BIOCHEMICAL DEFINITION OF DLA-A AND DLA-B GENE-PRODUCTS BY ONE-DIMENSIONAL ISOELECTRIC-FOCUSING AND IMMUNOBLOTTING

The serologically defined canine MHC gene products (DLA-A and DLA-B) w ere investigated by one-dimensional isoelectric focusing after deterge nt phase separation, and immunoblotting (1D-IEF). The animals studied (n = 36) represented three different breeds. Since DLA-A and DLA-B ant igens are MHC class I and class II molecules, respectively, cross-reac tive polyclonal antibodies specific for human class I or class II mole cules were utilized to visualize the protein pattern. The relative pos itions of the bands were correlated with the DLA-A and DLA-B antigens. In most cases DLA antigens of identical serological type did not diff er in their 1D-IEF pattern except for DLA-A9 and the serologically und efined DLA-B gene product DLA-B 'blank'. For DLA-A9, two subtypes (A9. 1 and A9.2) were identified in Beagle and associations of A9.1 with th e haplotype A9,B4 and of A9.2 with A9,B6 were seen. In contrast, the h aplotype A9,B6 in Labradors was associated with A9.1. Whereas all six serologically defined DLA-A antigens possessed distinct isoelectric po ints (IEP), not all DLA-B antigens could be differentiated by 1D-IEF: B5 as well as B13 coband with one B 'blank' specificity (BE1). Further more, a second B 'blank' (BE2) could be identified by 1D-IEF, underlin ing the advantage of this technique in the immunogenetic analysis of D LA gene products in dogs,