Characterization and quantification of solubilised HLA-DR antigens from circulating XXXX monocytes using an immunoblotting procedure.

@entity1 @entity1133 @entity3902 @entity6514 @entity6743 @entity214
An immunoblotting technique was modified to detect and biochemically characterize HLA-DR antigens expressed on circulating @entity1 monocytes. Membrane proteins of peripheral blood monocytes were solubilised using the mildly anionic detergent, @entity6743 . These solubilised proteins were resolved by @entity214 -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 @entity1133 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% @entity6514 and 2% @entity214 . A stronger reducing agent, 25 mM @entity3902 , 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.
