phosphate buffer, and other serum proteins in 0.5-1.0 M NaCI. Regenerate the column by washing with three bed volumes of regeneration buffer (0.2 M phosphate buffer, pH 7.4, made as described for equilibration buffer) and then with equilibration buffer. Fractions can be tested for the presence of IgG by SDS-PAGE. In addition, new products have been designed to isolate IgG by affinity chromatography using Protein A attached to cellulose disks. These may prove to be useful for some applications. It is critical to properly characterize antiserum prior to use. Antibody titer, specificity, subclass, and affinity can greatly affect subsequent experimental protocols. Common methods for antibody detection and characterization are outlined in Tables IV and V and discussed in detail elsewhere. 3,5,11 11 T. M. Timmons and B. S. Dunbar, this volume .
 P r e p a r a t i o n o f M o n o c l o n a l A n t i b o d i e s
By BONNIE S. DUNBAR and SHERI M. SKINNER The report by Kohler and Milstein 1 that a cell hybrid made by fusing normal spleen cells with malignantly transformed antibody-secreting cells (plasmacytoma or myeloma cell line) can provide a continuous source of antibody of predefined specificity has led to the explosion of the use of "monoclonal antibodies." Because this technology has been so widely used, several excellent texts and reviews have been written which describe this technology in great detail. 2-5 The properties of these antibodies as well as the advantages and disadvantages of their use as compared to polyclonal antibodies are summarized in Table I. These guidelines should assist in the choice of determining which type of antibodies are optimal for use in designated experiments. G. Kohler and C. Milstein, Nature (London) 256, 495 (1975). 2 R. H. Kennett, T. J. McKearn, and K. B. Bechtol, "Monoclonal Antibodies." Plenum, New York, 1980. 3 G. Kohler, "Hybridoma Techniques." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1980. 4 j. W. Goding, "Monoclonal Antibodies: Principles and Practice," 2nd Ed. Academic Press, New York, 1986. 5 H. Zola and D. Brooks, in "Monoclonal Hybridoma Antibodies: Techniques and Applications" (J. Hurrell, ed.), p. 1. CRC Press, Boca Raton, Florida, 1981.
METHODSIN ENZYMOLOGY,VOL. 182
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PREPARATION OF MONOCLONAL ANTIBODIES
TABLE I ADVANTAGES AND DISADVANTAGESOF MONOCLONALANTIBODIES
Major advantages Single homogeneous antibody to a defined antigenic determinant Specific antibody can be used to study functional domain of molecule Large quantities of antibody can be obtained since immortal cell lines can theoretically be developed Antibodies with low-affinity binding can be selected during screening procedures (these antibodies are designed for immunoaffinitychromatography) Major disadvantages Procedure is expensive and time consuming Well-equipped tissue culture facilities are needed Epitope recognized by antibody may be shared among many different antigens not related to antigen of interest Hybridoma cell lines are frequently unstable due to chromosome loss or may be lost because of tissue culture contamination
Antibody Screening Assays Prior to the generation o f monoclonal antibodies it is essential to first establish a sensitive and rapid antibody-screening assay. Because thousands of cell culture samples will usually have to be screened, adequate methods should be developed prior to setting up cell cultures. A variety of methods can be used to determine the presence, as well as the specificity, of an antibody. The method of choice will vary depending on the nature of the antibody to be detected (e.g., polyclonal vs monoclonal) or the nature of antigenic determinants to be recognized by that antibody (sequential, conformational, protein, carbohydrate, cell surface, glycolipid, etc.). These procedures which include immunocytochemical localization methods as well as biochemical methods are summarized in detail elsewhere. 3-6 Only the most c o m m o n l y used assays for screening large numbers of hybridoma cell lines are described here. These include enzyme-linked immunoassays and dot-blot immunoassays. (A variety of commercial kits are now available and methods are established for labeling antibodies for routine assays.) Equipment (e.g., the miniblotter manufactured by Immunetics, Cambridge, MA) has also been developed for the large-scale screening of antibodies by one-dimensional Western blots using monoclonal antibodies. Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. 6 B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987.
