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.
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.
Hashimoto
T
T
Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.
Amagai
M
M
Ebihara
T
T
Gamou
S
S
Shimizu
N
N
Tsubata
T
T
Hasegawa
A
A
Miki
K
K
Nishikawa
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001485:Basement Membrane / Q000276:immunology*; D002460:Cell Line; D002472:Cell Transformation, Viral; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D006825:Hybridomas / Q000382:microbiology; D015151:Immunoblotting; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins