Spatial Localization of Allospecific HLA Class I Epitopes by Antibody Blocking Studies

Abstract

A series of polymorphic and monomorphic monoclonal HLA class I antibodies was used in competitive blocking studies to explore the spatial relationships of the epitope sites recognized. This study asks whether four different polymorphic determinants present on A- and B-locus alleles have the same spatial position with respect to the epitopes recognized by the monomorphic antibodies (60.5, 71D9, H1DE, BB5). Cells (a B lymphoid cell line) were incubated with saturating quantities (approximately 2.0 µg/2 × 10⁵ cells) of “blocking” antibody (at 4°C). In the second step, a limiting amount of a fluorescein-isothiocyanate (FITC)-conjugated monoclonal was added without washing. Staining intensity was quantitated on an FACS, and percent blocking was calculated from the change in modal fluorescence in the presence and absence of blocking antibody. Maximal blocking was assessed by blocking each FITC-conjugated antibody by itself. Each antibody was tested as an FITC-conjugated probe and as a blocking antibody (unconjugated) on a single B-lymphoblastoid cell line (B-LCL) which reacted with all four polymorphic antibodies. The four polymorphic antibodies were P8.1, anti-B8; P5.3 and P7.1, anti-B27+; and P5.1, anti-A2+. None of the polymorphic antibodies except P5.3 and P7.1 blocked one another.