A method is described for the identification of antigens by monoclonal antibodies.
This is applicable whenever precipitating antibodies to the same antigens from a different
species are available. The method is based upon: 1) Separation and immunoprécipitation
of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis
in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody.
2) Coprecipitation of the monoclonal antibody with its antigen. 3) Subsequent passive
transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose
membrane. 4) Visualization of the blotted antibody using an enzyme-linked secondary
antibody and a chromogenic substrate. 5) Identification of the corresponding antigen
by comparisons to the immunoprecipitate pattern of the original immunoplate. To test
this method we have analyzed the detection of the antigens recognized by six previously
described monoclonal antibodies against platelet membrane proteins and von Willebrand
factor. Specific immunoblots were obtained in each case using small amounts of monoclonal
antibodies. Thus, the technique provides an alternative when epitopes are denatured
by SDS, and avoids the use of radioactively labelled monoclonal antibodies.
Keywords
Immunoblotting - Monoclonal antibodies - Crossed immunoelectrophoresis - Platelet
antigens
