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 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-alpha (TNFalpha) 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 were assayed for anti-TNFalpha activity utilizing ELISA. Two IgG1/kappa anti-TNFalpha antibodies and two IgM/kappa anti-TNFalpha 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 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 be composed of one to three different genes. These data support the conclusion that the cell clone is oligoclonal at the molecular level.
Takekoshi
M
M
Department of Molecular Life Science Tokai University School of Medicine, Bohseidai, Isehara 259-1193, Japan. mtakekos@is.icc.u-tokai.ac.jp
Maeda
F
F
Nagatsuka
Y
Y
Aotsuka
S
S
Ono
Y
Y
Ihara
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D008969:Molecular Sequence Data; D043025:Oligoclonal Bands; D016415:Sequence Alignment; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007136:Immunoglobulins; D043025:Oligoclonal Bands; D014409:Tumor Necrosis Factor-alpha