Because hormones circulate in low quantities in blood, their accurate measurement requires sensitive assays, usually in the form of a competitive immunoassay.
The original method is radioimmunoassay using an antibody directed against the hormone and a radiolabeled 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 the detection of low concentrations of hormones, which typically circulate in the picomolar (10–12) or nanomolar (10–9) range.
Nonradioactive tags (eg, chemiluminescence), sandwich-type assays, and ELISA methods have been developed for hormone measurement. Point-of-care or in-clinic instruments designed for hormone measurement are also in common use in veterinary practices.
Advances in point-of-care methods continue to be made. For example, a new assay platform using bulk acoustic wave technology is available to measure ACTH, cortisol, free T4, and canine and feline thyroid-stimulating hormone (TSH) concentrations.
Accurate measurement in veterinary species presents some unique challenges because normal concentrations of a given hormone can vary substantially between species. For example, normal total T4 concentrations in dogs and cats are ~4 times lower than those in humans.
Cross-reactivity is a limitation of concern. Protein and 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 humans), 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 the given species and that the laboratory has established normal ranges.