P r e p a r a t i o n a n d F u n c t i o n a l C h a r a c t e r i z a t i o n o f Monoclonal Antibodies against Glycoprotein Ib By LESLEY E. SCUDDER, EFSTATHIA L. KALOMIRIS, and BARRY S. COLLER
Introduction The glycoprotein Ib complex (GPIb complex) constitutes a nonintegrin platelet receptor for von Willebrand factor (see  in this volume). Monoclonal antibodies directed against the GPIb complex are useful tools for identifying and quantifying the GPIb complex, mapping functional domains of the receptor, and isolating the complex by immunoaffinity techniques.
Immunizations of Mice 1 Materials
Trisodium citrate solution, 40% Freund's complete adjuvant (Calbiochem, La Jolla, CA) Tris-EDTA buffer: 10 mM Tris-HCl, 0.15 M NaCI, 10 mM NaEDTA, pH 7.4 Tris-NaC1 buffer: 10 mM Tris-HC1, 0.15 M NaCI, pH 7.4 Mice: BALB/cBYJ (Jackson Laboratory, Bar Harbor, ME) 4-6 weeks old, either sex Method
Whole blood is collected from normal donors by venipuncture and placed into plastic tubes (Falcon 2059, Oxnard, CA) containing 1/100 vol 40% citrate. The blood is centrifuged at 700 g for 3.5 min at 22° and the supernatant platelet-rich plasma (PRP) transferred to another tube with a plastic pipette (13-711-5; Fisherbrand, Pittsburgh, PA). The PRP is then washed with a fivefold volume of Tris-EDTA buffer and sedimented at --1500 g for 8-10 min at 22 °. The supernatant platelet-poor plasma is decanted and the platelet pellet resuspended in a combination of the residual -0.1 ml of plasma and 0.2 ml Tris-EDTA buffer by gently aspirating the suspension into a plastic pipette tip (223-9000; Bio-Rad, Richmond, I B. S. Coller, E. I. Peerschke, L. E. Scudder, and C. A. Sullivan, Blood 61(1), 99 (1983). METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
PLATELET RECEPTORS" ASSAYS AND PURIFICATION
CA) and dispensing it back into the tube several times until the suspension appears homogeneous. After the second wash, the platelets are resuspended in 0.1 ml of Tris-EDTA buffer, and mixed 1 : 1 with the adjuvant with the aid of a device made by cutting two 18-gauge, Luer-lok metal needles close to their hubs and welding them together tip to tip. The platelet suspension and an equal volume of well-mixed complete adjuvant (making sure to resuspend the pellet of mycobacteria, which tends to settle) are each drawn up into a 1-ml plastic syringe. The syringes are joined at opposite ends of the Luer-lok fittings and the pistons simultaneously pushed to and fro until the suspension becomes thick and milky white. The suspension is withdrawn into one of the syringes, and the latter is then disconnected from the device and fitted with a 23-gauge needle for injection into the mice. In our study, ~ a BALB/c mouse receives six weekly intraperitoneal -0.2-ml injections containing - 3 x 108 fresh, washed platelets. The last injection is of - 5 x 108 platelets in Tris-NaCl buffer without either EDTA or adjuvant and is given intravenously into the tail vein. Each of the seven weekly platelet suspensions is obtained from a different donor. Three to 4 days after the tail vein injection, the mouse is sacrificed for the fusion. It should be noted that the immunization procedure detailed above is somewhat unconventional in that all six weekly injections of platelets include Freund's complete adjuvant. Most immunization schedules either use no adjuvant for cellular antigens or limit the complete adjuvant to only the first injection, z-5 We have no evidence that our technique is superior to the others. It is important to limit the volume administered in the intravenous injection given at the end of the schedule. We injected approximately 0.1 ml and found that this was not well tolerated; some animals, including the one from which we ultimately obtained an antibody, suffered what appeared to be a cardiorespiratory arrest, either on a temporary or permanent basis. Because we were interested in obtaining an antibody that inhibited platelet function, we tried to minimize the processing procedures so that the antigen would be as close to the native conformation as possible. The search for antibodies for different purposes may influence the choice of preparation technique. For example, if one wants to develop an antibody that reacts well in immunoblotting antigens separated by
2 j. W. Goding, "Monoclonal Antibodies: Principles and Practice." Academic Press, London, 1983. 3 G. Galfre and C. Milstein, M e t h o d s Immunol. 73, 3 (1981). 4 S. F. de St. Groth and D. Scheidegger, J. Immunol. M e t h o d s 35, 1 (1980). 5 H. Zola and D. Brooks, in "Monoclonal Hybridoma Antibodies: Techniques and Applications" (J. G. Hurrell, ed.). CRC Press, Boca Raton, Florida, 1982.
