production_of_monoclonal_antibodies

UNIT2.5 Production of Monoclonal AntibodiesHighly specific antibodies can be obtained by fusing immune B cells from the spleen withtumor cells to produce hybridomas,each of which will then secrete a single antibody.The desired antibody-producing hybridoma can be identified by a screening process.If this hybridoma is subjected to a cloning process in which clones are selected,suchthat all progeny are derived from a single cloned parental cell,a monoclonal antibodyis obtained.Monoclonal antibodies have high specificity and can be produced in largequantities.Thus,these biological reagents have been used extensively as probes in awide range of systems including the characterization of novel cell-surface and solubleproteins and carbohydrates,as enzyme catalysts,and for targeting in immunotherapy(see Commentary).This unit describes the production of monoclonal antibodies beginning with protocolsfor immunization(Basic Protocol1)and cell fusion and selection(Basic Protocol2).AnAlternate Protocol describes cell fusion and one-step selection and cloning of hybridomasutilizing a semisolid methylcellulose-based medium(ClonaCell-HY,a registered trade-mark of StemCell Technologies,Inc.;).Methods are providedfor screening primary hybridoma supernatants for antibodies of desired specificity(Sup-port Protocol1),establishment of stable hybridoma lines(Support Protocol2),cloningof these B cell lines by limiting dilution to obtain monoclonal lines(Support Protocol3),recloning of hybridoma cells in semisolid medium(Support Protocol4),and preparationof cloning/expansion medium(thymocyte-conditioned medium;Support Protocol5).Figure2.5.1summarizes these stages and notes the protocols in this and subsequent unitsin which they are detailed.Selection and cloning of hybridomas using the techniquesdescribed in Basic Protocol2require a major commitment of time and labor.However,the Alternate Protocol reduces the overall time necessary to produce monoclonal anti-bodies by18to20days.When successful,the monoclonal antibody may be an extremelyvaluable reagent that will be available in large quantities.Submission of monoclonal antibodies to the American Type Culture Collection(ATCC)for distribution to the scientific community is encouraged.Moreover,the ATCC servesas a repository for cell lines should the line be lost in the investigator’s laboratory due tounforeseen circumstances.NOTE:All protocols using live animals mustfirst be reviewed and approved by an Insti-tutional Animal Care and Use Committee(IACUC)and must conform to governmentalregulations for the care and use of laboratory animals.NOTE:All solutions and equipment coming into contact with living cells must be sterile,and proper aseptic technique should be used accordingly.NOTE:All culture incubations are performed in a humidified37◦C,5%CO2incubatorunless otherwise specified.Some media(e.g.,DMEM)may require altered levels of CO2to maintain pH7.4.Contributed by Wayne M.Yokoyama,Michelle Christensen,Gary Dos Santos,and Diane Miller Current Protocols in Immunology(2006)2.5.1-2.5.25Copyright C 2006by John Wiley&Sons,Inc.Induction of Immune Response2.5.1 Supplement74Production of Monoclonal Antibodies2.5.2Supplement 74Current Protocols in ImmunologyFigure 2.5.1Stages of monoclonal antibody production,with references to the Basic,Alternate,and Support Protocols in this unit (as well as subsequent units)that describe the steps.Induction of Immune Response 2.5.3Current Protocols in Immunology Supplement 74BASIC PROTOCOL 1IMMUNIZATION TO PRODUCE MONOCLONAL ANTIBODIESA wide variety of antigen preparations have been used successfully to produce mon-oclonal antibodies (see Critical Parameters for discussion of antigen preparation).The following protocol provides an immunization schedule for the production of most an-tibodies,although several different schedules can be used.In this protocol,emulsified antigen is injected intraperitoneally into the species of choice.A booster injection is administered 10to 14days after the primary immunization.Three days after the booster injection,the animals’spleens are ready for cell fusion (Basic Protocol 2).MaterialsAntigenComplete Freunds adjuvant (CFA;Sigma)Animal:pathogen-free