Characterization and quantification of solubilised HLA-DR antigens from circulating human monocytes using an immunoblotting procedure.
An immunoblotting technique was modified to detect and biochemically characterize HLA-DR antigens expressed on circulating human monocytes. Membrane proteins of peripheral blood monocytes were solubilised using the mildly anionic detergent, sodium deoxycholate. These solubilised proteins were resolved by SDS-PAGE and transferred electrophoretically to nitrocellulose. The HLA-DR antigen was detected using a polyclonal antiserum and two monoclonal anti-HLA-DR antibodies. Both immunoperoxidase and 125I autoradiography techniques were used for visualisation of the antigen. The resolution of HLA-DR reactive material was increased when proteins were renatured with 4M urea after blotting. Immunoprobing of a sample of solubilized membrane proteins showed three bands of HLA-DR antigenic reactivity at molecular weights 65kDa, 55kDa and 46kDa. After storage at -70 degrees C for 2 months, only the 46kDa HLA-DR antigen band was detectable. Nonetheless, the 2-chain HLA-DR molecule was found to be an extremely stable complex which could not be dissociated by boiling in sample buffer containing 5% 2-mercaptoethanol and 2% SDS. A stronger reducing agent, 25 mM dithiothreitol, was required to split the HLA-DR molecule into its alpha and beta subunit chains. Finally, in a study of circulating monocytes from normal subjects, the immunoblotting technique was shown to quantitate solubilised HLA-DR antigen in a reproducible manner.
Hassan
J
J
Department of Immunology, Trinity College, Dublin, Ireland.
Feighery
C
C
Bresnihan
B
B
Whelan
A
A
D004591:Electrophoresis, Polyacrylamide Gel; D006681:HLA-D Antigens / Q000032:analysis*; D006684:HLA-DR Antigens / Q000032:analysis*; D006801:Humans; D007163:Immunosorbent Techniques; D009000:Monocytes / Q000276:immunology*; D011487:Protein Conformation; D012995:Solubility; D013997:Time Factors
D006681:HLA-D Antigens; D006684:HLA-DR Antigens