Iscove's Dulbecco's minimal essential medium for 24 hr prior to hybrid selection. Colonies ... of specific IgG,K antibodyper 106 cells per ml per 24 hr. All sub-.
Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6376-6380, October 1983
Medical Sciences
Characterization of human hybridomas secreting antibody to tetanus toxoid (monoclonal antibodies/cell fusion/cell sorter)
JAMES W. LARRICK, KENNETH E. TRUITT, ANDREW A. RAUBITSCHEK, GEORGE SENYK, AND JANET C. N. WANG Cetus Immune Research Laboratories, 3400 West Bayshore Road, Palo Alto, CA 94303
Communicated by Hugh 0. McDevitt, July 5, 1983
We have selected a thioguanine-resistant lymphoblastoid cell line (LTR228) that forms human-human hybrids with high efficiency. Fusions with peripheral B cells consistently yield one colony per 105 cells plated. To produce antitetanus monoclonal antibodies, we withdrew blood from persons who had recently received booster injections of tetanus toxoid. T cells were separated from peripheral mononuclear cells by 2-aminoethylisothiouronium bromide-induced rosette formation, given 1,500 rads (1 rad = 0.01 gray), and cultured in a 1:1 ratio with nonrosetting cells. After 3 days of pokeweed mitogen stimulation, heterokaryons were produced by a plate-fusion technique and cultured in Iscove's Dulbecco's minimal essential medium for 24 hr prior to hybrid selection. Colonies appeared after 10-14 days in hypoxanthine/azaserine supplemented medium. A direct binding enzymelinked immunosorbent assay with specific tetanus toxoid inhibition identified positive wells. The hybridomas were cloned twice in soft agarose and by limiting dilution. The subcloned hybridomas double every 26 hr (vs. every 16 hr for LTR228) and produce 1-5 ,ug of specific IgG,K antibody per 106 cells per ml per 24 hr. All subclones (almost 200) continue to secrete antibody after 11 months
thioguanine (20 ,g/ml)-resistant mutant for its high fusing capacity. Our line is mycoplasma-free and doubles every 16 hr when grown in Iscove's Dulbecco's minimal essential medium (DME medium) (GIBCO) with 15% fetal calf serum. LTR228 has a typical lymphoblastoid nuclear EBV nuclear antigen staining pattern. Preparation of B Cells. Volunteers were vaccinated intramuscularly with tetanus toxoid (Wyeth, Philadelphia). Peripheral blood (80 ml) was drawn in heparin 9 days after vaccination. Lymphocytes were separated by Ficoll-Hypaque density gradient centrifugation. The lymphocytes were washed twice in Hanks' balanced salt solution and the T cells formed rosettes with 2-aminoethylisothiouronium bromide-treated sheep erythrocytes as described (13). The rosetted T cells (6 X 106) were exposed to 1,500 rads (1 rad = 0.01 gray) in a Gammacell 40 gamma-irradiator (Atomic Energy of Canada) and subsequently mixed with 6 x 106 nonrosetting B cells. The mixed cells were cultured at a density of 106 per ml in Iscove's DME medium/ 15% fetal calf serum/1% pokeweed mitogen (GIBCO). After 3 days the cells were again separated by Ficoll-Hypaque to obtain a population of primarily B-cell blasts. Preliminary experiments showed that B-cell blasts fused better than unstimulated lymphocytes. The irradiated rosette-forming T cells gave much less suppression than unfractionated lymphocytes. Fusion Protocol. Cells were combined and fused according to the protocol of Brahe and Serra (14) with slight modification. Cells were fused with 40% polyethylene glycol 4,000 (BDH)/ 10%0 dimethylsulfoxide/poly(L-Arg) (5 ,ug/ml) (Sigma) in Hanks' balanced salt solution without calcium and with magnesium. By using this technique, we consistently observed low cell death and formation of 3-8% heterokaryons. Cells were cultured 24 hr after fusion prior to addition of hypoxanthine (100 ,uM)/azaserine (8 ,g/ml) in Iscove's DME medium with 15% fetal calf serum. Fusions were plated at a density of 105 cells per microtiter well for hybrid selection. Cultures were subsequently fed every 3 days with selective medium. Hybrid colonies normally appeared after 10-14 days. Enzyme-Linked Immunosorbent Assays (ELISA). Culture supernatants were tested for anti-tetanus toxoid antibody and total Ig by the ELISA technique (15). Tetanus ELISA. Microtiter plates (Immulon II, Dynatech, Alexandria, VA) were coated with purified tetanus toxoid (10 /,g/ml) (Massachusetts State Laboratories, Boston) in sodium carbonate buffer (50 mM, pH 9.5) at 4°C overnight. Wells were blocked with 1% bovine serum albumin/0. 1% gelatin in phosphate-buffered saline (Pi/NaCI) for 2 hr at 37C. Culture supernatants and high-titer antitetanus human serum standards were diluted in Pi/NaCl and incubated for 1 hr at 37°C. Tetanus toxoid (10 ,g/ml) was added to duplicate wells to demonstrate
ABSTRACT
of continuous culture. Twelve representative subclones have near tetraploid amounts of DNA. From hybridomas grown in 5-liter spinner flasks, milligram quantities of the IgG,K antibody were purified by staphylococcus protein A affinity chromatography. Specific antibody from hybridoma cultures protected mice injected with 1,000 times the LD50 of tetanus toxin. Our cell line and associated techniques should permit the production of therapeutically important human monoclonal antibodies.