mouse,hamster,or rat (Armenian hamsters from Cytogen Research are recommended;see Critical Parameters for discussion of animal choice and UNIT 1.1)Incomplete Freunds adjuvant (IFA;Sigma),optional1-to 2-ml glass syringes with Luer-Lok tips,sterile3-way stopcock20-and 22-G needles,sterileAdditional reagents and equipment for handling and restraint of animals (UNIT 1.3)and intraperitoneal injection (UNIT 1.6)CAUTION:CFA is an extremely potent inflammatory agent,particularly if introduced intradermally or into the eyes.Profound sloughing of skin or loss of sight may occur.Self-injection can cause a positive TB skin test and lead to a granulomatous e gloves and protective eyewear when handling CFA.1.Prepare antigen using 2×106to 5×107cells or 1to 50µg protein or peptide per animal to be immunized in normal saline.The antigen may be in several different forms depending on the desired property of the MAb and the method of screening (see Critical Parameters for discussion of antigen preparation and screening assays).If cells are the immunogen,wash three times in serum-free medium before immunization.Plan the immunization of several animals (enough for several fusions)so that primed and boosted animals will be ready 3days before fusion (see Basic Protocol 2).To minimize the risk of introducing a pathogen into the rodent colony,screen cells for pathogens by antibody-production assay (UNIT 1.1).2.Draw up antigen into a sterile 1-to 2-ml glass syringe with a Luer-Lok tip.Connect syringe to a 3-way stopcock.pletely resuspend CFA to disperse the Mycobacterium tuberculosis bacilli,which settle to the bottom of the container with time.Draw up a volume of CFA equal to the antigen volume in a syringe and connect to the antigen-containing syringe.4.Emulsify antigen and CFA by discharging antigen into CFA,then discharging back and forth until a thickened mixture results.Test whether the emulsion is stable—a stable emulsion will not disperse when a drop of it is placed in water.See UNIT 2.4for further discussion of immunization.Figure 2.4.1illustrates the double-syringe device.5.Transfer all of the CFA/antigen emulsion to one syringe and remove the other syringe and stopcock.Attach a sterile 20-G needle to the syringe containing the emulsion.Production ofMonoclonal Antibodies2.5.4Supplement 74Current Protocols in Immunology6.Inject emulsion intraperitoneally into the animal using <0.2ml/mouse,0.5to 1ml/rat,or 0.2to 0.4ml/hamster.Be careful not to force the syringe plunger,since excessive pressure may dislodge the needle and spray the emulsion.Introduce the needle through the skin and tunnel the needle between the skin and peritoneal wall before entering the peritoneal cavity at a site distant from the dermal puncture site.Twirl needle before withdrawal to minimize leakage.Rats are generally anesthetized (UNIT 1.4)whereas mice and hamsters can be manipulated with one hand and do not require anesthetic.7.Boost animal after 10to 14days with approximately the same dose of antigen as in step 5.If cell fusion is planned for 3days after boosting,immunize with antigen alone in aqueous solution,or intact cells in suspension.If a fusion is not immediately planned,boost the animal with antigen emulsified in IFA (which does not contain Mycobacterium tuberculosis bacilli).Do not use CFA for the booster immunizations as this will cause intense inflammation and increased anti-TB antibody response.Blood can be collected from a tail bleed (UNIT 1.7)after the first intraperitoneal boost to obtain antibodies from the blood as a positive control in subsequent detection assays.If desired,antibody titers can be assayed by ELISA (UNIT 2.1)or immunoprecipitation (UNIT8.3),7to 10days after the primary and booster immunizations.BASICPROTOCOL 2CELL FUSION AND SELECTION OF HYBRIDOMAS While animals should be immunized as soon as the decision has been made to produce a monoclonal antibody and the antigen prepared,do not perform cell fusion until the screening assay (Support Protocol 1)has been perfected.Artifactual results that may arise from conditioned media must be identified before cell fusion,because after a fusion there is only a finite amount of time available to assay for the desired monoclonal antibody.Prior to cell fusion,the