BIOTINYLATION OF CELL-SURFACE MHC MOLECULES - A COMPLEMENTARY TOOL FOR THE STUDY OF MHC CLASS-II POLYMORPHISM IN CATTLE

Biotinylation of cell surface proteins is often used as an alternative to radioactive labelling procedures, but very little is known about t he labelling of the different allelic products of polymorphic antigeni c systems. In this report we demonstrate that NHS-LC-biotin labels bov ine MHC class II molecules with different efficiencies for several all elic forms of this polymorphic system compared to conventional metabol ic labelling with [S-35]methionine. This was shown after immuno-precip itation and one-dimensional isoelectric focusing (1D-IEF). The avidity of the monoclonal antibody Bo139 (ct-bovine MHC class II) was not aff ected after in situ biotinylation of bovine PBMC, as revealed by flow cytometric analyses, immunoblotting after SDS-PAGE and immunoprecipita tion. The biotinylation did not affect the apparent isoelectric points of polymorphic bovine MHC class II beta chains. This was demonstrated by double labelling of cells with [S-35]methionine and subsequent bio tinylation and comparison of the banding pattern after immunoprecipita tion and 1D-IEF. 1D-IEF of 11 unrelated animals resulted in the demons tration of 29 polymorphic bands of which eight were detected by both l abelling procedures, six only after biotinylation and 15 only after me tabolic labelling with [S-35]methionine. Hence, biotinylation alone ca nnot serve as an alternative for radioactive labelling of bovine MHC c lass II molecules but can reveal expressed allelic forms not detectabl e by metabolic labelling with [S-35]methionine.