Cell, Vol. 14,9-20, May 1978, Copyright 0 1978 by MIT Production of Monoclonal Antibodies to Group A Erythrocytes, HLA and Other Human Cell Surface Antigens-New Tools for Genetic Analysis C. J. Barnstable,* W. F. Bodmer,* G. Brown,t G. Galfre,* C. Milstein,$ A. F. Williamst and A. Ziegler* *Genetics Laboratory University of Oxford South Parks Road Oxford OX1 3QU, England TMRC Immunochemistry Unit Department of Biochemistry University of Oxford Oxford OX1 3QU, England *MRC Laboratory of Molecular Biology Hills Road Cambridge CB22QH, England Summary Antibody-secreting hybrid cells have been de- rived from a fusion between mouse myeloma cells and spleen cells from a mouse immunized with membrane from human tonsil lymphocyte preparations. Hybrids secreting antibodies to cell surface antigens were detected by assaying cul- ture supernatants for antibody binding to human tonsil cells. Six different antibodies (called W6/1, 126,132, 134, /45 and /46) were analyzed. These were either against antigens of wide tissue distri- bution (W6/32, /34, /45 and /46) or mainly on erythrocytes (W6/1 and W6/28). One of the anti-erythrocyte antibodies (W6/1) detected a polymorphic antigen, since blood group A, and Az erythrocytes were labeled while B and 0 were not. Antibodies W6/34, /45 and /46 were all against antigens which were mapped to the short arm of chromosome 11 by segregation analysis of mouse-human hybrids. Immunopre- cipitation studies suggest that W6/45 antigen may be a protein of 16,000 dalton, apparent molecular weight, while W6/34 and /46 antigens could not be detected by this technique. Antibody W6/32 is against a determinant common to most, if not all, of the 43,000 dalton molecular weight chains of HLA-A, B and C antigens. This was established by somatic cell genetic techniques and by immu- noprecipitation analysis. Tonsil leucocytes bound 370,000 W6/32 antibody molecules per cell at saturation. The hybrid myelomas W6/32 and W6/34 have been cloned, and both secrete an IgG, antibody. W6/32 cells were grown in mice, and the serum of the tumor-bearing animals contained >lO mg/ml of monoclonal antibody. The experiments established the usefulness of the hybrid myeloma technique in preparing mon- ospecific antibodies against human cell surface antigens. In particular, this study highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymor- phisms. Introduction Rodent-human somatic cell hybrids tend to un- dergo an early more or less random loss of human chromosomes and then become relatively kary- atypically stable (Weiss and Green, 1967; Bodmer 1971; McKusick and Ruddle, 1977). Thus human genes can be assigned to particular chromosomes by correlating the presence or absence of a partic- ular gene product either with a cytologically iden- tified chromosome or with a previously assigned marker in a series of such hybrids. Cell surface antigens have a number of advantages as markers in somatic cell genetics. First, they can be assayed quickly and quantitatively on a small number of cells. Second, they can be used to select for or against hybrids carrying particular chromosomes either by use of the fluorescence-activated cell sorter (Loken and Herzenberg , 1975) or by comple- ment-mediated cytotoxicity (Buck and Bodmer, 1974). A panel of monospecific antibodies to cell surface antigens would be particularly useful in such selective techniques, as well as for the rapid definition of the human chromosomal content of hybrids. The main difficulty is in producing suitable antibodies. At present, the only antigens suitable for genetic studies which can be assayed at the surface of human cells are antigens of the major histocompatibility (HLA) region, which is on chro- mosome 6 (van Someren et al ., 1974; Jones et al., 1976; Francke and Pellegrino, 1977); prmicro- globulin on chromosome 15 (Goodfellow et al., 1975); tissue-common antigens on chromosome 11 (Buck and Bodmer, 1974; Kao, Jones and Puck, 1977); an X-linked B cell antigen, SA-X (Buck and Bodmer, 1976); and an antigen on chromosome 7 (Aden and Knowles, 1976). A method is now available for producing mono- specific antibodies which involves the fusion of antibody-producing and myeloma cells to derive hybrid cell lines secreting monoclonal antibody. This system, developed for the production of mon- oclonal antibodies to sheep erythrocytes (Kohler and Milstein, 1975, 1976), has been extended to the production of antibodies to a variety of hap- tens, proteins and alloantigens (Galfre et al., 1977; Koprowski, Gerhard and Croce, 1977; Lemke et al., 1977; Pearson et al., 1977), as well as to cell surface antigens of rat cells (Williams, Galfre and Milstein, 1977). In the latter experiments, mice