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Research article - Peer-reviewed, 2012

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

Strage, Emma; Ström Holst, Bodil; Nilsson, Gunnar; Jones, Bernt; Lilliehöök, Inger

Abstract

Background 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. Objectives 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. Methods 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 degrees C, 28 degrees C, and -80 degrees C. Results The intra-assay CV in 1420 adjacent replicates (excluding position effects) was 2.04.2% and the inter-assay CV was 7.614%. The systematic and random position effect yielded a CV of 6.210%. 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 78105% and 86126%, respectively. Feline serum insulin concentration was stable for 24 hours at 20 degrees C, for 4 days at 28 degrees C, and for 15 months at -80 degrees C. Conclusions 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.

Keywords

Cat; ELISA; position effect; random; systematic; variation

Published in

Veterinary Clinical Pathology
2012, Volume: 41, number: 4, pages: 518-528
Publisher: WILEY-BLACKWELL