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.
Chen
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Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082, USA.
Tai
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HH
D000818:Animals; D000906:Antibodies; D000917:Antibody Formation; D000918:Antibody Specificity; D016572:Cyclosporine / Q000032:analysis* / Q000276:immunology; D005260:Female; D006241:Haptens / Q000737:chemistry / Q000276:immunology*; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D007124:Immunoenzyme Techniques; D008722:Methods; D011817:Rabbits; D012680:Sensitivity and Specificity
D000906:Antibodies; D006241:Haptens; D018796:Immunoconjugates; D016572:Cyclosporine