Requirements for the establishment of high-titered human monoclonal antibodies against tetanus toxoid using the Epstein-Barr virus technique.

Abstract

Epstein-Barr Virus (EBV) has been used in other laboratories to “immortalize” human B cell populations producing antibodies against a variety of haptens and antigens. In the present study we investigated the conditions necessary to achieve 100-fold higher amounts of stable monoclonal anti-tetanus toxoid antibody production than previously achieved. Peripheral blood mononuclear cells were enriched for B cells (> 85 to 90% slg+) by depletion of E+ cells and adherent monocytes. B cells from a multiply immunized (4 times) donor taken 2 to 3 wk after the last injection of tetanus toxoid (TT) contained the highest frequency of TT-binding cells and produced the greatest amount of anti-TT antibody 8 days after transformation with EBV. Antigen-binding cells were detected by biotinylated TT followed by FITC-conjugated avidin, and antibody was measured in culture supernatants with an ELISA technique using an affinity-purified human anti-TT immunoglobulin as standard. The method of preselecting antigen-binding cells prior to EBV transformation was critical. Negative preselection by removal of cells not binding antigen was most successful. B cells were allowed to bind TT and cap for 6 hr, after which time they had shed most of their surface Ig receptors. The non-TT-binding, slg+ cells were removed by rosetting with SRBC coupled to the F(ab′)2 portion of rabbit anti-human Ig. This technique resulted in a 6-fold increase in TT binding B cells and a 3-fold increase in anti-TT antibody production. Positive selection of TT-binding cells by rosetting with TT-coupled SRBC or by sorting “bright” (strongly binding) cells on the FACS was unsuccessful. Only a minority (2%) of the EBV-infected cells secreted antibody, as detected in a PFC assay, and bulk cultures quickly declined to 0 levels of anti-TT antibody production by 10 wk. Cloning of early cultures by limiting dilution on irradiated PBL feeder layers (cloning efficiency 30%) was necessary to obtain 3 clones producing relatively high amounts (1000 ng/ml) of anti-TT antibody of the IgM-kappa class. The monoclonality of this antibody was confirmed by isoelectric focusing. These clones have been stable to the present time (>6 mo) and were sub-cloned. In summary, we have outlined the immunization, preselection, and cloning techniques necessary to optimize the establishment of stable, long-term B cell clones producing relatively high amounts of anti-TT antibody. These techniques should be applicable to other protein antigens.