Covalent binding of detergent-solubilized membrane glycoproteins to 'Chemobond' plates for ELISA

An ELISA method is presented which is based on covalent binding of detergent-solubilized membrane proteins to surface-modified polystyrene plates (Chemobond plates). These plates carried 0.52-0.65 nmol of aldehyde groups per well (150 microliters) and allowed coupling of protein by Schiff base formation either at high pH and subsequent reduction with NaBH4 or by trapping reduced imines at pH 6-6.8 with cyanoborohydride. They bound 15 times the amount of normal plates. Sodium chloride (0.5 M) increased binding 2-3-fold. Binding was essentially resistant to elution by 1% sodium dodecyl sulfate. Reduction of uncoated plates with NaBH4 eliminated the high extent of binding. ELISA tests on Chemobond plates with a rabbit anti-band 3 antibody gave a ten-fold higher signal than plates to which band 3 protein was merely adsorbed. The use of an antigen-enzyme conjugate to detect bound antibody allowed to perform antibody binding and detection of bound antibody simultaneously in the presence of 0.05% Triton X-100. A competitive, one step ELISA system allowed determination of rabbit anti-band 3 antibodies in diluted serum with a sensitivity range of 0.02-0.4 microgram/ml.