单克隆抗体制备 PREPARATION OF MONOCLONAL ANTIBODIES

SECTION II PREPARATION OF MONOCLONAL ANTIBODIESThe preparation of monoclonal antibodies should be undertaken carefully, since the production of monoclonal antibodies is expensive and time-consuming. Figure 11.4.1summarizes the experimental procedures that must be carried out to prepare monoclonal antibodies. The various procedures are presented in individual protocols and described in sufficient detail to allow an individual with no prior experience to carry out a cell fusion,to produce monoclonal antibodies in ascites fluid, and to purify antigen-specific mono-clonal antibodies.isolation of spleen cells preparation of myeloma cellsfeeder cellscell fusionELISA screeningof hybridomasupernatantsexpansion and selection of cultures to be clonedfeeder cells freeze cellscloning by limiting dilutionisolation and expansion of clonesfreeze cellsrecovery of frozen cellsproduction of ascites fluidspurification of monoclonal antibodiesELISAscreeningof seraimmunization of miceFigure 11.4.1 Flow chart for preparation of monoclonal antibodies.Supplement 18Contributed by Steven A. Fuller, Miyoko Takahashi, and John G.R. HurrellCurrent Protocols in Molecular Biology (1992) 11.4.1-11.4.6Copyright © 2000 by John Wiley & Sons, Inc.11.4.1ImmunologyUNIT 11.4Immunization of Mice Antigen is prepared for injection either by emulsifying an antigen solution with Freunds adjuvant or by homogenizing a polyacrylamide gel slice containing the protein antigen.Mice are immunized at 2- to 3-week intervals. Test bleeds are collected 7 days after each booster immunization to monitor serum antibody levels. Mice are chosen for hybridoma fusions when a sufficient antibody titer is reached.BASICPROTOCOLPRODUCTION OF IMMUNE SPLEEN CELLS: IMMUNIZATION WITH SOLUBLE ANTIGEN Materials Phosphate-buffered saline (PBS; APPENDIX 2)Antigen Complete Freunds adjuvant Any strain mice, 6 to 8 weeks old Incomplete Freunds adjuvant 22-G needles 3-ml syringes with locking hubs (Luer-Lok, Becton Dickinson)Double-ended locking hub connector (Luer-Lok, Becton Dickinson)Sterile sharp scissors Sterile razor blades or scalpel blades Wooden applicator sticks 200-µl pipettor Additional reagents and equipment for ELISA (UNITS 11.2 & 11.3) and western blotting (optional; UNIT 10.8)1. Prepare an emulsion (200 to 400 µl/mouse) of equal volumes PBS containing 25 to 100 µg antigen and complete Freunds adjuvant. Using a 22-G needle, inject mice intraperitoneally. For each antigen, 3 to 5 mice are plete Freunds adjuvant contains mycobacteria—incomplete Freunds does not.An emulsion is most readily prepared by linking two locking syringes, one loaded with antigen and the other loaded with adjuvant, using a double-ended locking connector (see Fig. 11.4.2). Press syringe barrels back and forth, transferring contents from one syringe to the other , for 5 to 10 min until a stable emulsion is produced. For best antibody production, inject antigen in as small an emulsion volume as practicable.A stable emulsion is an oil-in-water emulsion which will not disperse when dropped into water . This is a useful check for the emulsification endpoint. Further , at the endpoint the emulsion will thicken noticeably.Mice may be restrained for immunization in the following manner: Place mouse on grilled cage top. Lift mouse by the tail (generally, when mice are lifted by the tail they will grab the bars of the cage top with their front feet, thus stabilizing themselves for restraint).Immobilize the mouse’s head by pinching together the skin at the base of the skull between thumb and forefinger . Turn hand over so that mouse is lying with its back against the palm.Wrap fourth finger around tail and stretch mouse over arched palm for intraperitoneal injection.CAUTION: Handle Freunds adjuvant carefully, since self-injection can cause a positive TB test and lead to a granulomatous reaction.2. Boost mice 3 weeks later by intraperitoneally injecting an emulsion (200 to 400 µl)of equal volumes PBS containing 10 to 50 µg antigen and incomplete Freunds adjuvant. The emulsion is prepared and injected as in step 1.Supplement 18Current Protocols