RESUMO
We describe a method for production of human monoclonal antibody by a combination of the capacity of Epstein-Barr virus (EBV) to transform human B lymphocytes with somatic cell hybridization, in which a new culture medium, GIT, is used. The transformed B cells from wells positive for anti-purified protein derivative (PPD) fused with a (mouse x human) heteromyeloma line (deficient in hypoxanthine-guanine phosphoribosyl transferase and ouabain-resistant) that had been cultured in GIT medium (Kudo et al. 1987) supplemented with geneticin (antibiotic G418) before cell fusion. The hybrids were selected in GIT medium containing HAT and ouabain (GIT-HAT-O) and cloned by limiting dilution technique by use of GIT medium. According to our method, we obtained higher fusion frequency (1/5.5 x 10(3) vs. 1/1.1 x 10(4)) and higher cloning efficiency (43.3-56.7% vs. 3.3-13.3%) compared with the regular method which used the culture medium containing fetal bovine serum (FBS). Six hybrid clones were consequently obtained and characterized. They produced large amount of specific antibodies (35-170 micrograms/ml) in GIT medium, while establishment of hybrid clones producing specific antibodies by the regular method was unsuccessful. This method will be applicable to any kind of human monoclonal antibody production.