Many attempts to produce human monoclonal antibodies have been made since the early mouse hybridoma work of Kohler and Milstein (1). Initial reports of successful fusions of human B cells and the U266 myeloma line (2) have not been confirmed in other laboratories. Problems with mycoplasma contamination of parent lines, suitable pre-fusion preparation of B cells, hybrid stability, and quantity of secreted monoclonal antibody have hampered most efforts with other human myeloma lines (3). Other approaches have also met with limited success. These have included mouse plasmacytoma by human B-cell hybridization (4, 5), direct Epstein-Barr virus (EBV) transformation of human B cells (6, 7), and fusion with transformed lymphoblastoid cell lines (8, 9), or combinations of the above (10, 11). We have developed an EBV-positive splenic lymphoblastoid cell line, LTR228, that forms stable human-human hybridomas with high efficiency. Here we describe the production and characterization of antitetanus human monoclonal antibodies. MATERIALS AND METHODS Cell Line. LTR228 is a lymphoblastoid cell line that originated in spleen cell culture (12). We selected a spontaneous 6The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; Pi/NaCl, phosphate-buffered saline.
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specific inhibition. Wells were washed with Pi/NaCl and developed with peroxidase-conjugated rabbit anti-human y-, ,u-, K-, or A-chain-specific antibodies (DAKO Labs, Accurate Chemicals, Westburg, NY) diluted 1/1,000 in P;/NaCl/1% bovine serum albumin. After a final wash, peroxidase substrate 2,2'-azino-di(3-ethylbenzthiazoline sulfonic acid) (ZYMED, South San Francisco, CA) was added and OD415 was read with a Titertek ELISA plate reader (Flow Laboratories). Ig ELISA. Ig levels of culture supernatants were measured by a chain-specific inhibition ELISA. Plates were coated with purified human Ig (10 pg/ml)/carbonate buffer, pH 9.5, at 40C overnight. Wells were blocked as described above. Supernatants or standards and affinity-purified peroxidase-labeled antiK, A, y, ,u, or a were mixed and added to the wells. Plates were processed in the same manner as the tetanus ELISA. Biosynthetic Labeling of Igs. Cells (20 106) were cultured overnight in 4 ml of methionine-free medium/1% dialyzed fetal calf serum/0.8 mCi of [3S]methionine (1 Ci = 3.7 10"' Bq; New England Nuclear). Cells were pelleted and Igs from the tissue culture supernatant were precipitated with chainspecific rabbit anti-human antibodies attached to Staphylococcus aureus (Cowan I strain). Immunoprecipitates were electrophoresed on a 5-15% polyacrylamide gradient gel in NaDodSO4 according to the procedures of Weber and Osborn (16). Gels were stained with Coomassie blue, soaked for 1 hr in EN3HANCE (New England Nuclear), dried, and fluorographed on Dupont Cronex film. Purification of' Monoclonal Antibody. Monoclonal antitetanus antibody was purified by staphylococcus protein A-Sepharose affinity chromatography (Pharmacia). Antibody was eluted with 100 mM acetate/150 mM NaCl, pH 2.3. Electron Microscopy. Logarithmic phase cells were washed twice in Pi/NaCl and fixed with 1% paraformaldehyde in Pi/ NaCl. Cells were attached by poly(L-Lys) to microscope slides, dehydrated, critical-point dried, and gold-coated prior to examination in an ISI-40 scanning electron microscope. Cell Sorter Methods. DNA histograms. Preparation of cells for DNA analysis was performed as described (17) with a slight modification. Cells (105) were sedimented in U-bottom microtiter wells, medium was aspirated, and cold 50% ethanol/Hanks' balanced salt solution was added. Cells were incubated for 30 min on ice, pelleted, and washed. The pellet was resuspended in 0.1 M Tris.HCI/0.1 M NaCl, pH 7/Hoechst-33342 at 0.4 ;g/ml (Calbiochem) and incubated at 22°C for 15 min. Cytofluorography was carried out on an EPICS V cell sorter (Coulter) equipped with a 5-W argon laser (Coherent Innova90). The laser output was adjusted to 100 mW UV, at 350-364 nm. A 408-nM-long pass filter shielded the detector. At least 5,000 cells were accumulated for each histogram. Cell sorter cloning. The EPICS V was sterilized with 0.05% NaOCI, and