in Molecular Biology11.4.2Immunization of Mice3-ml glass syringesdouble-endedLuer-Lok connectoror 3-way stopcockantigen and adjuvantFigure 11.4.2 Double-syringe device for preparation of antigen-adjuvant emulsions.3. Bleed mice 7 days after second immunization by cutting off 0.5 cm of the tail withsterile sharp scissors or a razor blade. Collect 100 to 200 µl blood into a 1.5-mlmicrocentrifuge tube. After clot formation, rim the clot with a wooden applicatorstick to dislodge the clot from the surface of the tube, but do not break up the clot.After clot retraction, transfer the serum into another microcentrifuge tube with a200-µl pipettor. If test bleeds are collected more than three times, it will be necessaryto cut the tail vein to obtain further samples rather than cutting off additional lengthsof the tail itself. This is done by nicking one of the lateral tail veins with a razor blade.The collection of blood may be facilitated by using a heat lamp to warm the mouse for 30sec to 1 min prior to cutting of the tail. Additionally, if blood flow from the cut tail is slow,the tail may be “milked” from base to the cut tip with thumb and forefinger.4. Determine the antibody titer in the serum by ELISA (UNITS 11.2 & 11.3). If desired,further characterize the antibody specificity by western blotting (UNIT 10.8).Antibody titer is operationally defined as that dilution of serum that results in 0.2absorbance units above background in the ELISA procedure.5. If the antibody titer is considered too low (≥1⁄1000) for cell fusion, mice can be boostedevery 2 weeks until an adequate response is achieved. Bleed the mice and test theserum with an ELISA.6. When the antibody titer is sufficient (>1⁄1000), boost mice by injecting 10 to 50 µgantigen in PBS intraperitoneally (200 to 400 µl), or intravenously (50 to 100 µl) viathe tail veins, 3 days before fusion but >2 weeks after previous immunization.In general, the higher the serum antibody titer, the more antigen-specific antibody-produc-ing hybridomas are obtained per fusion.If an antibody against a nonimmunodominant epitope is desired, the cell fusion may bedone at an earlier or later time, since the percentage of antibody-producing cells in thespleen directed at these less immunogenic regions of the antigen may vary with time in anunpredictable fashion.7. Perform cell fusion (UNIT 11.7) 3 days after the immunization (step 6).Immunology11.4.3 Current Protocols in Molecular Biology Supplement 18ALTERNATEPROTOCOLIMMUNIZATION WITH COMPLEX ANTIGENS (MEMBRANES, WHOLE CELLS, AND MICROORGANISMS)1. Prime the mice and boost intraperitoneally with adjuvant (i.e., complete Freunds for priming and incomplete Freunds for booster immunizations) as described for soluble antigen (see basic protocol, steps 1 and 2) or suspend antigen in PBS and inject. Use 1 to 2 × 107 cells for mammalian species or 108 to 109 bacterial or yeast cells.2. Bleed the mice and determine the antibody titer of the serum as described for soluble antigen (see basic protocol, steps 3 to 6).3. Perform cell fusion (UNIT 11.7) 3 days after final immunization.ALTERNATEPROTOCOLIMMUNIZATION WITH ANTIGEN ISOLATED BY ELECTROPHORESIS In some instances the antigen under investigation can be purified most conveniently by gel electrophoresis (UNIT 10.2). Mice can be immunized with protein antigens still contained in a polyacrylamide gel slice, as described in this protocol.Additional Materials 0.1 M KCl, cold Tissue grinder Additional reagents and equipment for denaturing (SDS) discontinuous gel electrophoresis (UNIT 10.2)1. Apply a protein mixture containing 10 to 50 µg of the desired protein antigen to an appropriate denaturing (SDS) discontinuous gel electrophoresis system (e.g., the Laemmli gel system) and complete the electrophoresis as described in UNIT 10.2.2. Soak gel 5 to 15 min in cold 0.1 M KCl. Protein bands will appear as white precipitates against a clear gel background.3. Cut out the appropriate bands from the gel with a razor blade or scalpel blade.4. Prepare gel suspension by homogenizing the gel slice in a minimum volume of PBS using a tissue grinder. Minimum volume is defined by adding successive 100-µl volumes of PBS until the homogenized gel is liquid.Alternatively, the gel may be air dried for 1 to 2 hr , smashed with a glass rod, and suspended in a minimum volume of PBS.5. Immunize each mouse with 200 to 400 µl gel suspension containing 10 to 50 µg antigen via an intraperitoneal injection.Amount of antigen is estimated from prior observation of the proportion of desired protein antigen to other antigens in the sample as determined by the relative intensity of stained bands on the polyacrylamide gel (see UNIT 10.6 for staining procedures).6. Boost mice after 3 weeks with 200 to 400 µl gel suspension containing 10 to 25 µg antigen.7. Bleed the mice and determine the antibody titer of the serum as described for soluble antigen (see basic protocol).Mice immunized repeatedly with polyacrylamide tend to form adhesions that can make aseptic removal of the spleen difficult.8. Perform cell fusion (UNIT 11.7) 3 days after final immunization.Supplement 18Current Protocols in Molecular Biology11.4.4Immunization of MiceCOMMENTARYBackground InformationThe stimulation of an effective humoral im-mune response in mice is critical to the produc-tion of monoclonal antibodies directed at aparticular antigen. The variety and quality ofthe monoclonal antibodies prepared is gener-ally directly proportional to the serum antibodytiter in the particular mouse used for cell fusion.Any means of antigen preparation, antigen de-livery, or immunization schedule that increasesantibody titer in the serum of the immunizedmouse will potentiate the isolation of hybrido-mas secreting monoclonal antibodies of inter-est. We have described two methods of antigenpreparation: (1) antigen emulsified in Freundsadjuvants (probably the most common tech-nique used) and (2) antigen isolated in apolyacrylamide gel slice and homogenized.Other preparation methods (e.g., adsorption ofantigen to supports such as aluminum hydrox-ide or aluminum phosphate, polystyrene beads,or nitrocellulose paper, and alternate sites ofinjection such as footpads) are discussed in thekey references.Critical ParametersIt is desirable to use antigen of the highestavailable purity for immunizations, particu-larly for primary immunizations. Contami-nants may be more immunogenic than the an-tigen of interest and as such may result in a lowspecificity antibody. Mice given primary im-munizations of highly pure antigen may beboosted with less pure material (containing aslittle as one-third specific antigen in a complexprotein mixture).TroubleshootingPoor success in raising an adequate antibodytiter to an antigen of interest can be attributedto several factors. Improperly prepared emul-sion when using Freunds adjuvant (i.e., theaqueous and oil phases separate upon standing)is ineffective in stimulation of an immune re-sponse. Contaminants in an antigen preparationmay be more immunogenic, necessitating amore homogeneous preparation of the desiredantigen. Other parameters that can be varied inan effort to produce a higher antibody titer andincreased specificity include presentation ofantigen (Freunds adjuvant emulsion versuspolyacrylamide gel slice), site of immunization (intraperitoneal versus footpad or tail vein),antigen dose, and frequency of immunization.Alternate immunization protocols are pre-sented in the key references below.Anticipated Results Isolation of high-quality monoclonal anti-bodies correlates with high-serum antibody tit-ers. A serum ELISA titer of 1⁄1000 is the mini-mum level before attempting a cell fusion.Titers for most antigens (particularly from ani-mals injected with highly purified antigen) will range from 1⁄1000 to 1⁄100,000 after 3 to 4 immu-nizations. Occasional serum samples will titer at greater than 106. The proportion of mono-clonal antibodies of IgG class rather than IgM class generally increases proportionally to the duration of the immunization schedule, al-though this can vary dramatically among dif-ferent antigens. [In general, IgG class antibod-ies are more suitable for immunoassays, west-ern blotting (UNIT 10.8), immunoaffinity chromatography (UNIT 10.11), and immunopre-cipitation (UNIT 10.16)].Time Considerations A primary immunization followed by two booster immunizations and test bleeds will oc-cupy 6 weeks. For many antigens, however, an adequate antibody response in the mice is achieved only after several months and multiple immunizations.Key References Hurrell, J.G.R., ed. 1982. Monoclonal Hybridoma Antibodies: Techniques and Applications. CRC Press, Boca Raton, ngone, J.J. and V an Vunakis, H., eds. 1986. Im-munological techniques, Part I: Hybridoma tech-nology and monoclonal antibodies. Methods En-zymol. 