SDS-polyacrylamide gel electrophoresis, it would probably be desirable to treat the antigen with sodium dodecyl sulfate (SDS) before immunizing. Screening Assay Our initial goal was to identify the ristocetin-dependent platelet receptor for von Willebrand factor (vWF) by preparing an antibody that would block the binding of vWF to ristocetin-treated platelets. We reasoned that such an antibody would also block the agglutination of platelets induced by ristocetin in the presence of vWF. We therefore devised a quick, easy, high-volume, and inexpensive screening assay employing formaldehydefixed platelets, normal plasma, and ristocetin. ~ Materials
Ristocetin: 50 mg/ml in Tris-NaCl buffer, pH 7.4 (Lenau, Denmark) Pooled normal plasma (PNP) (George King, Inc., Overland Park, KS) Formaldehyde fixing solution: Just before use, add 2 ml of 37% formaldehyde (F-79; Fisher, Pittsburgh, PA) to 98 ml of Tris-NaC1 buffer Formaldehyde-fixed platelets" One unit of fresh platelet concentrate is mixed 1 : 1 (v/v) with the fixing solution and stored at 4 ° for up to several months. For use: 8-10 ml of fixed platelets is washed three times in Tris-NaC1 buffer and resuspended to a count of 3-3.5 x 108/ml for platelet-rich buffer (PRB) Method
All steps are carried out at 22° . Add 50 tzl of PRB to each well of a round-bottom microtiter plate (Linbro, Hamden, CT), and then 50/zl of test culture supernatant, test serum, test ascites, or buffer. After 2 min, add 10/xl of PNP to each well and gently shake by sliding the plate with the right hand against a stationary left thumb in three or four smooth, short strokes while keeping the plate flat on the bench top. Add 3/zl of ristocetin to each well and shake gently again until uniformly cloudy (ristocetin tends to form a white precipitate at the bottom of the well). Attach the plate with rubber bands to a platform rotator (Tektator V; American Scientific Products, Edison, N J) and rotate for 8 min at - 2 8 0 rpm at 22°. The wells are then observed from the bottom with a magnifying mirror viewer (Cooke Microtiter Systems, Dynatech Laboratories, Alexandria, VA). Wells containing buffer or culture medium have marked agglutination (4 + rating), while those containing the supernatant of positive clones show less agglutination (0-3 + ). A positive control can be obtained by omitting the ristocetin or using plasma deficient in vWF (available from George King, Inc.).
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
Monoclonal Antibody Preparation 1-5 Materials
RPMI: RPMI 1640 culture medium supplemented with penicillin (1000 U/ml) and streptomycin (100 tzg/ml) (GIBCO, Grand Island, NY; or Flow Laboratories McLean, VA) RPMI-FBS: RPMI 1640 supplemented with I0 or 20% fetal bovine serum (FBS) (200-6140; GIBCO) NCTC 109 medium (M. A. Bioproducts, Walkersville, MD) P3X63-Ag8.653 nonsecretory BALB/c mouse myeloma cell line 6 N H 4 C I ( 0 . 8 3 % ) : Filter sterilize through 0.22-tzm filter (450-0020; Nalgene, Rochester, NY) and keep on ice Polyethylene glycol (PEG): Originally we used PEG from Koch-Light (Coinbrook, Bucks, England; Mr 1000) as a 35% PEG solution in RPMI. Autoclave pure PEG to sterilize and put in 3.5-ml aliquots. Store at 22 ° in the dark. To prepare the 35% solution, heat one aliquot to 56 ° and add to 6.5 ml warmed RPMI. Currently, we use 40% PEG (Mr 1500) from Boehringer Mannheim Corp., GmbH (Indianapolis, IN; #783641), which comes as a 50% (w/v) solution in 75 mM HEPES buffer. Mix one 4-ml vial of PEG with 1 ml RPMI just before use Note. There is evidence that many factors affect the fusion efficiency of different PEG preparations, and there may even be batch-to-batch differences with the same product. The pH of the solution may be important as well as contamination with aldehydes27'8; sterilization by pressure filtration rather than autoclaving may minimize the latter.
