Generation and Characterization of Monoclonal Antibodies Against the Intracellular Domain of Hemidesmosomal Type XVII Collagen (1)
Type XVII collagen, also termed bullous pemphigoid antigen 2 (BPAG2) or bullous pemphigoid antigen 180 (BP180), was first described as an autoantigen in patients with bullous pemphigoid and herpes gestationis. BP180/type XVII collagen is a transmembrane hemidesmosomal component with an unusual type II orientation with its N-terminus intracellularly located and with a large extracellular domain that spans the lamina lucida of the DEJ. Each collagen XVII molecule is a homotrimer consisting of three identical 180-kDa 1(XVII) chains. Ultrastructurally, type XVII collagen has a globular head (intracellular domain), a central rod (extracellular portion spanning the lamina lucida) and a flexible tail within lamina densa of the DEJ.
cDNA sequences coding for 4 overlapping fragments of the intracellular domain of human type XVII collagen were cloned into a prokaryotic expression vector and expressed in E. coli. The proteins, purified by gluthathione-affinity chromatography, migrated at 40, 45, 44, and 39 kDa when separated by SDS-PAGE
The immunized animals developed high levels of serum antibodies both recognizing the recombinant GST-fusion proteins and staining the basement membrane of human skin as revealed by ELISA and IF microscopy, respectively. Antibodies produced by one stable hybridoma cell line designated V58 were further characterized by IF microscopy on human skin sections using FITC-conjugated monoclonal antibodies to murine IgG subclasses. The isotype of this antibody is IgG2a.
This antibody recognized a 180 kDa band in extracts of cultured keratinocytes and reacted with fragment C encompassing aa 234-398 of the sequence of type XVII collagen.