121:1-947.Contributed by Steven A. Fuller and Miyoko Takahashi ADI Diagnostics Rexdale, Ontario John G.R. Hurrell Boehringer Mannheim Diagnostics Indianapolis, Indiana Current Protocols in Molecular Biology Supplement 1811.4.5ImmunologyUNIT 11.5Preparation of Myeloma Cells BASICPROTOCOLMyeloma cells are cultured with 8-azaguanine to ensure their sensitivity to the HA T selection medium (see UNIT 11.6) used after cell fusion (UNIT 11.7). One week prior to cell fusion, myeloma cells are grown in medium without 8-azaguanine. Cell culture conditions are adjusted such that the Sp2/0 cells are in the log phase of growth and exhibit high viability at the time of collection for fusion (UNIT 11.7).Materials Sp2/0 murine myeloma cell line (A TCC #CRL 1581)Complete culture medium 20 µg/ml 8-azaguanine Tissue culture flasks, 25 cm 2 or 75 cm 28% CO 2-in-air gas mixture Humidified 37°C, 8% CO 2 incubator Inverted microscope 1. Recover frozen cells from liquid N 2 storage, as described in UNIT 11.9.2. Grow Sp2/0 cells overnight in complete medium in tissue culture flasks at 37°C in a CO 2 incubator in 8% CO 2-in-air atmosphere with 98% relative humidity.3. Determine that the cells are growing by examining the cell cultures in the flasks with an inverted microscope and return culture flask to CO 2 incubator for continuation of cell growth.4. To ensure that the Sp2/0 cells remain aminopterin sensitive for the selection process following fusion, supplement the complete culture medium with 8-azaguanine at 20µg/ml during maintenance. One week prior to fusion, culture cells in medium without 8-azaguanine.A seeding cell density of 2.5 to 5 × 104 cells/ml works well with Sp2/0 cells.Sp2/0 cells will grow to a maximum density of 6 to 9 × 105 cells/ml, with a doubling time of 10 to 15 hr . When this density is reached, there is a rapid decline in cell viability. The Sp2/0 cultures are split every 2 to 3 days either by discarding an appropriate volume from the old flask and replacing with fresh medium or by transferring an appropriate volume of cells to a new flask and adding fresh medium. A 1-in-10 or 1-in-20 split is recommended.5. A total of 1 × 107 Sp2/0 cells (i.e., 1:10 ratio to immune spleen cells) is used for fusion. Cell viability at the time of collection should be greater than 95%. To ensure that cells are collected in log phase of growth, adjust the cell density to 2 × 105cells/ml the day before the fusion by adding fresh medium. Determine cell viability using the trypan blue exclusion method (see support protocol, below) on cells suspended in serum-free medium or PBS.SUPPORTPROTOCOLCELL VIABILITY TEST BY TRYPAN BLUE EXCLUSION This procedure is used to determine the number of viable cells present in the cell culture.A non-viable cell will have a blue cytoplasm; a viable cell will have a clear cytoplasm.Additional Materials Phosphate-buffered saline (PBS; APPENDIX 2) or serum-free complete culture medium 0.4% trypan blue solution Contributed by Steven A. Fuller, Miyoko Takahashi, and John G.R. Hurrell Current Protocols in Molecular Biology (1988) 11.5.1-11.5.3Copyright © 2000 by John Wiley & Sons, Inc.Supplement 1811.5.1Preparationof Myeloma CellsBinocular microscopeHemacytometer1. Centrifuge 1 ml cell suspension at 100 × g for 5 min.2. Resuspend the cell pellet in 1 ml PBS or serum-free complete culture medium.Serum proteins stain with trypan blue and can produce misleading results. Determinations must be made in serum-free solution.3. Mix 1 part of trypan blue solution and 1 part cell suspension (1⁄2 dilution).4. Using a binocular microscope, count the unstained (viable) and stained (dead) cells separately in a hemacytometer. Each of the four corner squares (composed them-selves of 16 smaller squares) have 1 mm sides and are 0.1 mm deep (0.1 mm 3). Count all cells within each of the four corner squares, including those that lie on the bottom and left-hand perimeters, but not those that lie on the top and right-hand perimeters.Count any clumps of cells as one cell. Calculate the mean number of cells per 0.1-mm 3volume. Multiply by 104 to obtain the number of cells/ml (i.e., cells/cm 3). Apply dilution factor for trypan blue (2×) to obtain the number of cells per milliliter of culture.5. Calculate the percentage of viable cells as follows:Viable cells (%) = Number of viable cellsTotal number of cells (dead and viable ) × 100REAGENTS AND SOLUTIONSComplete culture mediumDulbecco modified Eagle medium (DMEM), high-glucose formula (4.5 g glucose/liter; GIBCO/BRL #430-2100) supplemented to the indicated concentrations with the following additives:2.8 g/liter sodium bicarbonate (33.3 mM)4.8 g/liter HEPES (20 mM)10% fetal calf serum (v/v)10 ml/liter L -glutamine (2 mM)10 ml/liter sodium pyruvate (1 mM)10 ml/liter penicillin (50 IU/ml) and streptomycin (50 µg/ml)The last four additives are available as 100× solutions from GIBCO/BRL and other major suppliers of cell culture media. Penicillin and streptomycin are combined in one solution.Samples of fetal calf serum lots should be tested for ability to support efficient cell growth and cloning before a large purchase because there is much variability between lots of a given supplier. The fetal calf serum must be mycoplasma free. If low volume usage of fetal calf serum precludes testing of serum lots, purchase of mycoplasma-free, virus-free, low endotoxin sera from suppliers such as GIBCO/BRL, Flow Laboratories, or Sigma will generally provide satisfactory results. Horse or bovine serum is not an adequate substitute!Current Protocols in Molecular Biology Supplement 111.5.2ImmunologyCOMMENTARY Background Information The Sp2/0 cell line was chosen as the fusion partner for immune spleen cells because of its good rate of growth, the efficiency with which hybridomas are obtained after fusion, and, most importantly, because it does not synthesize or secrete any immunoglobulin heavy or light chains itself. The Sp2/0 myeloma cell line was developed by Schulman et al. (1978). Other commonly used cell lines are P3X63-Ag8.653(Kearney et al., 1979), which does not secrete immunoglobulins, and NS-1 (Kohler and Mil-stein, 1976), which produces only κ light chains.Critical Parameters Optimal growth of myeloma cells is density dependent. Cultures should be split at regular intervals to maintain >95% viability. Do not culture Sp2/0 cells longer than 1 month to avoid genetic drift and development of antibiotic-re-sistant contaminants. Maintain several aliquots of Sp2/0 cells in liquid nitrogen storage.Anticipated Results Proper care yields a healthy log phase myeloma cell culture able to sustain good pro-duction of hybridomas upon fusion.Time Considerations Depending on culture conditions, 105 cells can be expanded to the 107 cells required for fusion in 4 to 6 days.Literature Cited Kearney, J.F., Radbruch, A., Liesegang, B., and Ra-jewsky, K. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but per-mits the construction of antibody-secreting hy-brid cell lines. J. Immunol. 123:1548-1550.Kohler, G. and Milstein, C. 1976. Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur . J. Immunol. 6:511-519.Schulman, M., Wilde, C.D., and Kohler, G. 1978. A better cell line for making hybridomas secreting specific antibodies. Nature 276:269-270.Contributed by Steven A. Fuller, Miyoko Takahashi, and John G.R. Hurrell Allelix Inc.Mississauga, Ontario Supplement 1Current Protocols in Molecular Biology 11.5.3Preparationof Myeloma CellsUNIT 11.6Preparation of Mouse Feeder Cells for Fusion and CloningBASIC PROTOCOL Chilled sucrose solution is injected intraperitoneally into mice. When withdrawn, the solution contains feeder cells (macrophages and other cells) that are placed in the wells of microtiter plates 1 day prior to seeding of hybridomas from