A (50 × aminopterin): Dissolve 4.4 mg aminopterin in 500 ml distilled water. Filter sterilize and freeze 1-ml aliquots at - 2 0 ° HT (50 x hypoxanthine and thymidine): Dissolve 68.1 mg hypoxanthine and 19.4 mg thymidine in 100 ml distilled water. Adjust to pH 7.0, filter sterilize, and freeze 1-ml aliquots at - 2 0 ° HAT medium: Add 1 ml A and 1 ml HT to 50 mi RPMI-20% (v/v) FBS-10% (v/v) NCTC 109. Make fresh 2,6,10,14-Tetramethylpentadecane (Pristane) (Sigma Chemical Corp., St. Louis, MO) phosphate-buffered saline (PBS): 0.15 M NaC1, 0.01 M sodium phosphate, pH 7.4 Freezing solution: 10% (v/v) Dimethyl sulfoxide (DMSO) in 90% (v/v) FBS. Make fresh and filter sterilize 6 j. F. Kearney, A. Radbruch, B. Liesgang, and K. Rajewsky, J. Immunol. 123, 1548 (1979). 7 R. D. Lane, R. S. Crissman, and M. F. Lachman, J. ImrnunoL Methods 72, 71 (1984). 8 j. L. Kadish and K. M. Wenc, Hybridoma 2, 87 (1983).
Immunized mouse Ketamine, 100 mg/ml (Aveco, Fort Dodge, IA) Ethanol, 70% Two sterile 23-gauge needles attached to 1-ml syringe barrels Sterile flat-bottom, 96-well, microtiter plates with lids (Corning 25860, Coming, NY or Costar 3596, Cambridge, MA) Sterile, 24-well microtiter plates with lids (Costar 3524) Petri dishes, plastic pipettes (Falcon) Flasks, 75-cm 2 (Falcon 3024 or Coming 25110-75) Flasks, 25-cm 2 (25100-25; Corning) Hood (VBM-400 Sterigard hood; Baker Co., Inc., Sanford, ME) Dual-chamber, CO2 water-jacketed incubator (3325; Forma Scientific, Marietta, OH) Plastic tubes, 15 and 50 ml (Coming 25311 and Falcon 2070, respectively)
Method Myeloma Cells. Two to 3 weeks before the fusion, the myeloma cells are thawed and maintained in RPMI with 10% FBS. Most investigators believe it is vital that the cells be in the log phase of growth at the time of the fusion and this is achieved by diluting two or three 75-cm 2 flasks to -105 cells/ml 1 or 2 days before the fusion. On the day of the fusion, the cells are counted. Spleen Cells. If possible, use a separate room and hood for the mouse surgery. Keep scissors and forceps immersed in 70% ethanol. The immunized mouse is anesthetized with ketamine (10 mg ip) and then sacrificed by cervical dislocation. First, secure the mouse by the tail and let it stand on the bench top. Pinch the nape of the neck from behind with the thumb and forefinger. Quickly, depress the nape of the neck to the bench top as the tail is snapped horizontally away from the body. The neck can be felt to snap. Wash the abdominal area with 70% ethanol, pick up the skin to avoid penetrating the viscera, and cut into the abdominal cavity. Aseptically remove the spleen and rinse in a 50-ml tube containing 20-30 mi RPMI. This tube can then be brought to the tissue culture hood. Empty the tube contents into a sterile petri dish and aspirate the medium. Add 10 ml of fresh RPMI to rinse again and aspirate the supernatant medium again. Use the two fine needles to tease the spleen apart with a "knife and fork" action. Tease the pieces as small as possible, but try to work rapidly ( - 5 min). Pipette 8-10 ml RPMI into the dish, withdraw all the cells and tissue chunks, transfer to a 50-ml tube, and fill to 35 ml with RPMI. After 10 rain the large clumps will settle, and then transfer the top -32 ml of smooth cell suspension to a clean 50-ml tube, centrifuge at 500 g for 10
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
