containing 0-96 g agarose in 16 ml distilled water was heated to 95 °C until the gel

melted into a clear solution. The temperature was then decreased to 70 °C and 8 ml of the PAmicrosphere solution (12%, w/v) heated to 70°C were added. The solution was stirred for 30minand then poured into 100ml of peanut oil, stirred at 300rev./min at 70°C. Ten minutes later, thesolution was cooled with ice. The APAMB formed were purified from the oil by several extractionswith ether. Ether was removed by evaporation. The beads obtained ranged from 50-250fun indiameter. Fractions containing APAMB with diameters 50-150 j«n and 150-250/im were obtainedby passing the beads through appropriate sieves. APAMB with diameters ranging from 300—450 ^m were prepared by the same procedure but with a stirring rate of the peanut oil solution of100 rev./min. The APAMB were stored at 4°C in distilled water containing 0-05 % (w/v) sodiumazide.

Binding of polylysine—glutaraldehyde to the APAMBAPAMB (20ml) in 35 ml of distilled water were shaken for 24h with polylysine (40mg). The

polylysine-conjugated beads formed were washed free of unbound polylysine by repeated decanta-tion. The conjugated beads (20 ml in 35 ml H2O) were then shaken for 12 h with glutaraldehyde(2 ml of 50% solution) and the resultant polylysine-glutaraldehyde-conjugated beads were filteredand then washed with large amounts of distilled water.

Coupling of proteins to the APAMBAPAMB (1 ml) with or without a polylysine-glutaraldehyde spacer were shaken at 4°C for 24h

with appropriate proteins: GaRIgG, GaMIg, anti-Thy 1-2 and soybean agglutinin (10 mg proteinin 5 ml PBS). The remaining aldehyde groups were then quenched by shaking the beads for 12 hwith 0'05ml ethanol amine solution at pH7-2. The immuno-APAMB formed were then washedsuccessively with either PBS or Hanks' solution.

Determination of proteinsQuantities of proteins bound to the APAMB were determined by substracting the amount of

unbound proteins from that of total proteins measured according to the method of Lowry,Rosebrough, Farr & Randall (1951).

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152 5. Margel, M. Ofarim and Z. Eshhar

Fractionation of cellsThe immuno-APAMB were packed in a siliconized Pasteur pipette plugged with glass wool. The

beads were washed first with PBS to remove sodium azide and then with Hanks' solution containing5 % horse serum. Washed, viable cells (1 to 2 X 107) suspended in 1-2 ml of Hanks' + 5 % HS werefiltered through the immuno-beads (1 ml) at room temperature, at a rate of 1-3 drops/min. Thecolumn was then rinsed with Hanks' + 5 % HS till the eluate became cell-free. Non-adsorbed cellswere recovered by centrifugation at 500^* for 15 min and then resuspended in 1-2 ml of Hanks' +5 % HS. The beads were poured into a glass vial containing approximately 5 ml of Hanks' + 5 %HS and the adsorbed cells were then recovered by gently stirring the beads with a Pasteur pipette.Cells adsorbed to soybean agglutinin-conjugated APAMB were recovered by passing 0-2M-D-galac-tose in Hanks' solution (3-5 ml) through the column. The supernatant containing the cells wascentrifuged at 500 £ for 15 min, and the cells were then resuspended in 0-5— 1 ml of Hanks' + 5%HS. The immuno-beads were washed with PBS and then stored in PBS + 0-05 % (w/v) sodiumazide until reused.

Detection of cells bearing immunoglobulins (B cells) and Thy 1 -2 antigens (T cells) ontheir surface

Normal mouse B splenocytes were detected by the following method: cells (1 X 106 in 0-1 ml ofHanks' solution) were incubated for 1 h at 4°C with rhodaminated GaMIg (5/ig in 25^1 of PBS).After the addition of Hanks' + 5% HS (3 ml at 4°C) the cell suspension was washed at 4°C bycentrifugation at 500 #for 10 min. The cells were resuspended in 50filof Hanks' + 5% HS and theamount of fluorescent cells was immediately counted in a haemocytometer.

Normal mouse T splenocytes were detected by a similar procedure, using FITC conjugated toanti-Thy 1-2 instead of rhodaminated GaMIg.

