Detection of antibody secreting hybridomas with diazobenzyloxymethyl paper: An easy, sensitive and versatile assay

A sensitive and universal assay for the detection of monoclonal antibodies is described. One microliter of each hybrid cell supernatant is transferred to activated diazobenzyloxymethyl paper and, after coating the uncovered activated sites by incubation with bovine serum albumin, the paper is dipped in a solution of 125 I-labeled antigen. After incubation to allow for fixation of antigen to antibody present in the supernatants, the paper is rinsed and autoradiographed. As many as 96 wells can be tested on one paper and more than 20 papers can be treated in 1 day (∼ 2000 wells). The sensitivity and reliability of the method were tested with monoclonal antibodies against a subunit of scorpion ( Androctonus australis ) hemocyanin. Unlike other immunobinding assays the test depends only on antigen-antibody interaction and not on class or subclass of immunoglobulin. It is suitable for a variety of hybridomas.