partner (myeloma)cell line is expanded and a booster injection of antigen is administered to the primed animals.On the day of fusion,the spleens are harvested.Spleen cells and partner cells are washed,harvested,and mixed.Cell fusion is performed at 37◦C in the presence of polyethylene glycol (PEG).The resulting pellet is harvested and plated into tissue culture plates.After incubation with hypoxanthine,aminopterin,and thymidine (HAT)medium and feeding over ∼2weeks,the hybridomas are ready for screening (Support Protocol 1).Materials SP2/0-Ag14myeloma cell line (drug-marked,nonsecretory;ATCC #CRL 1581)Complete DMEM-10and -20media (APPENDIX 2A )with 10mM HEPES and 1mM sodium pyruvate Primed animal;mouse,hamster,or rat (10to 14days after primary immunization;(see Basic Protocol 1)Complete DMEM medium (APPENDIX 2A ),serum-free 50%polyethylene glycol (PEG),sterile Ammonium chloride solution (see recipe)Complete DMEM-20/HEPES/pyruvate/HAT (or HT)medium (see recipe)175-cm 2flasks Fine-mesh metal screen 50-ml conical polypropylene centrifuge tubes Beckman TH-4rotor or equivalent 96-well flat-bottom microtiter platesInduction of Immune Response 2.5.5Current Protocols in Immunology Supplement 74Additional reagents and equipment for animal euthanasia (UNIT 1.8),spleen removal (UNIT 1.10),and counting cells and assessing cell viability by trypan blueexclusion (APPENDIX 3B )Prepare myeloma cells (1week before fusion)1.One week before fusion,begin expansion of SP2/0-Ag14myeloma cell line (the fusion partner cell line)in complete MEM-10/HEPES/pyruvate (see Critical Pa-rameters).By the day cell fusion is to be performed,the following total number of myeloma cells must be available (in multiple 175-cm 2flasks containing 100ml each),depending upon the source of the primed animal:mouse spleen,1×108cells in two or three flasks;hamster spleen,2×108cells in three or four flasks;and rat spleen,5-10×108cells in ten flasks.Two mouse or hamster spleens,or one rat spleen,will provide enough cells for the fusion (see step 7).Boost primed animal (3days before fusion)2.Three days before fusion,boost primed animal(s)according to step 7of Basic Protocol 1.Prepare reagents and split myeloma cells (1day before fusion)3.One day before fusion,prepare all reagents and media,particularly 50%PEG.4.One day before fusion,split SP2/0-Ag14myeloma cells (from step 1)into fresh complete DMEM-10/HEPES/pyruvate medium.Vigorous growth of the SP2/0-Ag14cells is generally required for good fusion.Check myeloma cells and prewarm reagents (day of fusion)e an inverted microscope to check the SP2/0-Ag14myeloma cells to make sure they are growing vigorously (refractile and not pyknotic),they are not contaminated (no obvious bacteria or fungi),and there are enough cells for the fusion.It is better to postpone the fusion than to perform an ill-advised fusion,since the entire selection and screening effort will take ∼3weeks.6.Prewarm the following in a 37◦C water bath:Three 400-and three 600-ml beakers,each containing ∼100ml H 2O20ml sterile complete serum-free DMEM5ml sterile 50%PEG solution.Harvest spleen and prepare cells7.Sacrifice boosted animal(s)(UNIT 1.8)and aseptically harvest spleen(s)(UNIT 1.10).Do not use anesthetics for sacrifice.Instead,use cervical dislocation for mouse,or CO 2asphyxiation for mouse,hamster,or rat to avoid introducing an anesthetic into the bloodstream and therefore into the cultures.8.Transfer spleen to a sterile 100-mm-diameter petri dish filled with 10ml sterile complete serum-free DMEM.Perform all subsequent steps in a laminar flow hood.9.Tease spleen into a single-cell suspension by squeezing with angled forceps or by chopping with fine-tipped dissecting scissors.Remove debris and disperse cells further by passage through a fine-mesh metal screen.10.Transfer spleen cell suspension to a sterile 50-ml conical centrifuge tube and fillwith sterile complete serum-free DMEM.Do not use protein-or HEPES-containing medium because the PEG will precipitate proteins and HEPES can be toxic to cells during fusion.Production ofMonoclonal Antibodies2.5.6Supplement 74Current Protocols in Immunology11.Centrifuge 5min in TH-4rotor at 1500rpm (500×g ),room temperature,and discard supernatant.12.Lyse red blood