Method for Enzyme-Linked Immunoassay (ELISA) for Screening Monoclonal Antibodies There are currently large numbers of ELISA procedures for the detection of antigens and antibodies. Of the ELISA methods we have used, the avidin-biotinylated peroxidase complex (ABC) system has been the most successful primarily due to its sensitivity. 7'8 The reagents and antibodyscreening kits are available from a variety of sources, although we routinely use Vector Laboratories (Burlingame, CA). Antigen Binding to Microtiter Plates. To determine if a microtiter plate will optimally bind the antigen or cells of interest, it may be necessary to test a variety of plastics manufactured for this purpose. The capabilities of the spectrophotometric plate reader must also be considered in choosing a well shape. We routinely use a round-bottomed microtiter plate (Dynatech Immulon #2). It may require significant amounts of antigen to adequately coat the plate, although it may be possible to use a crude antigen preparation. It will be necessary to experiment with a variety of buffers, pH values, and antigen concentrations to find the optimal combination for maximum binding since different antigenic molecules will have distinct binding characteristics. Depending on the purity and nature of the antigen, one may need concentrations from 0.1 to 10 /xg/ml. A basic buffer (pH -9.6) works for most antigens, but this must also be experimentally determined. Some antigens, once bound to plastic wells, are stable enough to allow freezing of the plate containing the antigen-coating solution, or at a later step when the plate contains blocking solution (described below) although since some molecules may lose their antigenicity, this will have to be tested. All incubations should be carried out using a vibrating plate mixer for consistent results and intense color development. We use a microplate mixer (Fisher Scientific, Pittsburgh, PA), which may be placed in a cold room or an oven whenever necessary. Plates should be thoroughly washed between reagent treatments. This can be carried out inexpensively with no special equipment or, if available, a microtiter plate washing apparatus is preferable. To wash, pour solution over the face of the plate until the wells are filled. Shake the plate vigorously on the mixer and pour or pipet out wash solution. (Be careful not to mix samples from one well with another.) Three more such passes result in a very thorough washing, minimizing or eliminating background caused by nonspecific binding of reagents.
7 A. Voller, D. Bidwell, and A. Bartlett, in "Manual of Clinical Immunology" (N. R. Rose and H. Friedman, eds.), p. 506. Am. Soc. Microbiol., Washington, D.C., 1976. 8 D. W. Drell, D. M. Wood, D. S. Bundman, and B. S. Dunbar, Biol. Reprod. 30, 435 (1984).
PREPARATION OF MONOCLONAL ANTIBODIES
Antibody Detection. Antigen is diluted to a previously established concentration (we use 1 /zg/ml) in antigen-coating buffer (0.1 M Na2CO3/ NaHCO3, pH 9.6, for example), and 50 p.1 is added to each well. (Control wells include no antigen, no primary antibody, no secondary antibody.) Plates are incubated for 6 hr at room temperature or overnight at 4 ° on the microplate mixer. After coating, plates are thoroughly washed using assay buffer (20 mM PBS, pH 7.3, containing 0.15 M NaC1 and 0.05% Tween 20). Blocking of nonspecific binding sites is accomplished using blocking buffer [20 mM phosphate-buffered saline (PBS), pH 8.0, containing 0.15 M NaC1 and 2% nonfat dried milk] at 100/~l/well, and incubated overnight at 4 ° with shaking. Plates are again washed thoroughly and the primary antibody is applied. Dilutions of control or sample sera or other antibody-containing solutions are made using diluent (2% nonfat dried milk in assay buffer, pH 7.3). Plates are then incubated overnight at 4° . Thorough washing follows and the biotinylated second antibody is applied for 1 hr at room temperature. The dilution, made with the diluent solution described above, must be experimentally determined. For our work, a 1:200 dilution has been sufficient. After thorough washing, the plates are incubated for 30 min at room temperature with a freshly made mixture of 0.3% HzO2 in methanol (100/zl/well) to remove any endogenous peroxidase activity that might otherwise result in background color with subsequent reagents. After