RESULTS

A SEM picture of the PA microspheres used for the labelling and separationprocedures is shown in Fig. 1. The high specificity of the fluorescent PA microsphereswas demonstrated in two systems: (1) specific labelling of human RBC in a mixturecontaining human and turkey RBC (Fig. 2); (2) specific labelling of mouse Bsplenocytes (Fig. 3). In both cases, only the human RBC and the B splenocytes,respectively, were stained, indicating their specific labelling by the microspheres.

The binding capacity of the APAMB towards various antigens and antibodies isgiven in Table 1. Steric requirements can explain the significant increase (3- to 5-fold)in the binding capacity of the APAMB bound to the spacer arm polylysine-glutaral-dehyde. This increase leads also to the increase in the binding capacity of the beadsto cells. For example, the binding capacity of the APAMB conjugated to GaRIgGthrough the spacer polylysine—glutaraldehyde to human RBC (sensitized with RaHRBC) is 1-6 X 108RBC per 1 ml beads, while the binding capacity of the same beadsconjugated directly to GaRIgG is just 4-8X107 RBC per lml beads. Therefore,subsequent cell fractionation experiments were carried out with APAMB conjugatedto the spacer polylysine-glutaraldehyde.

Fig. 1. A SEM picture of the PA microspheres. Bar, 5 fim.

Fig. 2. Light (A) and fluorescence (B) micrographs of a mixture of human and turkey RBCsensitized with RaHRBC and labelled with FITC-labelled GaRIgG bound to PAmicrospheres. x 1400.

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Cell fractionation with immuno-microspheres

Fig. 1

Fig. 2CRL62

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154 5. Margel, M. Ofarim and Z. Eshhar

B-l

Fig. 3. Light and fluorescence micrographs of: A, mouse splenocytes; B, mousethymocytes, mixed with rhodaminated PA microspheres bound to GorMIg. X460. 1, lightpicture; 2, fluorescence picture.

Table 1. Binding capacity of APAMB to proteins

Proteins

GaMIgGaMIgAnti-mouse Thy 1-2Anti-mouse Thy 1-2GaRIgGGaRIgGSoybean agglutinin

APAMB (lml) weretemperature.

Spacer

Polylysine-glutaraldehyde—

Polylysine-glutaraldehyde—

Polylysine-glutaraldehydePolylysine-glutaraldehyde

shaken with excess quantities of the proteins in

Binding capacity(mg/ml beads)

2-412-42-49-82-36-05-9

PBS (5 ml) for 12 hat room

The APAMB preserved their physical and mechanical properties after the couplingof the proteins. Leakage of bound proteins from the beads to the supernatant underacidic and physiological conditions was not detected, as determined by using themethod of Lowry et al. (1951).

The efficiency of the cell separation is dependent on the size of the APAMB, asshown in Table 2. By passing a mixture containing approximately 50 % human RBCand 50% turkey RBC sensitized with RaHRBC, through a column of GctRIgG-bound beads with diameters ranging from 80-150/zm or 150-250 im the percentageof the adsorbed human RBC was 99% and 98%, respectively. On the other hand,

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Cell fractionation with immuno-microspheres 155

Table 2. Percentage of adsorbed human RBC and recovery of turkey RBCnon-adsorbed on anti-RIgG-conjugated APAMB of various sizes

Diameter of APAMB % of adsorbed human RBC % Recovery of turkey RBC

Km)90-150 99 60

150-250 98 80300-450 74 90

A mixture containing approximately 108 human RBC and 108 turkey RBC sensitized withRaHRBC was passed through a column containing 1 ml of GaRIgG-conjugated beads.

• • r

Fig. 4. A light microscopic picture of mouse spleen cells adsorbed to GaMIg bound toAPAMB. A. Control; immuno-beads without attached cells. X250. B and c. Immuno-beads labelled with the cells (arrows indicate the attached B lymphocytes), B, X 2 3 0 ; C,X920.

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156 S. Margel, M. Ofarim and Z. Eshhar

by carrying out the same experiment using beads with diameters of 300-450/im thepercentage of the adsorbed human RBC was only 74%. The recovery of the non-adsorbed turkey RBC is also dependent on the size of the APAMB: 60 %, 80 % and90 % were recovered with beads with diameters of 80-150, 150-250 and 300-450 [im,respectively. The smaller the beads, the higher the percentage of turkey RBC retainedin the interstices of the beads. APAMB of diameters ranging from 150-250 [im are themost suitable for cell fractionation and subsequent research was done with beads ofthese sizes.

