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.

Abstract

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 103 vs. 1/1.1 X 104) 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 (35-170pg/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. human monoclonal antibody ; GIT medium ; cell fusion

Production of a Human Monoclonal Antibody to a Synthetic Peptide by Active In Vivo Immunization Using a SCID Mouse Grafted with Human Lymphocytes.

“…The selective medium for hybridoma production was composed of GIT medium (Kudo et al 1987) supplemented with 2-mercaptoethanol (2 X 10-5 M), Lglutamine (2 mM), dihydroxyethylglycine (1.8 mg/ml) (Yabe et al 1986; Kudo et al 1988), IL-6 (6 ng/ml, GIBGO Life Technologies Inc., Gaithersburg, MD, USA) and 1/50 volume of concentrated HAT solution (Flow Lab., Irvin, Scotland).…”

“…The spleen cells, mainly consisting of human lymphoid cells, were washed with a serum-free RPMI 1640 medium three times, then mixed with washed SHM-D33 heteromyeloma cells at a ratio of 5 :1. The cell fusion was performed using PEG 1000 (Kudo et al 1988). Then, the fused cells were suspended in a warm GIT medium supplemented with 6 ng/ml IL-6 at the concentration of 10 X 104/ml myeloma cells and 50 X 104/ml spleen cells.…”

New Strategies to Establish Human Monoclonal Antibodies.

“…(Mouse x human) heteromyeloma (SHM-D33) and GIT culture medium used for cell fusion experiments (Kudo et al 1988). …”

“…Approximately 1 x 106 EBV-lymphoblastoid cells and an equal number of myeloma cells were fused using PEG as described previously (Kudo et al 1988). In order to detemine fusion frequency, various numbers of fused cells were put in each well of a 96-well plate.…”