Efficient generation in vitro, from human peripheral blood cells, of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization.

Abstract

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)

Mutations in the RB1 gene and their effects on transcription.

“…RB tumor cell lines (14,15) and Epstein-Barr virus (EBV)-transformed lymphocytes (34) were grown as described previously. Fresh surgical RB tumor specimens and cultured cells were lysed in guanidine thiocyanate and layered on a CsCl gradient.…”

B-Lymphocytes from melanoma patients and normal individuals react with melanoma cells but also with irrelevant antigens

“…It is known that antibodies reactive with tumour-associated and normal tissue antigens occur in healthy individuals (Houghton et al, 1980). Furthermore, monoclonal antibodies reactive with tumours and established autoantigens have previously been generated from unimmunised human individuals (Shoenfeld & Witz, 1986;Winger et al, 1983). Such findings have given rise to speculation that these autoantibodies play a role in the regulation of the idiotypic network of both B-and T-lymphocytes (Schoenfeld & Witz, 1986).…”

Immunological analysis of the specificity of the autologous humoral response in breast cancer patients

“…Thus, for example, clones of B lymphocytes secreting antibodies reactive with antigens such as DNA and thyroglobulin have been isolated from normal individuals (Winger et al, 1983). The significance of these anti-tumour antibodies in the defence of the patient against her own tumour may well be limited since even specific antibody may result from a secondary response to shed tumour antigens rather than a primary defence mechanism.…”