min at 22 °, and aspirate the supernatant. To lyse the red blood cells, add 5 ml of cold 0.83% (w/v) NH4CI to the pellet, place on ice for 10 min, add 5 ml of RPMI, and centrifuge at 500 g for 5 rain at 22°. The red pellet should become white. If not, the 0.83% NH4C1 step can be repeated. The final pellet should be gently resuspended in - 1 0 ml RPMI and a cell count obtained with a hemocytometer. Fusion. All steps are conducted at 22° and the steps involving cell manipulation are conducted in a hood. Add 4 x 108 spleen cells to 1 x 108 myeloma cells (4: 1) in a 50-ml tube and then centrifuge at 500 g for 5 rain. Aspirate the supernatant and gently tap the conical tube on the bench top to loosen the pellet. Add 3 ml of the 35% PEG or 1 ml of the 40% PEG using a wide-bore pipette (to avoid clogging by PEG and excess shear that would disrupt fused cells), mix gently by pipetting up and down two or three times, and immediately centrifuge at 500 g for 6 min. Do not aspirate supernatant. Add an additional 5 ml of RPM1, mix gently with the pipette, centrifuge again, but at 230 g for 6 rain, and aspirate the supernatant. The pellet is then resuspended in 50 ml of fresh RPMI-20% FBS-10% NCTC 109. The cells can receive HAT medium now or after an overnight incubation in a 75-cm 2 flask. There may be a slight advantage to the overnight incubation, since contaminating fibroblasts and other cells adhere to the flask. The next day the cells are mixed and decanted into a 50-ml tube, leaving unwanted cells attached to the flask. Add I ml of solution A and 1 ml of solution HT, mix, and aliquot 0.1-ml samples into five or six 96-well plates. Incubate for 4 days in a 37°, 5% CO2 incubator and then add 0.1 ml of HAT medium to each well. Clones should start appearing as "fuzzy spots" at the periphery of the well - 2 weeks after the fusion; at that time, 0.2 ml of medium is withdrawn from each well showing cell growth and 0.2 ml of RPMI-20% F B S - H T medium is added. After another 8-9 days, 0.2 ml of medium is withdrawn again and replaced with 0.2 ml of RPMI-20% FBS-HT. About 4 weeks after the fusion, the cells can be transferred to the wells of a 24-well plate. Pipette the well contents up and down several times to remove most of the cells and place in a well of a 24-well plate. Add an additional 0.5 ml of RPMI-20% F B S - H T and, in about 4 days, add 1 ml of RPMI-20% FBS. Expand the cells to 25-cm z flasks (10 ml) and then to 75-cm 2 flasks (50 ml) by adding increasing amounts of medium. We routinely grow cells in RPMI-10% FBS, but one can decrease the cost by switching to horse serum or lower concentrations of FBS. Because mold periodically and unpredictably appears in individual flasks, we keep duplicate flasks growing at all times. The cells need to be "split," i.e., diluted I : 5 or 1 : 10, every 3-4 days, thus permitting twice weekly attention. To ensure monoclonality, the cells must be subcloned either by the