laser.output was adjusted to 488 nM with 1,000 mW of power.. Scatter and fluorescence windows were set to ensure sorting of viable cells. Hybrids were cloned at one cell per well into U-bottom microtiter wells with Iscove's DME me.dium/20%' fetal calf serum. Surface antigen analysis. Cells were stained with fluorescent antibodies by the method of Loken and Stall (18). Fluorescein isothiocyanate-anti-Leu-10 and anti-DR, anti-y, .anti-, reagents were purchased from Becton Dickinson. Anti-Bi (Coulter) was developed with goat F(ab')2 from TAGO (Burlingame, CA) and ,u-chain specific goat anti-mouse F(ab')2 fragments (TAGO). Cloning and Reverse-Plaque Techniques. Limiting dilution (0.3 cells per well) cloning was performed in 96-well U-bottom plates (Costar, Cambridge, MA) in Iscove's DME medium/20% X
X
fetal calf serum. For soft agar cloning, 1,000 cells in 1 ml of 0.33% SeaPlaque
6377
agarose (FMC,. Rockland, ME), made in Iscove's DME medium/20% fetal calf serum, were placed over a bed of 4 ml of SeaKem agarose (04%) in 60-mm culture dishes (Falcon). To select for a nonproducer parent cell line or for high-producer hybrids a reverse-plaque technique was used. Protein Acoated sheep erythrocytes (1.0%) were added to the upper layer of soft agar according to the method of Gronowicz et al. (19). Clones secreting more antitetanus Ig had the largest plaques. A subclone of LTR228 secreting no Ig made no plaques.
RESULTS Table 1 summarizes our preparation of antitetanus human monoclonal antibodies; several points are noteworthy. (i) Peripheral blood from an immunized subject provided a suitable source of B cells. (ii) The eventual appearance of colonies in every well indicated that the fusion process produced at least one viable hybrid for every 120,000 cells plated. This efficiency of 1 in 5 x 104 LTR228 cells plated approaches that routinely achieved in the mouse system. (iii) Almost one-half of the wells with growing cells produced IgG or IgM (or both); 5% of the wells showed specific antitetanus activity. We initially cloned the antitetanus cells by limiting dilution and in soft agar. Well El produced the greatest number of antitetanus-positive clones, and we selected these cells for further study. Sublines were recloned by limiting dilution, cell sorter, and soft agar methods. All subsequent clones, about 200, have continued to produce antitetanus antibody. Morphological Characteristics of, Human Hybridomas. By light microscopy-the hybrids appeared to be larger than LTR228. However, scanning electron microscopy studies (Fig. 1) did not confirm this difference. Although the hybridomas secrete up to 5 A.g of specific antibody per ml, transmission electron microscopy shows both hybrids and the parent LTR228 to have very little rough enTable 1. Antitetanus human monoclonal antibody cell
fision protocol Procedure Immunization 0 Subject immunized intramuscularly with tetanus toxoid Pre-fusion preparation 9 After immuni- Withdraw blood; PWM stimulation zation Fusion 3 After PWM Fusion of 6 x i05 B-cell blasts and 6 x 106 LTR228 1 After. fusion Plate 120,000 per well; add hypoxanthine/ azaserine; feed every 3 days 14 Colonies appear 19 Tet ELISA 5+ wells, 2± wells 21 47 wells IgG', 39 wells IgM' 29 Growth in 100% of wells First cloning 0 A4, El, Gi, G3 200 wells each by lim. dil.; 2 x 60-mm dishes each by soft agar 16 After cloning Screen 200 soft agar and 800 lim. dil. clones: .11/48 El soft agar clones antitet positive; 3/100 El lim. dil. clones antitet positive Second cloning 16 After cloning All soft agar clones antitet positive; all lim. dil. clones antitet positive; 12/12 clones tetraploid by cell sorter PWM, pokeweed mitogen; tet, tetanus; lim. dil., limiting dilution. Day
Status
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Proc. Natl. Acad. Sci. USA 80 (1983)
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V-E54
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/
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0
1', 0
50 Time, hr
100
FIG. 2. Comparison of-growth rates of parent cell line (closed sym-
bols) and four antitetanus hybridomas (open symbols). Doubling time of parent is 16 hr; doubling time of hybrids is 26 hr.
