Validation of a chemiluminescent enzyme immunometric assay for plasma adrenocorticotropic hormone in the dog.

Abstract

The concentration of canine adrenocorticotropic hormone (ACTH) is usually determined by radioimmunoassay. However, chemiluminescent assay techniques have many advantages for clinical endocrine testing. The objectives of this study were to validate a commercially available chemiluminescent assay for determination of canine ACTH concentration and to determine whether protease inhibitors are appropriate for use in the chemiluminescent assay system. Biological specificity was evaluated by treatment of 3 dogs with ovine corticotropin-releasing hormone (CRH) followed by serial measurements of ACTH and by comparison with a previously validated immunoradiometric assay. All samples were collected both in the presence and absence of aprotinin, a protease inhibitor. The assay was further evaluated by measurement of intra-assay precision, interassay precision, and recovery after dilution. Baseline ACTH concentrations ranged from 5.6 to 15.3 pg/mL, and maximum ACTH concentrations of 158 to 1240 pg/mL were observed 30-60 minutes after CRH administration. Plasma samples collected with aprotinin had significantly lower ACTH concentrations than did samples collected without aprotinin. The intra-assay coefficients of variance (CVs) ranged from 4.1 to 8.2%, and interassay CVs ranged from 4.6. to 14.8%. Recovery after dilution with canine plasma ranged from 93.4 to 103.0% of predicted concentration; however, inadequate recovery was observed with other diluents. There was a high correlation with the immunoradiometric assay (r =.925) but a significant negative bias (-32.9, 95% confidence interval -50.8 to -14.9). This chemiluminescent assay is a valid technique for measurement of ACTH in canine plasma. ACTH concentration measured by chemiluminescence is lower than that measured by immunoradiometry. Aprotinin decreases the measured concentration of ACTH, and this effect should be taken into account when interpreting results. Diluents supplied with the kit should not be used for dilution of canine samples.