cell fusion (UNIT 11.7) or cloning (UNIT 11.8) procedures.Materials0.34 M sucrose solution, sterile and chilledMice (any strain)70% ethanolHA T medium, chilledSterile phosphate-buffered saline (PBS; APPENDIX 2)10-ml syringe, sterile18-G needle, sterile50-ml conical centrifuge tube, sterileDissecting boardForceps, sterileScissors, sterile96-well microtiter plates8% CO 2-in-air gas mixtureHumidified CO 2 incubatorAdditional reagents and equipment for estimating cell viability by trypan blue exclusion (support protocol, UNIT 11.5)1. Just prior to sacrificing a mouse, fill a 10-ml syringe with 8 ml chilled sucrose solution and attach 18-G needle.To avoid macrophages adhering to plastic surfaces, it is important to use chilled solutions to optimize cell harvest.2. Chill the 50-ml conical centrifuge tube in ice.3. Kill mouse by cervical dislocation.This is accomplished by firmly holding a thick pencil or similar rod-shaped object to the neck of the mouse just behind the skull and quickly and firmly pulling the tail.4. Immerse the mouse in a 100-ml beaker containing 70% ethanol.5. Lay out mouse on dissecting board.6. Snip skin at diaphragm level and pull skin back, exposing the lower part of the rib cage and abdomen.With forceps pull skin from underlying tissue at the diaphragm level and snip with a scissors. With forceps or sterile gloved hands, pull skin back at both sides of the incision to expose the lower part of the rib cage and abdomen.Care must be taken not to tear or cut the peritoneal membrane.7. Insert the needle into the peritoneal cavity at the base of the sternum and rest the tip of the needle over the liver. Inject sucrose solution. Gently squeeze the abdomen two or three times.8. Harvest the peritoneal feeder cells by withdrawing as much solution as possible into the syringe.Care must be taken not to puncture the digestive organs, which may lead to fecal contamination of the feeder cells.Supplement 1Contributed by Steven A. Fuller, Miyoko Takahashi, and John G.R. Hurrell Current Protocols in Molecular Biology (1988) 11.6.1-11.6.3Copyright © 2000 by John Wiley & Sons, Inc.11.6.1ImmunologyEnough peritoneal feeder cells can usually be isolated from one mouse to seed ∼100 to 300wells. However , some mice do not yield effective feeder cells. Depending on the total number of wells that must be seeded with mouse feeder cells, an appropriate number of mice must be killed. Peritoneal exudate feeder cells can be prepared up to 3 days prior to use.9. Transfer the feeder cell–containing sucrose solution into the 50-ml centrifuge tube.10. In a sterile fume hood, add 20 ml chilled HA T medium.11. Centrifuge at 100 × g for 5 min at room temperature.12. Resuspend the pellet in 1 ml chilled HA T medium and perform cell viability test by trypan blue exclusion as described in the support protocol, UNIT 11.5.13. Suspend the cell pellet in chilled HA T medium at 1 × 105 cells/ml.14. Add 100 µl cell suspension to each of the 60 inner wells of the 96-well plates. The peripheral 36 wells are filled with sterile PBS.Plates having 24 wells may be used. If this is the case, add 1 ml cell suspension/well.15. Incubate plates overnight at 37°C in a CO 2 incubator in 8% CO 2-in-air with 98%relative humidity.REAGENTS AND SOLUTIONS The following solutions are sterilized by filtration through a 0.22-µm membrane. A suitable sterilization system is a disposable filter unit (e.g., Nalgene #120-0020). Glass-distilled water should be used for all preparations.0.34 M sucrose solution 58.2 g sucrose H 2O to 500 ml Filter sterilize and store at 4°C in 100-ml aliquots HAT (hypoxanthine/aminopterin/thymidine) medium Complete culture medium (see reagents and solutions, UNIT 11.5) supplemented to the indicated concentrations with the following additives: 20% (v/v) fetal calf serum 0.1 mM nonessential amino acids 100 µM hypoxanthine 0.4 µM aminopterin 16 µM thymidine These additives may be purchased in concentrated and sterile solutions from the major suppliers of cell culture media and reagents. Concentrated solutions of hypoxanthine and thymidine (HT) and aminopterin may also be prepared in the laboratory (see following