Fractionation of mouse splenocytes into B and T cells was achieved with APAMBconjugated to either GaMIg, anti-Thy 1 -2 or soybean agglutinin. The lectin, soybeanagglutinin, binds specifically to B cells and not to T cells, as reported previously(Reisner & Sharon, 1980). Therefore, cells adsorbed to the soybean agglutinin-derivatized beads are predominantly B cells and those that do not adsorb are mainlyT cells. Fig. 4 is a light-microscope picture demonstrating the labelling of mouse Bsplenocytes with GaMIg-conjugated APAMB. A more quantitative result illustratingthe efficiency of the cell fractionation through the various immune-APAMB is shownin Table 3. In the control experiment mouse splenocytes were passed through acolumn containing GaRIgG bound to APAMB. The composition of the B and T cellsremained almost constant: 53% and 43 % of B and T cells before fractionationcompared to 51 % and 45% after fractionation. A very pure population of T cells(97—98 %, Fig. 5) was obtained in the non-adsorbed cell fraction passed through theGaMIg-conjugated beads. However, the cells eluted from these immuno-beads for-med small aggregates, which made cell counting difficult. The efficiency of the

Table 3. Cell fractionation of mouse splenocytes with immuno-APAMB

Immuno-beads

GcrRIgG-APAMB(control)

GaMIg-APAMBGaMIg-APAMBAnti-Thy 1-2-APAMBAnti-Thy 1-2-APAMBSoybean agglutinin-

APAMB

Soybean agglutinin-APAMBf

Beforefractionation

A

B cells T

53

6058575957

561710

' cells

43

4040404038

418190

PercentageA

rAdsorbedB

——403784

84

_

of cells

After

cellsT

•*

545416

16

fractionationA

Non -adsorbedB

51

00

868415

17107

• \

cellsT

45

9897131477

819090

Mouse splenocytes (1 to 2 (X 107) cells) were passed at a flow rate of 1-3 drops/min. through acolumn containing APAMB conjugated with either GaMIg, anti-Thy 1-2 or soybean agglutinin.

• Small aggregates of cells make cell counting inefficient,f The non-adsorbed cells were passed through the immuno-beads three times.

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Cell fractionation with immuno-microspheres

B-l

157

Fig. 5. Fluorescence and light micrographs of mouse splenocytes passed through acolumn of GaMIg bound to APAMB and detected with FiTC-labelled anti-Thy 1-2antibody, A-1 and A-2: control, light and fluorescence pictures of non-fractionated cells.B-l and B-2: light and fluorescence pictures of the non-adsorbed fractionated cells. X690.

separation obtained with the anti-Thy 1-2 and soybean agglutinin-conjugated beads,as shown in Table 3, is also satisfactory, although not as efficient as with theGaMIgG-conjugated beads. The main advantage of the lectin(soybean agglutinin)-conjugated beads is the ability to elute the adsorbed cells by washing the column withan appropriate sugar, i.e. D-galactose, which has high affinity towards soybeanagglutinin, eliminating several subsequent procedures. In all the experimentsdescribed in Table 3 the viability of the fractionated cells was unaffected and therecovery was between 80% and 100%.

DISCUSSION

In the present and a previous publication (Margel, Beitler & Ofarim, 1982) thepotential use of PA microspheres as a new tool for mapping cell surface receptors hasbeen demonstrated. Several authors also described the use of polymeric microspheres,especially with magnetic properties, for cell separation (Jovin & Jovin, 1980; Margeletal. 1979; Molday et al. 1977). However, we found that the use of microspheres forcell separation is limited. The size of the microspheres is much smaller than that ofthe cells to be labelled: 0-2 /im compared to approximately 5 pm. During the labelling

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158 5. Margel, M. Ofarim and Z. Eshhar

procedure each cell can be labelled with hundreds or thousands of microspheres, ashas been shown by SEM pictures (Margel et al. 1979; Rembaum, Yen & Volksen,1978). Each microsphere can bind through the aldehyde groups to many receptors.It is extremely difficult to remove the microspheres from the cell surface and to obtaina pure cell population. On the other hand, the agarose—polyacrolein microspherebeads can be very useful for cell fractionation. The cell size is smaller than that of theAPAMB: 5 pan compared with an average size of 200 pan. Each bead can be labelledwith hundreds of cells (e.g. the capacity of the APAMB to human RBC is 16 X108cells/ml bead). Assuming that the density of the bead is 1 g/cm3, each gram ofbeads with an average radius of 100 pan contained 2 5 X 10s beads. Therefore, eachbead is labelled with 640 cells (1-6 X 10Y2-5 X 105). However, by gentle stirring ofthe labelled beads the cells detach from the bead's surface and a pure cell populationcan be obtained.

