Configuration of antibodies for assay of urinary cortisol in dogs influences analytic specificity

Abstract

Whether the variation in the reported urinary corticoid-to-creatinine ratio in dogs is affected by the application of 2 commonly applied anticortisol antibodies was investigated. Free-catch morning urine samples of 50 healthy dogs were analyzed in duplicate with the use of 2 different polyclonal antibodies (antibody A and B) raised in different rabbits. Antibody A was raised against cortisol-3-carboxymethyl-oxime and antibody B against cortisol-21-hemisuccinate linked to BSA. Enzyme immunoassays were applied by using corresponding biotinylated labels. To examine possible cross-reactions with conjugated and nonconjugated cortisol metabolites, EIA measurements were performed with urine samples both before (directly assayed) and after diethyl-ether extraction, as well as after reversed-phase HPLC. Although the results correlated (P < 0.001), urinary corticoid concentrations and accordingly the urinary corticoid-to-creatinine ratios were 8 times higher when using antibody A than when using antibody B (mean ± SD corticoid concentrations, 223 ± 131 vs 29 ± 12 nmol/L; P < 0.001). Irrespective of the antibody used, extraction significantly decreased measured corticoid concentrations (antibody A, 158 ± 120 nmol/L; antibody B, 15 ± 8 nmol/L; P < 0.001), but the decrease was conspicuous when antibody A was used. Antibody A cross-reacted significantly with polar (eg, conjugated) metabolites, clearly depicted in the chromatogram by 3 additional peaks in earlier fractions well separated from cortisol. In contrast the assay that used antibody B was specific, showing only 1 major peak in the fractions eluting authentic cortisol. In summary, the study indicates that the configuration of the antibody considerably influences the analytic specificity of cortisol assays and underlines the pivotal importance of assay validation for each species and sample material.