recipes).100× HT solution Weigh 340.3 mg hypoxanthine and 96.9 mg thymidine; add water to 250 ml. Heat to 70°C to dissolve. Filter sterilize and store in 20-ml aliquots at −20°C. Thaw at 70°C for 10 to 15 min.1000× aminopterin solution Weigh 17.6 mg aminopterin. Add 60 ml water and dissolve by adding 0.1 M NaOH dropwise. Titrate with HCl to pH ∼8.5. Adjust volume to 100 ml and filter sterilize.Make 100× working solution by diluting stock in complete culture medium. Store in 5-ml aliquots at −20°C.Aminopterin precipitates at low pH and is light sensitive.Supplement 1Current Protocols in Molecular Biology 11.6.2Preparation of Mouse Feeder Cells for Fusion and CloningCOMMENTARYBackground InformationTo maximize the yield of hybrids from thefusion and cloning procedures, feeder cells arerequired to be cocultured with the hybrids,while hybrid cell density is low. Mouse perito-neal cells, most of which are macrophages,have been found to be convenient and effectivefeeder cells which are a source of solublegrowth factors for hybridoma cells.Critical ParametersFeeder cells such as peritoneal cells providebest support of hybridoma growth when used1 to 3 days after harvest. Use of chilledsolutions is necessary for optimum cell harvest,to prevent macrophages adhering to plastic sur-faces.Anticipated Results From 1 to 3 × 106 peritoneal feeder cells are harvested from one mouse. The number of feeder cells will be enough to seed 100 to 300wells.Time Considerations Peritoneal feeder cells from one mouse can be processed in 1 hr or less.Contributed by Steven A. Fuller, Miyoko Takahashi, and John G.R. Hurrell Allelix Inc.Mississauga, Ontario Current Protocols in Molecular Biology Supplement 111.6.3ImmunologyUNIT 11.7Fusion of Myeloma Cells with Immune Spleen Cells BASICPROTOCOLFreshly harvested spleen cells and myeloma cells are copelleted by centrifugation and fused by addition of polyethylene glycol solution to the pellet. Cells are centrifuged again and the PEG solution diluted by slow addition of medium. Fused cells are centrifuged,resuspended in selection medium, and aliquoted into 96-well microtiter plates. Hybrido-mas are grown to 10 to 50% confluence and then assayed for production of antigen-spe-cific antibody.Materials Any strain immunized mouse (UNIT 11.3)Sp2/0 murine myeloma cells in active log phase (Am. Type Culture Collection #CRL 1581; UNIT 11.5)Diethyl ether 70% ethanol Dulbecco modified Eagle medium (DMEM) with supplements Sterile polyethylene glycol (PEG) solution HA T medium (UNIT 11.6)HT medium Phosphate-buffered saline (PBS; APPENDIX 2)15- and 50-ml centrifuge tubes Glass desiccator or metal can with lid Dissecting board 10.5-cm scissors (Irex #IR-105), sterile 10.5-cm forceps (Irex #IR-1393), sterile 60- and 100-mm petri dishes Stainless-steel strainer (Cellector; GIBCO #1985-8500), sterile 3-cc glass syringes with 26-G needle 5-ml serological pipets 37° C water bath Stopwatch 8% CO 2-in-air gas mixture Humidified CO 2 incubator Polyvinyl or polystyrene 96-well microtiter plates Inverted microscope Additional reagents and equipment for estimating cell viability by trypan blue exclusion (UNIT 11.5) and for detection of antibodies (UNIT 11.4)Preparation of myeloma and spleen cells 1. Just prior to sacrificing the mouse, transfer 1 × 107 Sp2/0 murine myeloma cells (prepared as described in UNIT 11.5) to a 50-ml centrifuge tube. Check the percentage of viable cells using the trypan blue exclusion method (support protocol, UNIT 11.5).2. Sacrifice the mouse by anesthetizing with diethyl ether in a closed container (e.g., a glass desiccator or a metal can with a lid).At this point, a blood sample may be collected from the mouse by severing the blood vessels of one forelimb. Collect the blood with a Pasteur pipet and place the blood in a microcen-trifuge tube.3. Immerse the mouse in a beaker containing 70% ethanol and lay out on a dissecting board.Contributed by Steven A. Fuller, Miyoko Takahashi, and John G.R. Hurrell Current Protocols in Molecular Biology (1988) 11.7.1-11.7.4Copyright © 2000 by John Wiley & Sons, Inc.Supplement 111.7.1Fusion of Myeloma Cells with Immune Spleen Cells。