limiting dilution technique 2-5'9 or by growth in soft agar. 2-5 Additional evidence for monoclonality can be obtained from subclass and light chain typing, by immunodiffusion, or by isoelectric focusing. Freezing Clones To avoid losing valuable clones, they should be frozen in several different aliquots as soon as their density reaches -105/ml (usually 5-10 ml in flask). The cells are pelleted at 1000 g for 8 min at 22 ° and the culture supernatant aspirated and saved. The cells are resuspended in the freezing solution at - 5 x 105 cells/ml and frozen at a rate of - l°/min in a standardized biological freezing tray (Ro36-8c15, Union Carbide, Indianapolis, IN). T h e y are stored indefinitely in the fluid phase of liquid nitrogen. For cells that are likely to be in storage for a number of years, it is important to thaw aliquots of cells every year or so to prepare flesh cells for refreezing. Ascites Production To obtain higher titers of antibody than can be achieved in culture medium, the hybrid cells can be grown as an ascites tumor in the abdominal cavity of a mouse. B A L B / c mice are primed intraperitoneally with 0.5 ml of Pristane approximately 10-14 days before receiving the cells, which seems to be the best pretreatment period, l°m The cells are washed twice in PBS and injected intraperitoneally into 6-week-old B A L B / c mice. Each mouse gets 0.1-0.2 ml usually containing 1-5 x 106 cells. In 2-4 weeks, the animal is anesthetized with ketamine (0.5-1 mg), the abdominal area is cleansed with alcohol, and the ascites drained through a 20-gauge needle into a 17 x 100-mm plastic tube (2059; Falcon). The fluid is heated to 56 ° for I0 min to defibrinate the ascites and then centrifuged at I100 g for 10 min at 22 °. The supernatant is saved frozen at - 2 0 ° until ready for purification. If desired, the procedure can be performed in a sterile hood. Purification of Antibodies Materials
Phosphate buffer: 0.1 M NaPO4, 0.05% (w/v) N a N 3, pH 8.0 Citrate buffers: 0.1 M sodium citrate, 0.05% (w/v) NAN3, pH 6.0, 4.5, 3.5, and 3.0 N o t e : Filter buffers through 0.45-/zm membrane and deaerate 9 H. A. Coller and B. S. Coller, Hybridoma 2, 91 (1983). to N. Hoogenraad, T. Helman, and J. Hoogenraad, J. Imrnunol. Methods 61, 317 (1983). I1 B. R. Brodeur, P. Tsang, and Y. Larose, J. Immunol. Methods 71, 265 (1984).
PLATELET RECEPTORS" ASSAYS AND PURIFICATION
Protein A-Sepharose Cl 4-B, 5-I0 ml (Pharmacia, Piscataway, N J), or 5-10 ml protein A-agarose (Boehringer-Mannheim, Indianapolis, IN) Glass column: 0.8 x 20 cm (Bio-Rad) Crystalline ammonium sulfate (Schwarz/Mann, Cambridge, MA)
Method Sample Preparation from Culture Supernatant. One or 2 liters of culture supernatant (containing - 5 - 2 0 mg of monoclonal antibody) is stirred on ice while ammonium sulfate is slowly sprinkled in until 50% saturation (29.1 g/100 ml solution) is achieved. Stirring is continued on ice for 1 hr and then the solution is centrifuged for 20 min at 3000 g at 4°. After decanting the supernatant, the pellet is resuspended in as small a volume as possible of the phosphate buffer and dialyzed against an excess of the same phosphate buffer. If desired, one can check for the completeness of dialysis by adding a drop of the dialysate to a solution of saturated BaC12 ; a white precipitate indicates the presence of residual ammonium sulfate. If the antibody solution contains precipitate after dialysis, the suspension should be clarified by centrifugation before applying to the protein A column. Sample Preparation from Ascites. Heat-defibrinated ascitic fluid is thawed at 37°, centrifuged at 1100 g for 10 min if any particulate matter remains, and diluted 1 : l with 0.1 M NaPO4-0.05% NaN s , pH 8.0 buffer. Chromatographic Purification. ~2Protein A-agarose or protein A-Sepharose C1 4B (5-10 ml) is washed in