Human Hybridomas and Monoclonal Antibodies by Jerry W. Shay (auth.), Edgar G. Engleman, Steven K. H.

By Jerry W. Shay (auth.), Edgar G. Engleman, Steven K. H. Foung, James W. Larrick, Andrew A. Raubitschek (eds.)

Soon after Kohler and Milstein defined using somatic mobilephone hybridization for the construction of murine monoclonal antibodies of wanted specificity, this particularly easy method turned largely utilized. certainly, construction of murine monoclonal antibodies is now thought of regimen via immunologists and nonimmunologists alike. besides the fact that, as heterologous proteins, mouse monoclonal antibodies have one significant challenge: they're immunogenic in guy and, for that reason, their use in vivo is significantly constrained. An noticeable strategy to this challenge is to provide human hybridomas with a similar thoughts used for the creation of rodent hybrids. regrettably, the historical past of human hybridomas has been marked by means of noticeable and sometimes exasperating tech­ nical difficulties, and the 1st stories of hybrids secreting human immu­ noglobulin of wanted specificity didn't seem till 1980. those studies have been met with preliminary enthusiasm, however it quickly grew to become obvious that whereas human lymphocytes could be fused, their frequency, point of Ig synthesis, and balance have been such that creation of human antibodies with this technique used to be neither regimen nor sensible. still, a adequate variety of investiga­ tors continued, and through the following five years particularly effective B-cell fusion companions in addition to greater equipment of Epstein-Barr virus transformation have been constructed. iteration of human T -T hybrids has additionally been completed, even if difficulties of chromosomal balance stay a considerable situation, extra so than with B-cell lines.

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Plenum Press, New York, pp. 81-100. HUMAN HYBRlDOMAS AND MONOCLONAL ANTIBODIES 19 Jongkind, j. , 1979, Isolation of human fibroblast heterokaryons with two-color flow sorting (FACS II), Exp. Cell Res. 120:444-448. Keeler, P. , 1977, A fluorescence enhancement assay of cell fusion,I Cell Sci. 28:167-177. , 1975, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495-497. Larrick, j. , and Buck, D. , 1984, Practical aspects of human monoclonal antibody production, Bio Techniques 1:6-14.

Polykaryons containing more than two nuclei are only rarely viable, and, when coupled with the rate-limiting step of obtaining sufficient numbers of sensitized lymphocytes, cell fusion proct>dures may be of critical importance. Most fusion protocols recommend using a higher ratio of spleen to myeloma cells (10:1) and plating out the fusion products in a large number of multiwell dishes containing HAT medium. , 1983) and to increase the number of these cells by inducing cell growth as described in the previous section.

The lymphocytes and Iymphoblastoid cell lines were obtained from peripheral blood of donors immunized with TT or from colorectal carcinoma patients. bSecreted Ig was quantitated by a solid-phase ELISA system. " Less differentiated cells with small amounts of RER, moderately developed Golgi, and extended nuclear chromatin were considered "Iymphoblastoid" (LCL). dDetermined by limiting dilution. 'Human hybridomas obtained by fusion of KR-4 or KR-12 with Iymphoblastoid cell line B6 were injected subcutaneously (sc) into irradiated (350 R) or unirradiated BALB/c nude mice (107 cells per mouse).

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