cells (RBC)by resuspending pellet in 5ml ammonium chloride solution.Let stand 5min at room temperature.13.Add 45ml sterile complete serum-free DMEM,and centrifuge as in step 11.14.Resuspend pellet in 50ml sterile complete serum-free DMEM.Centrifuge as in step 11.Repeat DMEM addition and centrifuging once (each repeat is a wash).15.While spleen cells are being washed,separately harvest the SP2/0-Ag14myeloma cells (from step 5)by transferring the cells to 50-ml conical centrifuge tubes.Centrifuge as in step 11.Resuspend myeloma cells in DMEM and pool all cells into one 50-ml conical centrifuge tube.Wash myeloma cells three times as in step 14.16.Separately resuspend the spleen and myeloma cells in 10ml complete serum-free DMEM.Count cells and assess viability in each cell suspension using a hemacytometer and trypan blue exclusion (APPENDIX 3B );there should be nearly 100%viability of both suspensions.17.On basis of cell counts (from step 16),calculate the amount of complete DMEM-20/HEPES/pyruvate needed to plate cells at ∼2.5×106total cells/ml.Prewarm this amount of complete DMEM-20/HEPES/pyruvate in 37◦C water bath.Prepare 96-well flat-bottom plates by labeling them sequentially:one plate is required for each 10ml of final cell suspension.Perform cell fusion 18.Mix SP2/0-Ag14myeloma and spleen cells at a 1:1ratio in a 50-ml conical cen-trifuge tube.Fill the tube with complete serum-free DMEM.Other cell ratios work.Successful fusions have been performed with a ratio of myeloma/spleen cells as low as 1:20.19.Centrifuge cell mixture 5min at 500×g ,room temperature.20.While cells are in the centrifuge,prepare three 37◦C double-beaker water baths in the laminar flow hood by placing a 400-ml beaker (from step 6)containing 100ml of 37◦C water into 600-ml beaker containing 75to 100ml of 37◦C water.Place the tubes of prewarmed 50%PEG solution and complete serum-free DMEM (from step 6)into two of the 37◦C water baths in the hood.21.Aspirate and discard supernatant from the mixed-cell pellet (from step 19).22.Perform the cell fusion at 37◦C by placing the tube containing the mixed-cell pellet in one of the double-beaker water baths in the laminar flow ing a 1-ml pipet,add 1ml prewarmed 50%PEG to the mixed-cell pellet drop-by-drop over 1min,stirring the cells with the pipet tip after each drop.Stir for an additional ing a clean pipet,add 1ml prewarmed complete serum-free DMEM to the cell mixture drop-by-drop over 1min,stirring after each drop.Repeat once with an additional 1ml of prewarmed complete serum-free DMEM.25.With a 10-ml pipet,add 7ml prewarmed complete serum-free DMEM drop-by-drop over 2to 3min.Macroscopic clumps of cells should be obvious at this point.26.Centrifuge 5min at 500×g ,room temperature.Induction of Immune Response 2.5.7Current Protocols in Immunology Supplement 7427.While the cells are in the centrifuge,rewarm the beaker water baths to 37◦C andplace in the hood.Place prewarmed complete DMEM-20/HEPES/pyruvate (from step 17)in the beaker water bath.28.Discard the supernatant (from step 26).Place tube in the beaker water bath.29.With a clean 10-ml pipet,forcefully discharge 10ml prewarmed complete DMEM-20/HEPES/pyruvate to the cell pellet.30.Repeat step 29until the total volume of prewarmed complete DMEM-20/HEPES(calculated in step 17)is added.If necessary,allow clumps to settle and disrupt with the pipet tip.Further warming of cell suspension is no longer required.If the total volume exceeds 50ml,gently aspirate and transfer to another sterile container such as a tissue culture flask.31.Gently aspirate 10ml of cell suspension with a 10-ml pipet.Add 2drops (100to 125µl)of suspension to each well of a 96-well flat-bottom plate (continue until entire suspension is plated).Incubate overnight in a humidified 37◦C,5%CO 2incubator.Vigorous pipetting of the cell suspension should be avoided at this point,as the newly formed hybrids are unstable.Moreover,the vigorous addition of cells to the wells with repeating micropipettor is not e a pipet aid and hold the 10-ml pipet at a 45◦angle with the tip 1to 2cm above the well,bracing the pipet with a finger from the opposite hand.To avoid introducing contaminants,do not hold hands above the plate.A steady,even flow of drops from the