washing as before, the plates are treated with the premixed avidinbiotinylated horseradish peroxidase complex (Vector Laboratoriesl Burlingame, CA, Vectastin kit) (50/zl/well) for 1 hr at room temperature with shaking. After washing, substrate solution is added (50/zl/well) and incubated for 30 min at room temperature with shaking. Our best results have been achieved with a solution of 0.8 mg/ml o-phenylenediamine dihydrochloride (OPD) (Bethesda Research Laboratories) in 0.1 M citrate/phosphate buffer (pH 5.0) containing 0.3% H202. The OPD substrate can be made at 5 × concentration and stored frozen in small aliquots for later use. The citrate/phosphate buffer (without H202) can be made ahead of time and stored frozen (as can the diluent and the blocking solution), but H202 must be added just prior to solution use. After substrate incubation, the plates are read at 450 nm on a microtiter spectrophotometer. It is important to first scan an untreated plate to blank, and subsequently an uncoated well on the treated plate as the reagent blank. As the reaction cannot be stopped it is important to standardize the time of the substrate reaction for reading consistently between assays.
Immunoblotting. Another type of assay uses immobilization of the antigen on a nitrocellulose membrane. The antigen(s) may be electrophoretically transferred to the membrane from a gel using methods outlined in the chapter on protein blotting in this text,9 allowing probing of the antigen in various states of denaturation, chemical alteration, or purification. Production of Monoclonal Antibodies Once antibody-screening assays have been established, monoclonal antibody production may be undertaken. Hybridoma antibody-producing cells are made by fusing mouse myeloma cells with mouse lymphocytes. This results in a cell line proliferating indefinitely and secreting an immunologically homogeneous product. By screening the resulting hybridomas, one may select those producing useful antibodies.
Immunization Procedure The intradermal immunization procedures normally used for polyclohal antibody production in laboratory animals such as rabbits are difficult to use in mice. Therefore other methods have been developed such as subcutaneous injections with adjuvants, nitrocellulose implants (containing antigen), and intrasplenic injections (refer to reviews in Refs. 2-6 for more detail). Generally, subcutaneous immunizations using antigen emulsified in Freund's complete adjuvant are used as the primary injection. (See protocols for antigen preparation in  on polyclonal antibodies.) A variety of immunization strategies can also be used to help in obtaining the type of antibodies desired. For example, a longer immunization time should result in a better chance of obtaining IgG rather than IgM immunoglobulins. If limited antigen is available, an alternative immunization procedure can be followed. Fifty to one hundred micrograms antigen in 0.2 ml Complete Freund's adjuvant is injected into the hind foot pad of the mouse. After 10-12 days, the popliteal lymph node in the mouse hind leg will be swollen, and can easily be dissected from the surrounding fat pad. These cells are fused with myeloma cells using the procedure described for spleen cells (omitting the red blood cell lysis step). 10
Spleen Cell Preparation The spleens are removed from immunized mice (24-72 hours after boost) using sterile conditions and are placed into Dulbecco's minimal 9 T. Timmons and B. Dunbar, this volume . 10 R. Conitti, G. Rocchetti, P. Gnocci, E. Monandi, and Y. M. Galante, J. Immunol. Methods 99, 25 (1987).
PREPARATION OF MONOCLONAL ANTIBODIES
essential media (or other standard MEM) which has been supplemented with 2 mM glutamine, 100 IU/ml penicillin, and 100/zg/rnl streptomycin. The connective tissue splenic capsule is removed and the tissue is minced and cells dispersed. The cell suspension is allowed to settle for 10 min on ice and the supernatant then centrifuged (1600 rpm for 6-7 min). The supernatant is discarded and red blood cells in the pellet are lysed by suspension of the pellet in 5 ml/spleen of 0.83% NH4CI solution. Incubation on ice for 10 min completes this step. Equal volumes of medium are then added and the cells washed twice. Viable cells are counted using dye exclusion and a suspension of approximately 100 million cells/spleen is made.
