Use of monoclonal antibodies for quantitative analysis of antigens in normal and neoplastic tissues

Abstract

We describe two methods of analyzing tissues for cell-surface antigens identified by monoclonal antibodies. The first method is a binding assay, in which a membrane fraction prepared by differential centrifugation of a tissue homogenate is incubated with 125I-labeled antibody. The specificity of the observed binding is established by competition with unlabeled antibody. The second method, double-determinant immunoassay, involves two monoclonal antibodies that recognize two distinct epitopes of the antigen molecule. An immunoabsorbent prepared from one of the antibodies is incubated with a detergent lysate of the tissue. The immunoabsorbent is then washed and incubated with the 125I-labeled second antibody. In both assays the amount of radiolabeled antibody bound is proportional to the amount of antigen in the test sample, and the limit of detection is approximately 10 pg of antibody, corresponding to less than 1 fmol of antigen.