pipet will allow the most efficient delivery of cell sus-pension or medium to the e a fresh pipet to withdraw additional cell suspension.As an optional step to minimize fibroblast overgrowth,permit the fibroblasts in bulk-fused cell suspension to adhere overnight to tissue culture flasks before seeding the 96-well plates.Many investigators select their hybridomas under bulk conditions—i.e.,they incubate large numbers of cells per well in larger plates or flasks.This makes feeding easier,but allows fast-growing hybridomas to overgrow the others.Since nonproducing hybridomas tend to grow faster,especially in the hamster-mouse fusions,hybridomas are isolated initially in multiple small wells in this protocol.The primary hybridomas tend to be monoclonal.This is especially important when screening procedures are used that require differential reactivities,e.g.,to different cell lines by flow cytometry analysis or to different antigen preparations.In those cases,multiple hybridomas per well will obscure the reactivity of the MAb of interest.Monitor and feed cells32.After one day of incubation,check wells under an inverted microscope.If seededwith the appropriate number of cells,there should be a nearly confluent monolayer of highly viable cells on the bottom and obvious clumps of cells.33.Add 2drops complete DMEM-20/HEPES/pyruvate/HAT to each well with a 10-ml pipet (see step 31).Place in humidified 37◦C,5%CO 2incubator.Use a separate pipet for each microtiter plate and keep the same covers with each plate to ensure that each plate remains a separate unit and to avoid spreading contamination.It cannot be overemphasized that it takes practice and meticulous attention to possible sources of contaminants to keep these plates sterile during the subsequent 2-to 3-week feeding and monitoring schedule.If plates become contaminated,discarding them is advised.Alternatively,contamination in one or two wells may be treated by aspirating the contents of the contaminated well with a sterile Pasteur pipet attached to a vacuum flask,rinsing the well with 70%ethanol,and wiping with a sterile cotton swab.Wash twice with ethanol.Finally,blot the well dry with the sterile cotton swab and blot the appropriate area of the cover with a sterile cotton swab soaked in 70%ethanol.Do not open contaminated plates while other plates are in the hood.Production ofMonoclonal Antibodies2.5.8Supplement 74Current Protocols in Immunology34.On days 2,3,4,5,7,9,and 11,aspirate half the volume of each well using a sterile,short Pasteur pipet attached to a vacuum flask,holding pipet at a 45◦angle and touching tip to surface of supernatant at the point where the liquid meets the opposite wall of the well.Feed the cells by adding 2drops complete DMEM-20/HEPES/pyruvate/HAT from a 10-ml pipet (see steps 31and 33)to each well,and return to humidified 37◦C,5%CO e a separate Pasteur pipet for each plate to minimize spreading contamination.Since the frequency of successful viable hybridoma formation is ≤10−5,when HAT is added,profound cell death should be apparent at days 2and 3and the remaining viable cells should not be readily apparent until they have expanded.By day 7to 9for mouse-mouse fusions,day 11for rat-mouse fusions,and day 14for hamster-mouse fusions,clusters of hybridoma cells should become visible under the inverted microscope.If profound cell death is not apparent on days 2and 3,check the medium containing HAT and the parental cell line by incubating an aliquot of the parental myeloma line with the medium containing HAT.The feeding schedule is not rigid except for the first 4days,when it is necessary to remove the toxic products of cell death.Thereafter,feedings will depend on the actual number of cells deposited in the wells,efficiency of fusion,and appearance and growth of hybridomas.Do not allow wells to become yellow (acidic)for more than a day.Examine plates daily,even if cells are not scheduled to be fed,and feed plates if acidic wells are noted.35.On day 14,repeat feeding as outlined in step 34except use complete DMEM-20/HEPES/pyruvate/HT to feed cells.Return to 37◦C,5%CO 2incubator.Cells do not require more than one change of complete DMEM-20/HEPES/pyruvate/HT.After this change,the aminopterin (from prior