Myeloma Cell Preparation Numerous myeloma cell lines are now commercially available. The optimal cell line should be HGPRT negative, not produce or secrete its own immunoglobulin, and its growth should be stable (preferably in the absence of feeder cells). We have used p3U1 cell lines with routine success. H In carrying out a fusion, the cells are concentrated by centrifugation (1600 g for 6-7 min). It is important to concentrate one-tenth as many myeloma cells as spleen cells. The cells are resuspended in medium containing 15% fetal calf serum, and a viable cell count is made.
Fusion Procedure For cell fusion, the HAT selection method is routinely used. 12Briefly, HAT (hypoxanthine, amonopterin, thymidine) selection utilizes the functional complementation of two different (parental) metabolic defects to produce complete function and therefore survival in the hybrid cells. It is not necessary to select against the growth of unfused lymphocytes since they will not grow in tissue culture. One must select only against the unfused tumor cells. The use of HAT prevents them from growing unless "rescued" by fusion with the lymphocytes which supply the enzyme HGPRT (hypoxanthine-guanine phosphoribosyltransferase), a critical metabolic enzyme which is missing in the cell line. Cell Fusion Protocol. Myeloma and spleen cells are combined at a ratio of 1:10 in a 50-ml conical centrifuge tube ( - 1 0 ml). They are centrifuged at 1600 rpm for 6-7 min and the supernatant discarded. The cell pellet is gently dislodged and 2 ml of PEG 1000 (pH 8.0) is added. The pellet is resuspended and centrifuged (1600 rpm for 3 min). (Note: The time and efficiency of fusion may depend on the batch of PEG. You may have to try u D. W. Yelton, B. A. Diamond, S.-P. Kwan, and M. D. Scharff, Curr. Top. Microbiol. Immunol. 81, 1 (1978). n M. L. Gefter, D. H. Margulies, and M. O. Scharff, Somat. Cell Genet. 3, 231 (1977).
several lots for best results.) With PEG still in the sample, 5 ml of serumfree medium is slowly added and the pellet gently resuspended. Centrifugation is then carried out at I000 rpm for 6 min. The supernatant is discarded and 10-20 ml of serum-containing growth medium is added. The fused cells are resuspended, pipetted into a Petri dish, and incubated for 1-3 hr (5% CO2, 37°). The fused cells are pipetted back into a conical centrifuge tube and centrifuged at 1000 rpm for 10 min. Sufficient HAT medium is added to dilute the cell suspension to 10 6 cells/ml (e.g., approximately 100 ml/ spleen). HAT medium stock can be made using 136/zg/ml hypoxanthine, 0.9% ~g/ml aminopterin, and 3.88 /xg/ml thymidine in the Dulbecco's medium described above. This solution may then be stored in the dark in frozen aliquots. When needed, 1 ml of this stock may then be diluted to 100 ml in the above-mentioned Dulbecco's medium. Cells (2 ml/well) are pipetted into cell culture trays (usually Costar or Linbro 24-well trays are optiomal for initial cloning) and placed in the incubator. The incubator is monitored for 2 weeks at which time one can begin screening the media for secretion of antibodies (one should be able to visualize hybridoma colonies by days 10-14). After 7-10 days, wells can be examined for hybrids and medium can be replaced by HT medium. (HT medium is simply HAT medium without aminopterin.)
Subcloning Hybridomas Hybridomas can be subcloned 1'3 by microscopically selecting and pipetting out individual colonies. Alternatively, they can be subcloned using limiting dilution in which cells are diluted such that, statistically, there should only be one viable cell per well. For example, following subcloning, if more than 37% of wells have no growth, there is a reasonable probability that wells with growth will contain single clones. 4 Multiple subclonings should be carried out to better guarantee the monoclonality of a cell line. At each stage of the subcloning procedure, some cells should be frozen and stored to ensure that a cell line will not be lost.
