Analytical biochemistry

Use of different hapten-protein conjugates immobilized on nitrocellulose to screen monoclonal antibodies to abscisic acid.

PMID 2937340


The dot-immunobinding method for screening antibodies to proteins on sheets of nitrocellulose has been modified to allow monoclonal antibodies (McAb) to the hapten abscisic acid (ABA) to be screened. Several methods for conjugating ABA to proteins using new bifunctional coupling reagents, specific for hapten keto groups, are described. Hybridomas secreting McAb with a defined specificity for the hapten can be identified by screening supernatants against the carrier protein and other hapten-protein conjugates with different conjugation bridges or modified hapten structure. Inhibition of binding to conjugates by free hapten is used to determine the relative avidity of the McAb for free and bound hapten. All of these tests could be done with no more than about 50 microliter of antibody solution. Dot immunobinding is a useful alternative to radioimmunoassay for screening McAb to haptens.