addition of HAT medium)is apparently diluted out enough so that the cells can survive without additional HT.36.On day 15and subsequently,feed wells as noted using complete DMEM-20/HEPES/pyruvate without HAT or HT.The hybridomas are ready for screening when most of the wells containing growing cells demonstrate 10%to 25%con-fluence and when those with denser populations turn yellow within 2days after feeding (see Support Protocol 1).If the screening assay requires a [3H]thymidine incorporation assay (APPENDIX 3D ),be aware that the large amount of thymidine in complete DMEM-20/HEPES/pyruvate with HAT and HT will serve as a cold-label inhibitor of [3H]thymidine incorporation.At least 3to 4changes of complete DMEM-20/HEPES/pyruvate without HT are required to dilute out excess thymidine.ALTERNATEPROTOCOLCELL FUSION,SELECTION,AND CLONING OF HYBRIDOMAS USING A SEMISOLID MEDIUM (CLONACELL-HY)Traditionally,monoclonal antibody development has involved selecting hybridomas in suspension cultures,identifying cultures that produce antibodies specific for the targeted antigen,and cloning the specific antibody-producing hybridoma(s)by at least one round of culture under limiting-dilution conditions (as described in Basic Protocol 2and Support Protocols 1to 3).This approach is laborious and time consuming and may result in the selection of identical,duplicate clones.This section describes protocols for the use of a methylcellulose-based medium system,ClonaCell-HY ,for cloning and selection of mouse hybridomas.Performing hybridoma selection and cloning simultaneously in ClonaCell-HY reduces the time and reagents necessary to obtain a monoclonal hybridoma producing antibody against the antigen of interest.Cultures do not need feeding or maintenance during the selection process.This approach also allows all daughter cells to remain together during the selection process,decreasing the number of clones that need to be tested for antibody production.Selection of duplicate hybridomas,a common occurrence with hybridoma cloning in suspension cultures,is avoided.An additionalInduction of Immune Response 2.5.9Current Protocols in Immunology Supplement 74advantage is that smaller,slow-growing clones,which can easily be lost due to overgrowth by larger,faster-growing hybridomas in traditional liquid suspension culture,remain physically separated in semisolid medium from the larger,faster-growing clones,and can thus be isolated and screened separately.NOTE:All solutions and media should be prewarmed to 37◦C unless otherwise indicated.MaterialsMyeloma cell line (e.g.,SP2/0,X63Ag8.653;available from ATCC)ClonaCell-HY Monoclonal Antibody Production Kit (StemCell Technologies,Inc.)containing:Medium A—ClonaCell-HY Pre-Fusion Medium and Hybridoma Expansion Medium,500mlMedium B—ClonaCell-HY Fusion Medium,500mlMedium C—ClonaCell-HY Hybridoma Recovery Medium,100mlMedium D—ClonaCell-HY Hybridoma Selection Medium containing HAT,90ml Medium E—ClonaCell-HY Hybridoma Growth Medium containing HT,500ml Polyethylene glycol—ClonaCell-HYPEGSolution,pretestedforcellfusion,1.5ml Immunized mouse,1to 4days after final antigen boost (Basic Protocol 1)3%(v/v)acetic acidLiquid nitrogen (optional)Fetal bovine serum (FBS)containing 20%(v/v)DMSO15-and 50-ml conical polypropylene centrifuge tubes100-mm petri dishesFine-mesh metal screenLow-speed tabletop centrifuge3-ml and 12-ml syringes25-and 75-cm 2tissue culture flasks16-G blunt-ended hypodermic needles96-and 24-well tissue culture platesCryotubes (e.g.,Nunc)Liquid nitrogen freezer (Dewar flask and canes to accommodate cryotubes;optional)Additional reagents and equipment for determining cell viability by trypan blue exclusion (APPENDIX 3B ),animal euthanasia (UNIT 1.8),spleen removal (UNIT 1.10),preparing a single-cell suspension of splenocytes (UNIT 3.1),counting cells using a hemacytometer (APPENDIX 3),assaying for antigen production from hybridoma clones by ELISA (UNIT 2.1),flow cytometry (Chapter 5),or immunoblotting (UNIT 8.10),and cryopreservation of cells (APPENDIX 3G )Prepare myeloma cells1.Culture the parental myeloma cells in 25-cm 2tissue culture flasks with Medium A (Pre-Fusion Medium from ClonaCell-HY kit)for at least 1week prior to fusion to ensure they are well