Freezing Hybridoma Cells We have used the procedure previously described 5 to freeze cells. Hybridoma cells are washed once in culture medium and suspended at a concentration of 6 x 106/cells/ml HT medium with 50% fetal calf serum. To this suspension, add dropwise an equal volume of medium containing 30% DMSO. Gently mix while slowly adding medium. Transfer 2-ml aliquots to 2-ml freezing vials and freeze using conventional tissue culture techniques. Cells can be stored in liquid nitrogen at -190 to -150 °.
PREPARATION OF MONOCLONAL ANTIBODIES
Cells are thawed by removing them from the freezer and placing them in a 37° water bath. Immediately after thawing, the contents are diluted by adding dropwise an equal volume of HT medium. After 15 min, another 6 ml of medium is added over the next 10 min and the cells are left at room temperature for 15 min. The cells are then washed twice in HT medium and placed into the incubator. Characterizing Subclasses of Monoclonal Antibodies It is generally necessary to determine the subclass of the antibody. For example, if you are not interested in obtaining IgM subclasses, you may be able to eliminate these from the cultures and reduce screening numbers. Also, some subclasses do not bind protein A, etc. 9 Because subtyping kits are now available commercially (e.g., Bethesda Research Laboratories) this is easily done using the ELISA assay. Because some subtyping reagents are not specific for immunoglobulins from different strains of mice, it is helpful to use mice, as spleen donors for antibody production, which are compatible with the antibody-subclassing reagents. In Vitro Immunization and Fusion of Peripheral L,ymphocytes
A number of laboratories have updated the use of in vitro immunization methods. ~3'14 These methods were developed because frequently adequate numbers of antigen-specific B lymphocytes are not stimulated during in vivo immunization procedures. As discussed by Reading, 15this failure may be due to tolerance (antigen-specific nonresponsiveness) or to an antigen hierarchy response (selective responsiveness to one or a few components of the immunogen preparation). Effective hybridoma formation may therefore be achieved if in vitro immunization methods are used. 13-22Immunization takes place in a matter of days rather than weeks or months. Also, because the normal in vivo regulation of the immune response is not a factor, it may be possible to produce antibodies against molecules normally considered only weakly immunogenic. Although these 13 D. Grarecos, M. Astier, and M. Semeriva, J. lmmunol. Methods 103, 169 (1987). ~4 S. A. Danielsson, S. A. Muller, and C. A. K. Borrebaeck, Immunology 61, 51 (1987). 15 C. L. Reading, this series, Vol. 121, p. 18. 16 B. Sharma and P. I. Terasaki, Cancer Res. 34, 115 (1974). J7 R. L. Lundah and D. J. Raidt, Cell. Immunol. 9, 60 (1973). 18 M. Schelling, Hybridoma 5, 159 (1986). i9 C. A. K. Borrebaeck and S. A. Moller, J. Immunol. 136, 3710 (1986). 20 C. A. K. Borrebaeck, Trends Biotechnol. 4, 147 (1986). 21R. L. Pardue, R. C. Bardy, G. W. Perry, and J. R. Dedman, J. CellBiol. 96, 1149 (1983). 22 C. L. Reading, J. Immunol. Methods 53, 261 (1982).
