Cloning and Expression of Human Anti-Tumor Necrosis Factor-a Monoclonal Antibodies from Epstein-Barr Virus Transformed Oligoclonal Libraries

• Published in: Journal of biochemistry (Tokyo) Vol. 130; no. 2; pp. 299 - 303
• Main Authors: Takekoshi, Masataka, Maeda, Fumiko, Nagatsuka, Yasuko, Aotsuka, Satoshi, Ono, Yasushi, Ihara, Seiji
• Format: Journal Article
• Language: English
• Published: Oxford University Press 01-08-2001
• Subjects: EBV; Fab; human monoclonal antibody; oligo clone; TNFα
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a002986

Peripheral blood was obtained from a healthy human volunteer and transformed 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-α (TNFα) activity. RNA from two positive clones was applied to RT-PCR using antibody-specific primers, and the light (κ and λ) and heavy chain genes (γ and μ) were cloned into the plasmid vector pFabl-His2. The antibodies produced in Escherichia coli as Fab fragments were assayed for anti-TNFα activity utilizing ELISA. Two IgGl/kappa; anti-TNFα antibodies and two IgM/κ anti-TNFα antibodies were isolated. 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 ELJSA-testing. Ten clones randomly selected from light (κ and μ) and heavy (γ and μ) chain genes in the oligoclonal cell library 1D5 were se-quenced, and each gene (κ, λ, γ, and μ) was found to be composed of one to three different genes. These data support the conclusion that the cell clone is oligoclonal at the molecular level.