Validation of an ELISA assay for measurement of the metabolite of serotonin, 5-Hydroxyindole acetic acid (5-HIAA), in canine urine

Objectives Serotonin (5-hydroxytryptophan), implicated in a number of canine diseases, has a very short half life in the serum. Urine concentration of its breakdown product 5-hydroxyindole acetic acid after an 8 hour fast is a more reliable measure of circulating serotonin in humans. This study aimed to validate a commercially available ELISA assay to measure 5-hydroxyindole acetic acid concentrations in canine urine by comparing the analytical performance with the gold standard liquid chromatography tandem mass spectrometry Methods Urine was collected from 26 dogs undergoing routine diagnostic investigations at one referral centre and rapidly processed and stored prior to testing for 5-hydroxyindole acetic acid using the two methods. Deming regression and Bland-Altman analyses were used to compare the results between the two methods Results The ELISA demonstrated acceptable precision and repeatability (coefficient of variation <20%). There was good agreement between the two methods (bias 0.92 µmol/L; 95% limits of agreement -6.44 to 8.29 µmol/L), although the ELISA was not tested at values close to the upper end of the claimed analytical measurement range limit. The ELISA was likely to be very reliable at low concentrations but may exceed acceptable error limits at high concentrations. Clinical significance A commercially available ELISA was validated to measure urine 5-hydroxyindole acetic acid. This less invasive method than blood sampling for serotonin should give a more reliable indication of long term serum serotonin concentrations. More studies of normal and diseased dogs are needed to confirm these findings before applying them in a clinical setting.