The APAMB fulfill the essential requirements of effective insoluble supports.They are stable, and the non-specific interaction between the cells and the beads, andthe toxicity of the beads towards the cells, are negligible. Amino ligands can be boundcovalently to the beads in a single step and under physiological pH. Moreover, leakageof the bound ligands to the supernatant was not detected under acidic and physiologi-cal pH values. The APAMB were shown previously to have a potential use for affinityseparation (Margel, 1982) and haemoperfusion (Marcus, Ofarim & Margel, 1982). Afurther application of the APAMB, as an effective insoluble support for cell fractiona-tion, has been demonstrated in this paper.

The research was supported by the G. Schmidt Fund for Industrial Research at the WeizmannInstitute of Science and by the Ministry of Science and Development, National Council forResearch and Development, Israel.

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BOEHMER, U. V., SHORTMAN, K. & NOSSAL, G. J. V. (1974). The separation of different cellclasses from lymphoid organs. J. cell. Physiol. 83, 231-242.

BRANDTZAEGL, P. (1973). Conjugates of immunoglobulin G with different fluorophores: I.Characterization by anionic exchange chromatography. Scand.J. Immun. 2, 273-290.

FAGUET, G. B. (1974). Lymphocyte purification: an improved method. Qualitative and quanti-tative evaluation. Biomedicine 21, 153-157.

HULETT, H. R., BONNER, B. A., SWEET, R. G. & HERZENBERG, L. A. (1973). Development andapplication of a rapid cell sorter. Clin. Chem. 19, 813-816.

JOVIN, T. M. & JOVIN, J. A. (1980). Cell separation. Trends Biochem. Set. 5(8), 214-219.JULIUS, M. H., SIMPSON, E. & HERZENBERG, L. A. (1973). A rapid method for the isolation of

functional thymus-derived murine lymphocytes. 3, 640-644.LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement

with the folin phenol reagent. J. biol. Chem. 193, 265-275.MARCUS, L., OFFARJM, M. & MARGEL, S. (1982). A new immunoadsorbent for hemoperfusion:

agarose-polyacrolein microspheres beads. In vitro studies. Biotnat., Med. Devi, Art. Org. 10(3),157-171.

MARGEL, S. (1982). Agarose polyacrolein microspheres beads, new effective immunoabsorbent.FEBS Letts 145(2), 341-344.

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Cell fractionation with immuno-microspheres 159

MARGEL, S., BEITLER, U. & OFFARIM, M. (1982). Polyacrolein microspheres as a new tool in cellbiology.7. CellSci. 56, 157-175.

MARGEL, S., ZISBLATT, S. & REMBAUM, A. (1979). Polyglutaraldehyde: a new reagent for couplingproteins to microspheres and for labeling cell surface receptors. II. Simplified labeling methodsby means of non-magnetic and magnetic polyglutaraldehyde microspheres. J. Immun. Meth. 28,341-353.

MOLDAY, R. S., YEN, S. P. S. & REMBAUM, A. (1977). Application of magnetic microspheres inlabeling and separation of cells. Nature, Land. 268, 437-438.

NICOLA, N. A., BURGESS, A. W., METCALF, D. &BATTYE, F. L. (1978). Separation of mouse bonemarrow cells using wheat germ agglutinin affinity chromatography. Aust.jf. exp. Biol. med. Set.56, 663-679.

REISNER, Y. & SHARON, N. (1980). Cell fractionation by lectins. Trends Biochem. Sci. 5(2), pp.29-31.

REMBAUM, A., YEN, S. P. S. & VOLKSON, W. (1978). Labeled cells. Chemtech. 8, 182-190.ROTHSTEIN, A. M., FINK, P., GRIDLEY, T., RAULET, D. H., BEVAN, M. J. & GEFTER, M. L.

(1979). Properties and applications of monochlonal antibodies directed against determinants oftheThy-1 locus.7. Immun. 122(6), 2491-2497.

{Received 20 October 1982 -Accepted 3 December 1982)

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