the phosphate buffer and the pH 3.0 citrate buffer before being poured into a 0.8 x 20 cm column. The column is eluted at 4 ° at rates up to 30-60 ml/hr depending on the manufacturer's recommendations. After the column is reequilibrated with the phosphate buffer, the sample is applied and the column eluted with the phosphate buffer until the optical density at 280 nm of the eluate returns to near baseline. The antibody, which is bound to the protein A, can then be eluted from the column by stepwise elutions with the citrate buffers at pH 6.0, 4.5, 3.5, and finally at pH 3.0. The IgG subclass will determine the pH at which the antibody elutes. Because IgG~ binds least well to protein A, it is important that the pH of the sample and the buffer in the column be 8.0 before applying the sample. Even then, we have had protein A columns that did not completely bind IgG~ antibodies, but did delay their elution. Thus, it is important to assay all of the column fractions for antibody until the behavior of the antibody on that particular column is determined. There may be a benefit in using a 1.5 M NaCl, 0.01 M NaPO4, pH 8.0 buffer in the binding step, but we have not made direct comparisons. In 12 p. L. Ey, S. J. Prowse, and C. R. Jenkin,
addition, a 3 M NaC1, 1.5 M glycine, pH 9.0 buffer is also said to improve the binding of IgG~ to protein A columns. A propriety buffer system is also available that is said to improve the yield of IgG~ antibodies (BioRad). Antibodies of IgG2a subclass usually do not elute until pH 4.5. Because we have observed that most of the IgG in fetal bovine serum elutes at pH 6, stepwise elutions with pH 6.0 and 4.5 buffers may yield a purer preparation than that obtained with just a single pH 4.5 elution. The column can be reequilibrated with the phosphate buffer and used again. The protein elution pattern is monitored by optical density at 280 nm and each of the citrate buffer fractions is adjusted to pH 7.0 -+ 0.2 with 1 M Tris-HCl, pH 8.5. The fractions with protein are pooled and dialyzed against Tris-NaCl buffer containing 0.05% NAN3, pH 7.4. Antibody concentration is estimated by absorption at 280 nm, assuming .Al~ , 1 2 8 0 n m = 14. The purified antibody can be assayed for functional activity in the vWF screening assay. To establish the titer, serial dilutions are tested until no activity is present. We store antibody at 4 ° and have found that it retains activity for months to years under these conditions. Iodination Procedure Materials
Sephadex G-25 (Pharmacia) and disposable glass column (737-2240); alternatively, 10-ml columns of BioGel P-6 can be obtained prepacked (Econo-Pac 100G; Bio-Rad) 125I (carrier-free NaI in 0.1 N NaOH; New England Nuclear, Boston, MA) Iodogen (Pierce, Rockford, IL) Glass scintillation vials with caps (2.5 x 5.5 cm) Tris buffer, 0.05% NaN 3, pH 7.4 Tris buffer, 0.05% NAN3, pH 7.4 with 0.2% (w/v) bovine serum albumin (BSA; Sigma, St. Louis, MO) PBS, pH 7.4 PBS, pH 7.4 with 1% BSA (Sigma) Saturated solution of sodium thiosulfate TCA solution, 100% (w/v) (trichloroacetic acid, A-322; Fisher Scientific) Method
A Sephadex G-25 or BioGel P-6 column with a bed volume of 9-12 ml is prerun with 50 ml of the Tris-NaC1-NaN 3 buffer containing 0.2% BSA at 22°. The column is equilibrated and eluted with the Tris-NaC1-NaN3 buffer without the albumin until the optical density nears baseline.
