Journal of Immunological Methods 1989-03-31

Antibodies to cyclosporine A (CsA) by a novel route and their use to monitor cyclosporine levels by radioimmunoassay (RIA).

N A Cacalano, W L Cleveland, B F Erlanger

Index: J. Immunol. Methods 118(2) , 257-63, (1989)

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Abstract

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.