Characterization of class I bovine lymphocyte antigens (BoLA) by one-dimensional isoelectrofocusing

BoLA class I antigens were characterized in a group of British and Dutch Friesian cattle by one-dimensional isoelectric focusing (1D-IEF) and the results compared with serology using alloantisera and microcytotoxicity. For IEF analysis, non-stimulated peripheral blood mononuclear cells (PBM) were metabolically labelled with 35S methionine, detergent lysates were prepared and MHC molecules precipitated with the monoclonal antibodies (mAbs) W6/32 or B1.1G6. Staphylococcus protein A precipitated antigens were separated on a vertical slab gel under denaturing conditions. The banding patterns seen for the W6/32 precipitated molecules obtained by 1D-IEF were compared with the serological specificities. Characteristic banding patterns were observed for most serological specificities as well as workshop undefined haplotypes. These patterns were seen both in families and the outbred population. In families IEF haplotypes segregated with serotypes. Additional MHC class I products were suggested by variable banding patterns for different w10 haplotypes and when using the different mAbs. A pulse chase experiment with a w12 animal also suggested more than one expressed product. The w2 and w5 specificities were not precipitated by either W6/32 or B1.1G6 and w6.2 and w6.4 were precipitated by W6/32 but not by B1.1G6. These results show that 1D-IEF is useful for BoLA typing. For the characterization of class I antigens, however, much depends on the mAbs used.