P L A T E L E T RECEPTORS: ASSAYS A N D P U R I F I C A T I O N
Iodogen is dissolved in dichloromethane at 0.5 mg/ml and then 50/zl is used to coat the bottom of an uncapped glass scintillation vial (2.5 × 5.5 cm). The Iodogen is very gently swirled to ensure even coating as the dichloromethane evaporates. After evaporation, a stream of nitrogen is passed over the bottom of the vial and the vial is capped and stored at - 2 0 °. Although some vials retain their activity for several years under these conditions, others do not, and so we currently make freshly coated vials for each iodination. One millicurie of ~25I(in 10/~1) is then added to I ml of antibody solution (usually 1 mg/ml in 0.15 M NaCI, 0.01 M Tris/HC1, 0.05% sodium azide, pH 7.4), and after brief mixing the mixture is added to the Iodogen-coated vial. The vial is then capped, swirled intermittently for - 10 min, and then the reaction is terminated by applying the contents of the reaction vial to the equilibrated Sephadex G-25 column. After the first peak of 125I-labeled protein elutes, the column is stopped to avoid eluting the free 1251 peak. The free 125I peak is then eluted into saturated sodium thiosulfate and saturated sodium thiosulfate is passed through the column to eliminate any volatile 1251. The individual fractions are characterized further by their optical density at 280 nm, TCA precipitability, and specific activities. In addition, each sample is tested by polyacrylamide gel electrophoresis to be certain that the radioactivity migrates with the antibody. Trichloroacetic acid precipitability is performed by adding 1/xl of 125Ilabeled protein to 0.9 ml P B S - 1% BSA. After mixing, 0.1 ml of 100% TCA is added, the solution is incubated on ice for 1 hr, and then centrifuged at 12,000 g at 22 ° for 3 min. Finally, 0.1 ml of the supernatant is removed and the radioactivity counted in a 3' counter. Binding Studies Materials Sucrose solution, 30% (w/v) (Fisher) Microcentrifuge tubes, 0.4 ml (223-9502; Bio-Rad) Microcentrifuge tubes, 1.5 ml (223-9501; Bio-Rad) Microcentrifuge (Beckman Instruments, Irvine, CA; or 5413, Eppendoff, Hamburg, Germany) ~25I-labeled antibody Platelet-rich plasma (PRP) Method Binding studies are performed at 22° using PRP prepared from whole blood collected into 1/100 vol of 40% sodium citrate. Platelet-rich plasma
(0.2 ml) is placed in a 1.5-ml microtube and J25I-labeled antibody or a labeled/unlabeled mixture is added. After incubating the samples long enough for the reaction to reach equilibrium, duplicate 0.1-ml aliquots of PRP are gently layered over 0.1-ml samples of 30% sucrose contained in 0.4-ml microtubes. Layering of PRP is facilitated by fitting the pipette tip (223-9000; Bio-Rad) with a - 3 - c m piece of microbore tubing (i.d. 0.050 in., 14-170-15E; Fisher Scientific). The duplicate microtubes are centrifuged at 22° for 3 min at 12,000 g, removed immediately from the centrifuge, and placed upright. The tip of each tube, containing the platelet pellet, is amputated with a dog nail cutter into a glass test tube (12 × 75 mm; Fisherbrand, Pittsburgh, PA) and the remaining portion of the microtube is rapidly placed into a second glass tube. Both tubes are counted in a y counter. The fraction of radioactivity bound is calculated by dividing the counts in the pellet by the sum of the counts in the pellet and the supernatant. Although the performance of binding studies is remarkably simple, their interpretation is not nearly as straightforward. In general, one wants to know the affinity of the antibody for the antigen and/or the total number of sites to which the antibody can bind. The mathematical rationales for using different equations and graphic formats to determine these parameters has been dealt with at great length by many authors and will not be reviewed here, but a few practical points will be emphasized. 1. As pointed out by Klotz, 13 if one chooses to use the method of Scatchard, it is imperative that data points be obtained at concentrations of the antibody high enough so that the free antibody concentration is considerably greater than the Ko. 2. Values for the number of antibody molecules bound at intermediate ratios of bound/free antibody concentrations are considerably more reliable than values obtained at either very high or very low bound/free ratios. Thus, preliminary experiments may be required to determine where this range is. 3. It should be fully appreciated that not all of the radioactivity added to a sample represents homogeneously labeled and active antibody molecules. Depending on the precise nature of the experiments, corrections may have to be made for (a) iodine that is not conjugated to antibody (or any other protein) as determined by the precipitability of the preparation with 10% TCA (usually