methods may have some benefits, the drawbacks are the production of predominantly IgM subclasses and the difficulty of standardizing the system. It is generally recommended that the beginner become well versed in methods for conventional preparation of monoclonal antibodies prior to initiating this method. Because human splenic and tonsillar tissue is largely unavailable, it is preferable to use peripheral blood lymphocytes for production of human monoclonal antibodies. This has been successful only recently. Procedures were developed by Danielsson et al. 14using an elegant separation scheme in which lymphocytes were divided into several subpopulations. These were activated and reconstituted to give a population with a specific B:T cell ratio. Careful use of a number of support substances during induction and immunization resulted in several hundred cells/10 6 B cells which secreted antigen-specific antibodies. The technique is quite intricate, but appears to produce B cells which are amenable to fusion for production of human monoclonal antibodies. Antiidiotypic Antibodies If a homogeneous antibody (e.g., a myeloma-produced antibody) is used as an antigen, certain portions of the molecule may be recognized as antigenic by the responding immunized host. The portion of an antibody molecule which recognizes its antigenic determinants is a set of unique sites termed "idiotype." These sites are made up of particular amino acid sequences in the hypervariable portion of the variable region of the antibody. The antibodies produced by the host against these sites are therefore termed antiidiotype. Antiidiotypic antibodies have internal images of the original immunogen, and therefore are identified operationally as, antibodies which have activities which mimic those of the original immunogen. 23-26Antiidiotypic antibodies have been described which mimic such proteins and molecules as insulin 23 and alprenolol. 27 23 K. Sege and P. A. Peterson, Proc. Natl. Acad. Sci. U.S.A. 75, 2443 (1978). 24 A. Nisonoff and E. Lamoyi, Clin. Immunol. lmmunopathol. 21, 391 (1981). 25 B. F. Erlanger, W. L. Cleveland, N. H. Wasserman, B. L. Hill, A. S. Penn, H. H. Ku, and R. Sarangarajan, in "Molecular Basis of Nerve Activity" (J. P. Changeux, F. Hucho, A. Maelicke, and E. Neumann, eds.), p. 523. de Gruyter, Berlin, 1965. 26 B. F. Erlanger, W. L. Cleveland, N. H. Wasserman, H. H. Ku, B. L. Hill, R. Sarangarajan, R. Rajagopalan, E. Cayanis, I. S. Edelman, and A. S. Penn, Immunol. Rev. 94, 23 (1986). z7 A. B. Schreiber, P. O. Couraud, C. Ande, B. Vray, andA. D. Strosberg, Proc. Natl. Acad. Sci. U.S.A. 77, 7385 (1980).
PROTEIN BLOTTING AND IMMUNODETECTION
While this technology is just becoming feasible, ithas been demonstrated that the antiidiotypic strategy is a potentially powerful approach for the preparation of monoclonal antibodies to receptors which are difficult to isolate in quantities sufficient to be used as antigens. 26 Single-Chain Antibodies via Genetic Engineering Another method of generating homogeneous antibodies to take advantage of or improve their specificity is the genetic engineering of singlechain antibodies. 2s'29 These recombinant molecules consist of the two antibody variable regions connected by a linear peptide. While this technology is in its infancy, the potential for low-cost, high-volume production of highly specific antibodies is considerable. 3° Acknowledgment We wish to acknowledge the expert clerical help in manuscript preparation given us by Ms. Suzanne Mascola. 28 S. Cabilly, A. D. Riggs, H. Pande, J. E. Shively, W. E. Holmes, M. Rey, L, J. Pery, R. Wetzel, and H. L. Heynehey, Proc. Natl. Acad. Sci. U.S.A. 81, 3273 (1984). 29 M. A. Boss, J. H. Kenten, C. R. Wood, and J. S. Emtage, Nucleic Acids Res. 12, 3791 (1984). 30 A. Klausner, Biotechnology 4, 1041 (1986).
 P r o t e i n B l o t t i n g a n d I m m u n o d e t e c t i o n
By THERESE M. TIMMONS and BONNIE S. DUNBAR Polyacrylamide gel electrophoresis (one- and two-dimensional) has become one of the most widely used techniques for the analysis and characterization of complex protein mixtures. 1-5 These gels can be stained directly and proteins visualized by several different methods. 2'3 However, because access to proteins within the matrix is limited, the information i D. Garfin, this volume . 2 B. S. Dunbar, H. Kimura, and T. M. Timmons, this volume . 3 C. R. Merril, this volume . 4 B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. B. D. Hames and D. Rickwood, "Gel Electrophoresis of Proteins: A Practical Approach." IRL Press, Washington, D.C., 1988.
METHODS IN ENZYMOLOGY, VOL. 182
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