A sensitive enzyme immunoassay for cyclosporin A using antibodies generated against a novel hapten

A simple and sensitive enzyme immunoassay for cyclosporin A has been developed. Cyclosporin A (CsA) antibodies were generated in rabbits using a novel hapten (CsA-C6) derived from ozonolysis of a double bond at residue 1 followed by reaction with 6-amino hexanoic acid. The antibodies generated from CsA-C6 recognized the spacer arm between the hapten and the carrier protein. A heterologous bridge strategy for haptenenzyme conjugate was used to improve the sensitivity. A hemisuccinyl ester of cyclosporin A alcohol (CsA-C4) was synthesized, conjugated to horseradish peroxidase and used as the enzyme labeled hapten. A high sensitivity enzyme immunoassay for CsA was developed. The concentration that gave 50% inhibition of binding was 1 ng/ml (8.3 x 10(-10)M) of CsA. The specificity of the antibodies was examined against various metabolites. Metabolites with structural modifications at distal residues 4, 8 and 9 showed the least crossreactivity, whereas metabolites derived from residue 1 exhibited full crossreaction as expected.