Because hormones circulate in low quantities in blood, accurate measurement of these substances requires sensitive assays, usually in the form of a competitive immunoassay. The original method is radioimmunoassay employing an antibody directed against the hormone and a radio-labeled form of the hormone. The labeled hormone competes with unlabeled hormone for antibody-binding sites. A standard curve containing known amounts of hormone is used for comparison to calculate the concentration of hormone in patient samples. The use of radioactive tags permits detection of low concentrations of hormone, which typically circulate in the pico- (10–12) or nanomolar (10–9) range. In recent years, nonradioactive tags (eg chemiluminescence), “sandwich-type” assays, and ELISA methods have been developed for hormone measurement and are in increasingly frequent use. Point-of-care or in-clinic instruments designed for hormone measurement are also in common use in veterinary practices.
Accurate measurement in veterinary species presents some unique challenges, because normal concentrations of a given hormone can vary significantly between species. For example, normal total T4 concentrations in dogs and cats are ~4 times lower than those in people. Concern about cross-reactivity is important; protein/polypeptide hormones vary in amino acid composition and in other structural ways (eg, patterns of glycosylation) across species. As a consequence, antibodies made against a particular hormone may not recognize that material from another species. Finally, although steroid hormones are structurally identical across species (cortisol in dogs is identical to that in people), substances present in the serum of a given species can sometimes interfere in an assay, leading to inaccurate results. Overall, it is important that a laboratory providing measurement of a particular hormone in a species demonstrate that the assay is valid in that species and that the laboratory has established normal ranges.