FIG. 1. Scanning electron microscope photographs-of LTR228 (A) and antitetanus hybridomas (B).
doplasmic reticulum (data not shown). Other workers have also noted that the presence of rough endoplasmic reticulum may not correlate with the capacity of B-cell lineage cell lines to secrete Ig (20). -Growth Characteristics of Human Hybridomas. The hybridomas double every 25-28 hr as compared to every 16 hr for the parent (Fig. 2). They can easily be, adapted to growth in serum-free Iscove's medium supplemented with bovine serum albumin (700 ,ug/ml)/transferrin (10 ,g/ml)/insulin (0.2 unit/ ml). Under such conditions, the time required for hybrid cultures to reach confluence is 30% longer than in Iscove's DME medium/15% fetal calf serum. Stability of Human Hybridomas. After two rounds of cloning, all sublines continue to secrete antitetanus antibody. Eight
months in culture and expansion of cells in 4-liter vessels has not altered the secretion of specific antibody. Furthermore, cell sorter histograms showed 12 representative clones all to have nearly tetraploid DNA levels (Fig. 3). Chromosome studies yield similar conclusions (Fig. 4). LTR228 has a median of 48 chromosomes; hybrids have 91. Quantity of Monoclonal Antibody Secreted. Fig. 5 shows the levels of antitetanus Ig produced by 12 subclones. Spent hybridoma culture medium (24 hr) of 106 cells contains 1-2.5 tkg of specific antibody per ml. A reverse-plaque technique was used to select for high-producer clones. With this method, we have been able to. derive sublines that secrete twice as much Ig (data not shown). Biochemical Characterization of Monoclonal Antitetanus Antibodies. Fig. 6 shows the results of chain-specific immunoprecipitation carried out on [35S]methionine-labeled culture
supernatants. LTR228 produces an IgM, K antibody; the hybrid subclones secrete both an antitetanus IgG, K and the parent Ig. The IgG antibody binds to staphylococcus protein A with high affinity. We have used staphylococcus protein A affinity chromatography to purify milligram quantities of monoclonal antitetanus Ig. We have purified antibody from both serum-supplemented and serum-free cultures. Surface- Phenotype of Parent and Hybridoma Cells. B-cell lineage surface markers were measured on hybrid and LTR228 cells by cell sorter methods. It was hoped that one or more of the B-cell lineage surface markers might correlate with increased secretion of Ig. Fig. 7 shows plots of forward-angle light scattering (cell size) vs. logarithm fluorescence for surface Igs, DR, Leu-10 (a D-region-associated antigen) and B1 (a B-cell differentiation antigen) (21). When compared with LTR228, the hybrids express less BL, more DR, more Leu-10, and approximately equivalent amounts of surface Ig. The parent LTR228 also stained with several anti-y reagents, despite the fact that IgM was the only Ig internally labeled and immunoprecipitated (see Fig. 6). The plots also show the cloned hybrids to be more heterogeneous in size than the parent cell lines. -Hybrids- cloned by the cell sorter for increased or decreased expression of B1, DR, or Leu-10 antigens failed to secrete a level of Ig different from the parent (data not shown).
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FIG. 3. DNA histograms of parent LTR228 (left peak) to 12 antitetanus-producing hybridomas (right peaks). Note that all 12 are near tetraploid.
.A
Medical Sciences: Larrick et al.
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Proc. Natl. Acad. Sci. USA 80 (1983)
6379
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Clone FIG. 5. Production of specific antitetanus monoclonal antibodies by 12 representative clones. Values are for 106 cells at 24 hr of spent culture medium. 4. -, `.iol-
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FIG. 4. Representative metaphase chromosome spreads of our antitetanus hybridoma (A) and.parent cell line LTR228 (B).
