Human monoclonal antibody to purified protein derivative of tuberculin produced by hybrids constructed with Epstein‐Barr virus‐transformed B lymphocytes and mouse myeloma cells

Abstract

A method of producing human monoclonal antibody by combining somatic cell hybridization technology with the capability of Epstein-Barr virus (EBV) to transform human B lymphocytes is described. Peripheral blood lymphocytes from a donor with positive tuberculin skin test reaction were transformed by EBV and then tested for antibody production to mycobacterial purified protein derivative (PPD) by an enzyme-linked immunosorbent assay. Two EBV-transformed lymphoblastoid cell lines making IgM antibodies to PPD were obtained. One of these cell lines was fused by polyethylene glycol with a murine hypoxanthine-guanine phosphoribosyl transferase-deficient myeloma cell line that had been selected for resistance to ouabain. The human-mouse hybrids were selected in ouabain-containing HAT medium and 11 heterohybridomas producing IgM antibody to PPD were obtained. One of these was cloned by limiting dilution with efficiency at least 20-fold higher than parent EBV-transformed cell line. Heterohybridoma subclones reached levels of IgM antibody as high as 75.0 micrograms/ml of culture medium, whereas IgM production of EBV-transformed B cell clones ranged between 3.0 and 4.0 micrograms/ml.

Highly efficient procedure for production of human monoclonal antibodies: Establishment of hybrids between Epstein-Barr virus-transformed B lymphocytes and heteromyeloma cells by use of GIT culture medium.

“…The fusion efficiency in the GIT culture system was much better than that reported previously (Garzelli et al 1986;Kozbor et al 1986;Roder et al 1986). …”

Interleukin-1-Like Activity Produced by Hybrids Constructed with Epstein-Barr-Virus-Transformed Human B Lymphocytes and Mouse Myeloma Cells

“…Approximately 2 x |04 cells exposed to EBV were seeded in 0.2 ml of culture medium in each well of a 96-well plate (Sterilin Ltd., Feltham, UK). After 3 weeks of incubation at 37°C in a humidified atmosphere in the presence of 5% CO,, the cultures were screened for lgM and IgG antibody production to purified protein derivative of tuberculin (PPD) by an en zyme-linked immunosorbent assay (Elisa), as previously re ported [12).…”

“…We previously reported, however, that when EBV-transformed B cells are fused with mouse myeloma cells, the resulting human-mouse heterohy brids can be cloned more efficiently and produce larger amounts of human monoclonal antibody [12], It is therefore of interest to know whether humanmouse hybrid cells constructed with EBV-trans formed B lymphocytes and mouse myeloma cells also maintain the capacity of secreting biologically im portant factors, other than human monoclonal anti body. In this paper, we report that one of such hetero hybrids, coded HY 05-20, temporarily secreted a fac tor resembling human IL-I in biochemical and bio logical properties.…”

Epstein-Barr Virus-Transformed Human B Lymphocytes Endocytose Autologous and Heterologous Red Blood Cells

“…These B-cell lines, coded GL23 and RB, were polyclonal and secreted IgM, IgG, and IgA, as tested by ELISA. Cloned EBVtransformed B cells, coded BA-G2, isolated in the course of previ ous experiments from a tuberculin skin test-positive healthy donor and producing IgM human monoclonal antibody to purified pro tein derivative of tuberculin [3] were also used. All the B-cell lines employed in this study expressed Epstein-Barr nuclear antigen (EBNA) [14] and did not secrete agglutinating or complement-fix ing antibody to human autologous (when available) and allogeneic (0, Rh-) RBC, as well as to sheep or mouse RBC.…”