Further analyses of epitopes for human monoclonal anti-basement membrane zone antibodies produced by stable human hybridoma cell lines constructed with epstein-barr virus transformants

Takashi Hashimoto, Masayuki Amagai, Tamotsu Ebihara, Shinobu Gamou, Nobuyoshi Shimizu, Takuichi Tsubata, Akira Hasegawa, Keizaburo Miki, Takeji Nishikawa

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Abstract

We previously established Epstein-Barr virus (EBV)-transformed bullous pemphigoid (BP) patient lymphoblastoid cell lines, which produced human monoclonal anti-basement membrane zone antibodies. In the present study, we established two independent human-human hybridomas by fusion of these EBV transformants with a human B-cell line. These hybridomas, designated 5E-HY-4B and 10D-HY-8B, were very stable and showed a high yield of monoclonal antibody (MoAb) secretion. Each cell line was tetraploid and showed combined rearranged segments of immunoglobulin heavy-chain gene derived from both an EBV transformant and a parent cell. Immunoblot analysis showed that the 5E-HY-4B MoAb recognized the 230-kDa BP antigen but that the 10D-HY-8B MoAb did not show any reactivity. In contrast, both MoAbs precipitated the 230-kDa BP antigen with immunoprecipitation. These results indicate that the two MoAbs reacted with different epitopes on the 230-kDa BP antigen: a continuous epitope for the 5E-HY-4B MoAb and a conformation-dependent epitope for the 10D-HY-8B MoAb. This speculation was confirmed at the molecular level by the result that the fusion protein produced by a partial cDNA for the 230-kDa mouse BP antigen reacted with the 5E-HY-4B MoAb but not with the 10D-HY-8B MoAb. Furthermore, the study of the reactivity with fusion proteins of a series of deleted clones restricted the epitope for the 5E-HY-4B MoAb within the region with 114 amino acid residues in the C-terminal domain of the 230-kDa BP antigen.

Original languageEnglish
Pages (from-to)310-315
Number of pages6
JournalJournal of Investigative Dermatology
Volume100
Issue number3
DOIs
Publication statusPublished - 1993 Mar

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Dermatology
  • Cell Biology

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