Monoclonal Antibody Protects Mice from Lethal Tetanus Toxin Injection. Outbred Swiss-Webster mice (20 g) were injected intraperitoneally (0.5 ml) with various -doses of tetanus toxin mixed with serum-free medium conditioned by either hybridoma or LTR228 cells. Doses of toxin were 1-1,000 times the LD' . Results in Table2 show that the monoclonal antibody is protective in vivo against the lethal effects of tetanus toxin.
DISCUSSION Several workers have shown that EBV-infected B-cell lines can be used to produce human monoclonal antibodies (8, 9, 11, 22). Although human myeloma lines are available,- they have not been as useful in producing hybridomas as EBV-infected B-cell lines. Even under optimal conditions, human myeloma cell lines fuse with- lower frequency, grow more slowly, and are unstable. producers of Ig (unpublished data). We have developed an EBV-infected B-cell line that greatly facilitates production of human hybridomas. By using- this line we have produced and purified-a specific human monoclonal antibody, IgG,K, that protects mice against tetanus toxin. The cell line we developed produces hybrids with a high frequency (1 per 5 x 104 parent cells) and has been used to produce human monoclonal antibodies to other antigens (unpublished re-
suits). Using this technique, we have shown that.multiple clones are stable producers of antibody over a period of 11 months. We have been able to boost antibody production from less than 1 jig/ml to almost 10 pg per 106 cells per 24 hr. It is noteworthy that the parent line LTR228 and all antitetanus hybridomas clone with atieast 10% efficiency in soft agar and by limiting dilution. This characteristic allowed us to clone these cells frequently and to select for high producers by the reverse-plaque technique. Because the cells grow rapidly in soft agar, it is often possible to pluck positive clones after 3 days. We have also used the reverse-plaque technique to select for nonproducer cell lines, and initial experiments suggest these lines produce hybrids as efficiently as LTR228. We have constructed a ouabain/6-thioguanine-resistant derivative of LTR228 and have fused this line to EBV-transformed B cells secreting anti-blood group A antibody (unpublished results). These hybrids and cells from three successive subclones produce an IgM anti-blood group A Ig similar to the parent lymphoblastoid line. Kozbor et al. (11) have also used this technique to immortalize rare B-cell clones. The reasons why EBV-transformed lines (8, 9, 11, 22) such as LTR228 have to date proven superior to myeloma cell lines 1
2
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FIG. 6. Chain-specific immunoprecipitation of parent LTR228 (lanes 2) and antitetanus hybridoma (lanes 1). Reduced samples have-been resolved by NaDodS04/polyacrylamide gel electrophoresis. Anti-y precipitates the antitetanus Ig. Anti-,u precipitates parental IgM in supernatants from hybrid and LTR228. Anti-K precipitates both.antibodies from hybrid supernatant but only the parental Ig in the.case of LTR228. Anti-A fails to react with either hybrid or parent supernatant.
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DR
Leu-10
Bi
FIG. 7. Cell sorter plots of forward light scattering (vertical) vs. logarithm offluorescence (horizontal) for several B-cell lineage surface markers. (Left) LTR228. (Right) Hybrid.
for generation of human B-cell hybridomas are unclear. A rapid growth rate and a high cloning efficiency both in soft agar and by limiting dilution are important characteristics of LTR228. Selection for subclones that efficiently form heterokaryons ("fusibility") also improves the chances of successful hybridoma outgrowth. Tetraploid human hybrids are not inherently unstable, but certain differentiated functions including production of Igs are often lost after fusion (23, 24). The available human myeloma lines may be in a state of differentiation unable to form antibody-producing hybrids with more immature B-cell blasts. Although a myeloma-plasmacytoma parent might be expected to produce more Ig, our efforts to correlate surface B cell differentiation markers and Ig secretion rates have to date been unsuccessful (Fig. 7). However, the ease with which hybrids of LTR228 clone and form reverse plaques has permitted us to select for high-secreting clones approaching the production of mouse hybridomas. One of the antitetanus hybrids has been adapted to growth Table 2. Protection of mice against tetanus toxin
Mediumt Antitetanus hybrid Parent cell line 3/3 0 3/3 3/3 1 1/2 3/3 0/3 10 3/3 0/3 100 2/3 0/3 1,000 Results indicate surviving mice (at day 4)/total mice injected. * 1 unit = LD50.. to.5 ml of -spent medium was mixed with tetanus toxin and injected
Toxin, units*
intraperitoneally.
