Equine insulin elisa

Serum insulin concentrations were measured in duplicate at the end of the experimental phase using an equine-optimized ELISA (Mercodia Equine Insulin ELISA; Mercodia AB, Sylveniusgatan 8A, Uppsala, Sweden; inter-assay coefficient of variation: 7.7%) previously validated for use in horses.

All blood samples were collected into evacuated tubes (Vacuette 9 ml, Greiner Bio-One GmbH, Kremsmünster, Austria) containing lithium heparin and immediately placed on ice for 5 minutes before centrifugation (10 min, 2700 × g). Plasma was separated, frozen rapidly and stored at -80°C until later analysis of plasma insulin and glucose concentrations. Plasma glucose concentrations were measured enzymatically with an automated clinical chemistry analyser (YSI 2300 Stat Plus Analyzer, YSI Incorporated, Yellow Spring, Ohio). Endogenous concentration of plasma insulin was measured using a commercialised equine-optimised ELISA (Mercodia equine insulin ELISA, Mercodia AB, Uppsala, Sweden) and insulin concentrations were verified with a commercial kit (Mercodia animal insulin control; low, medium and high, Mercodia AB, Uppsala, Sweden) [31 (link)]. All analyses were performed in duplicate.

Plasma glucose concentrations were measured enzymatically with an automated clinical chemistry analyzer (YSI 2300 Stat Plus Analyzer, YSI Incorporated, Yellow Spring, Ohio). Endogenous equine plasma insulin concentrations from the OST were determined using a commercial equine-optimized ELISA (Mercodia Equine Insulin ELISA, Mercodia AB, Uppsala, Sweden) evaluated for use in horses [19 (link)] and insulin levels were controlled with a commercial kit (Mercodia Animal Insulin Control (Low, Medium, High), Mercodia AB, Uppsala, Sweden). Plasma insulin concentrations from the continuous rate infusion of recombinant human insulin during the EHC procedures were determined using a commercial human ELISA (Mercodia Insulin ELISA, Mercodia AB, Uppsala, Sweden) and a commercial kit (Mercodia Diabetes Antigen Control (Low, High)/Human, Mercodia AB, Uppsala, Sweden) was used as a control. All analyses of plasma glucose and insulin were performed in duplicate. The mean intra-assay CVs for glucose, equine insulin (equine-optimized ELISA) and human insulin (human ELISA) were 0.4, 2.7 and 2.7 % respectively, as determined from duplicate analyses.

All horse owners provided informed written consent. Thirty-five client owned horses and ponies previously diagnosed with ID by referring veterinarians using an oral sugar test [21 (link)] were enrolled in the study. The diagnosis of ID was based on blood samples obtained between 60 and 90 minutes after oral administration of the syrup and analyzed for insulin (Mercodia equine insulin ELISA, Mercodia AB, Uppsala, Sweden). To be eligible to participate in the study the insulin concentration had to be > 90 µIU/mL (insulin concentrations > 45 µIU/mL is considered diagnostic for ID). In addition, 26 clinically healthy horses and ponies owned by the Swedish University of Agricultural Sciences were included in the study. The horses were divided into 3 major groups: warmblood horses (warmbloods and Standardbreds), Icelandic horses and ponies (pony crossbreds, Welsh ponies, Gotland ponies and Shetland ponies). Criteria for inclusion were no ongoing episode of laminitis based on clinical examination and normal plasma ACTH adjusted for the season. All horses in the study were fed a hay or haylage diet supplemented with minerals. Horses were housed in individual box stalls and allowed daily turnout in a dirt or sand paddock. None of the horses had been on grass pasture for at least 2 months before testing.