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) were investigated by one-dimensional isoelectric focusing after detergent phase separation, and immunoblotting (1D-IEF). The animals studied (n = 36) represented three different breeds. Since DLA-A and DLA-B antigens are MHC class I and class II molecules, respectively, cross-reactive polyclonal antibodies specific for human class I or class II molecules were utilized to visualize the protein pattern. The relative positions of the bands were correlated with the DLA-A and DLA-B antigens. In most cases DLA antigens of identical serological type did not differ in their 1D-IEF pattern except for DLA-A9 and the serologically undefined 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 the haplotype A9,B4 and of A9.2 with A9,B6 were seen. In contrast, the haplotype A9,B6 in Labradors was associated with A9.1. Whereas all six serologically defined DLA-A antigens possessed distinct isoelectric points (IEP), not all DLA-B antigens could be differentiated by 1D-IEF:B5 as well as B13 coband with one B 'blank' specificity (BE1). Furthermore, a second B 'blank' (BE2) could be identified by 1D-IEF, underlining the advantage of this technique in the immunogenetic analysis of DLA gene products in dogs.