Proc. Natl. Acad. Sci. USA 80 (1983) in nude mice (unpublished results). Inoculation of 107 cells subcutaneously produces tumors within 3-4 weeks; After removal from the mouse and subsequent in vitro culture, this clone has permitted the production of ascites with a high titer of human monoclonal antibody. When nude mice were injected with adapted cells (2 x 107), ascites containing 0.5 mg of specific antibody per ml formed within 2 weeks. Production of purified antigen-specific monoclonal antibody in milligram quantities will make it possible to use human antibodies when murine products are either ineffective or immunogenic. Passively administered human antibodies can be used to treat hemolytic disease of the newborn (Rh), tetanus, rabies, hepatitis B, Gram-negative sepsis, and snakebite. Future uses may include radioimaging with tracer-labeled antibodies, toxin-antibody conjugates to modulate immunological diseases, and antibodies to eliminate toxins or drugs. Human proteins should be less antigenic and, in some cases, more specific, and they should have longer in vivo half-lives because of the correct species-specific carbohydrate side chains and Fc sequences. Human Fc sequences may also augment the bioactivity of antibodies binding to macrophages or lymphocytes in vivo. We gratefully acknowledge the expert technical assistance of Hanna Hutchins and Howard Weintraub. Bradley Dyer and David Buck provided helpful discussions. We are indebted to Dr. Wally Laird for his in vivo testing of our antibodies and to Dr. Jon Kosek for the scanning electron microscope photographs. Joan Murphy and Dianne Jacobs gave superb secretarial help. Tim Culp prepared the graphics. 1. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495-497. 2. Olsson, L. & Kaplan, H. S. (1980) Proc. Nati. Acad. Sci. USA 77, 5429-5431. 3. Sikora, K., Alderson, T., Phillips, J. & Watson, J. V. (1982) Lancet i, 11-13. 4. Lane, H. C., Shelhamer, J. H., Mostowski, H. S. & Fauci, A. S. (1982) J. Exp. Med. 155, 333-338. 5. Butler, J. L., Lane, H. C. & Fauci, A. S. (1983)J. Immunol. 130, 165-168. 6. Steinitz, M., Klein, G., Koskimies, S. & Makela, 0. (1977) Nature (London) 269, 420-422. 7. Kozbor, D. & Roder, J. C. (1981) J. Immunol. 127, 1275-1280. 8. Croce, C. M., Linnenback, A., Hall, W., Steplewski, Z. & Koprowski, H. (1980) Nature (London) 288, 488-489. 9. Chiorazzi, N., Wasserman, R. L. & Kunkel, H. G. (1983)J. Exp. Med. 156, 930-935. 10. Kozbor, D., Roder, J. C., Chang, T. H., Steplewski; Z. & Koprowski, H. (1982) Hybridoma 1, 323-328. 11. Kozbor, D., Lagarde, A. E. & Roder, J. C. (1982) Proc. Natl. Acad. Sci. USA 79, 6651-6655. 12. Levy, J. A., Viroloinen, V. & Defendi, V. (1968) Cancer 22, 517524. 13. Madsen, M. & Johnson, H. E. (1979)J. Immunol. Methods 27, 6174. 14. Brahe, C. & Serra, A. (1981) Somatic Cell Genet. 7, 109-115. 15. Engvall, E. & Perlmann, P. (1971) Immunochemistry 8, 871-880. 16. Weber, K. & Osborn, M. (1969)J. Biol. Chem. 244, 4406-4411. 17. Taylor, I. W. & Milthorpe, B. K. (1980)J. Histochem. Cytochem. 28, 1224-1232. 18. Loken, M. R. & Stall, A. M. (1982)J. Immunol. Methods 50, R85R112. 19. Gronowicz, E., Coutinho, A. & Melchers, F. (1976) Eur. J. Immunol. 6, 588-590. 20. Kozbor, D., Dexter, D. & Roder, J. C. (1983) Hybridoma 2, 716. 21. Stashenko, P., Nadler, L. M., Haroy, R. & Schlossman, S. F. (1980) J. Immunol. 125, 1678-1685. 22. Shoenfeld, Y., Rauch, J., Massicotte, H., Datta, S. K., AndreSchwarts, J., Stollar, B. D. & Schwartz, R. S. (1983) N. Engl. J. Med. 308, 414-420. 23. Bengtsson, B. D., Nabholz, M., Kennett, R. H. & Bodmer, W. F. (1975) Somatic Cell Genet. 1, 41-64. 24. Stanbridge, E. J.,, Der, C. J., Roerson, C. J., Nishimi, R. Y., Peehl, D. M., Weissman, B. E. & Wilkinson, J. E. (1982) Science 215, 252-259.