adapted to this medium.Seed cells at a density of ∼5×104cells/ml and passage every 2days.Suggested maximum cell density is 4×105cells/ml.The parental myeloma cells must not secrete any of their own immunoglobulin chains.They should be mycoplasma-free and efficiently fuse to form stable hybridomas that continuously secrete specific monoclonal antibodies.Parental myeloma cells that meet these criteria (such as SP2/0and X63Ag8.653)are widely available.2.Calculate the cell growth rate at every passage (APPENDIX 3A ).The day before the fusion,count the viable cells and split cells so that there will be at least 2×107parental myeloma cells available the next day.The recommended cell density for fusion is 2×105cells/ml.Only 100ml of these cells will be needed,but 200ml should be cultured to ensure an adequate supply.Production of Monoclonal Antibodies2.5.10Supplement 74Current Protocols in Immunology 3.Harvest the required number of parental myeloma cells in a 50-ml conical polypropylene centrifuge tube.Centrifuge 10min at 300×g ,room temperature or 37◦C,and remove the supernatant.Wash three times,each time by adding 30ml of Medium B (Fusion Medium),centrifuging again as before,and removing the supernatant.Resuspend final pellet in 25ml Medium B.This step may be performed simultaneously with or subsequent to spleen cell preparation (steps 6to 10)to ensure that the myeloma cells do not sit for an extended period of time.It is important to remove all the serum adhering to the cells by washing with serum-free Medium B.If the serum is not removed,the PEG will not fuse the cell membranes and the fusion frequency will drop drastically.4.Count live cells using a viability stain (APPENDIX 3B ).Viability of parental myeloma cells should be >95%.5.Calculate the volume of cell suspension that contains 2×107cells,to be used in step 11.Place cells at room temperature or 37◦C.Harvest spleen and prepare spleen cells 6.Sacrifice immunized animal(s)(UNIT 1.8)and aseptically remove spleen(s)(UNITS 1.8&1.10).Place spleen in a sterile 100-mm petri dish containing 5ml Medium A (Pre-Fusion Medium).IMPORTANT NOTE:The ClonaCell-HY kit has been optimized for use with mouse hybridomas.Hybridomas from other species have not been tested.Do not use anesthetics for sacrifice.Instead,use cervical dislocation or CO 2asphyxia-tion to avoid introducing anesthetic into the bloodstream and therefore into the cultures.It is important to collect blood from the animal to obtain serum as a source of antibodies for a positive control in subsequent screening assays.Blood can be collected from a tail bleed (UNIT 1.7)after the first intraperitoneal boost (see Basic Protocol 1,step 7)or from the heart at the time of spleen harvest.To collect blood from heart use a sterile Pasteur pipet and place blood into a 1.5-ml microcentrifuge tube.Let blood stand at room temperature with the cap off to allow blood to clot.After 30min,remove the blood clot with a sterile Pasteur pipet and place tube at 4◦C.The next day,centrifuge tube for 15min at 400×g,4◦C or room temperature.Collect serum and add sodium azide to a final concentration of 0.1%.Store at −20◦C 7.Disaggregate the spleen into a single-cell suspension (UNIT 3.1).Transfer the spleen to a fine-mesh metal screen placed on top of a 50-ml conical centrifuge tube,and use the plunger of a 3-m1syringe to gently grind the cells out of the spleen.Rinse the screen with Medium B to help cells pass through the screen.Pipet the cells up and down in the tube with a 10-ml pipet to break up lumps.Try not to cause the solution to foam.Only the spleen membrane should be left in the screen.See UNIT 3.1for additional dis-cussion of the above procedure.Other spleen disaggregation methods may also be used.8.Centrifuge cell suspension 10min at 400×g ,room temperature or 37◦C,and remove supernatant.Wash the splenocytes three times,each time by adding 30ml Medium B,centrifuging 10min at 400×g ,room temperature or 37◦C,and discarding the supernatant.Resuspend the final cell pellet in 25ml Medium B.It is important to remove all the serum adhering to the cells by washing with serum-free Medium B.If the serum is not removed,PEG will not fuse the cell membranes and the fusion frequency will drop drastically.。