Validation of an enzyme‐linked immunosorbent assay for measurement of feline serum insulin

Abstract

Feline insulin has been measured previously using assays developed for measuring human insulin. As feline insulin differs from human insulin, it is important to validate the assay before use. The aims of this study were to validate an ELISA, the Mercodia Feline Insulin ELISA, intended for measuring feline insulin and to determine the stability of feline insulin in serum. Validation of the ELISA, which uses monoclonal antibodies that recognize both human and feline insulin, included evaluation of coefficients of variation (CVs), patterns of variation, and consistency after dilution and spiking with feline insulin. Stability was evaluated by measuring insulin in feline serum samples stored at 20°C, 2-8°C, and -80°C. The intra-assay CV in 14-20 adjacent replicates (excluding position effects) was 2.0-4.2% and the inter-assay CV was 7.6-14%. The systematic and random position effect yielded a CV of 6.2-10%. When 3 feline serum samples were set at fixed positions and analyzed on 8 plates, microplate effects and interaction were significant for all 3 samples. Recovery upon dilution and spiking was 78-105% and 86-126%, respectively. Feline serum insulin concentration was stable for 24 hours at 20°C, for 4 days at 2-8°C, and for 15 months at -80°C. The Mercodia Feline Insulin ELISA can be used for measuring serum feline insulin. Recovery after spiking and dilution was acceptable. As in many ELISAs, intra-assay CV for adjacent replicates was low, whereas the position and between-assay CVs were considerably higher.