This paper describes a simple protocol for the efficient generation of large numbers of human monoclonal antibody-producing cells. This system is based on initial limiting-dilution culture after Epstein-Barr virus exposure of highly enriched precursors selected from peripheral blood mononuclear cells. Precursors can be enriched by using rosetting or panning approaches. Antibodies to erythrocytes, a mouse mammary carcinoma, DNA, and sperm antigens, produced without any deliberate immunization, are described. Large-scale human monoclonal antibody production may be facilitated by a combination of this protocol with a human cellular fusion system. For efficient precursor analysis and short-term (2 months or more) monoclonal antibody production, however, the system described here may be sufficient.Two general systems-the hybridoma approach (1-3), and the Epstein-Barr virus (EBV) transformation approach (4-6)-have been used for immortalizing human antibody-producing cells. A major obstacle to date, however, to the efficient production of human monoclonal antibodies of predetermined specificity, using either of these techniques, has been the problem of procuring a sufficient number of cells of the desired specificity for statistically probable immortalization.Thus, for either the hybridoma approach (7,8) or the EBV approach (6), relatively heroic in vivo immunization schedules and time constraints after priming have been considered necessary to yield sufficient (re)circulating precursors. Alternatively, tonsillar cells have been the tissue of choice for in vitro immunization prior to fusion (9); also, draining lymph nodes from solid cancer-compromised patients (10) or mononuclear cell infiltrates of the tumor mass (11)
The specificity repertoire of B lymphocytes from 14 multiple myeloma patients has been studied using the technique of Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) coupled with clonal analysis by limiting dilution. We find that up to 100% of the B cells from myeloma patients undergoing EBV transformation secrete IgM specific for determinants on the F(ab')2 region of autologous and/or heterologous monoclonal immunoglobulin. In normal individuals 0.02-0.73% of the transformed B cells secrete IgM specific for F(ab')2 determinants. Two patients with monoclonal gammopathy of undetermined significance had only a weak reactivity to F(ab')2 fragments. The number of anti-F(ab')2 B cells was up to 145-fold greater in patients than in normal donors. The majority of antibodies from patient clones recognized determinants shared among 3-12 different F(ab')2 fragments, whereas those originating from normal donor B cells saw determinants expressed on only one or two of the panel of test F(ab')2 fragments. There was a preference for autologous M components and a high proportion of antiidiotypic reactivity in five of eight patients so analyzed. We speculate that these findings indicate the existence of an anti-F(ab')2 immunoregulatory network mediating patient immunodeficiency network mediating patient immunodeficiency, thereby creating an abnormality that may enable the progression of multiple myeloma.
A monoclonal antibody-producing hybridoma cell line has been raised against merozoites of Babesia divergens. This antibody is strongly reactive against merozoites in an enzyme-linked immunosorbent assay (ELISA) and is also positive in an indirect fluorescent antibody test (IFAT) on preparations of live merozoites. The antibody recognizes an antigen, of molecular weight (Mr) 50-60 K by Western blot analysis on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Merozoite neutralization assays show that the antibody significantly inhibits merozoite invasion of bovine erythrocytes.
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