Antibodies to cyclosporine A (CsA) by a novel route and their use to monitor cyclosporine levels by radioimmunoassay (RIA).
Anti-cyclosporine antibodies were generated in rabbits, using an antigen derived from CsA. CsA was linked to carrier proteins by means of a photoactive cross-linking reagent, 4-benzoylbenzoic acid (BBa), an alpha, beta-unsaturated ketone. In the presence of CsA, photolysis of BBa results in hydrogen abstraction and random insertion into the cyclosporine molecule, generating a population of CsA with carboxyl groups at various positions. Immunization with CsA-BBa-bovine serum albumin gave rise to high affinity antibodies to CsA, with Kd = 9.8 +/- 2.8 x 10(-11) M, as determined by Scatchard analysis. Specificity was determined by competition experiments in a radioimmunoassay (RIA), using a panel of six cyclosporine derivatives, substituted at different positions. The derivatives could be arranged into three groups according to their affinities. One derivative with the lowest affinity had a bulky O-t-butyl-D-serine in place of D-alanine in position 8. Serum CsA levels in 25 transplant patients were measured by RIA, and compared to levels determined with a commercially available polyclonal antibody, which is routinely used clinically. The rabbit antiserum gave values that correlated well with the results using the commercial antibody.
Cacalano
N A
NA
Department of Microbiology, Columbia University, New York 10032.
Cleveland
W L
WL
Erlanger
B F
BF
D000818:Animals; D000906:Antibodies / Q000032:analysis; D000918:Antibody Specificity; D001577:Benzophenones; D003524:Cyclosporins / Q000097:blood* / Q000276:immunology / Q000627:therapeutic use; D005260:Female; D016027:Heart Transplantation; D006801:Humans; D015166:Monitoring, Immunologic / Q000379:methods; D010782:Photolysis / Q000379:methods; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods; D011933:Reagent Kits, Diagnostic
D000906:Antibodies; D001577:Benzophenones; D003524:Cyclosporins; D011933:Reagent Kits, Diagnostic; C058818:4-benzoylbenzoic acid