Cloning and expression of human anti-tumor necrosis factor-alpha monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries

Peripheral blood was obtained from a healthy human volunteer and transforme d with Epstein-Barr virus (EBV). This produced an oligoclonal cell library in culture medium that was screened by ELISA for anti-human tumor necrosis factor-alpha (TNF alpha) activity. RNA from two positive clones was applied to RT-PCR using antibody-specific primers, and the light (kappa and lambda ) and heavy chain genes (gamma and mu) were cloned into the plasmid vector pFab1-His2. The antibodies produced in Escherichia coli as Fab fragments we re assayed for anti-TNF alpha activity utilizing ELISA. Two IgG1/kappa anti -TNF alpha antibodies and two IgM/kappa anti-TNF alpha antibodies were isol ated. DNA sequence analysis showed that the VL and VH gene families of IgM and IgG were the same. Both the antibodies showed almost the same activity on ELISA-testing. Ten clones randomly selected from light (kappa and lambda ) and heavy (gamma and mu) chain genes in the oligoclonal cell library 1D5 were sequenced, and each gene (kappa, lambda, gamma, and mu) was found to b e composed of one to three different genes. These data support the conclusi on that the cell clone is oligoclonal at the molecular level.