Plasma interleukin-6 concentration in Standardbred racehorses determined by means of a novel validated ELISA.

Abstract

To evaluate plasma interleukin 6 (IL-6) concentration in Standardbred racehorses by means of a novel ELISA following validation of the assay for use with equine plasma samples. Plasma samples obtained from 25 Thoroughbreds for use in assay validation and from 319 Standardbred racehorses at rest 2 to 2.5 hours prior to warm-up and racing. A sandwich ELISA was developed with equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to enhance sensitivity. The assay was validated for specificity, sensitivity, precision, and accuracy by use of both recombinant and endogenous proteins. For the assay, cross-reactivity with other human and equine cytokines was very low or absent. Serial dilution of plasma samples resulted in proportional decreases in reactivity, indicating high specificity of the method. Partial replacement of detection antibody with capture antibody or pretreatment of samples with capture antibody caused assay signals to significantly decrease by 55%. The inter- and intra-assay precisions were ≤ 13.6% and ≤ 9.3%, respectively; inter- and intra-assay accuracies were within ranges of ± 14.1% and ± 8.6%, respectively, at concentrations from 78 to 5,000 pg/mL, and the sensitivity was 18 pg/mL. Plasma IL-6 concentration varied widely among the 319 Standardbreds at rest (range, 0 to 193,630 pg/mL; mean, 6,153 pg/mL; median, 376 pg/mL). This ELISA method proved suitable for quantification of IL-6 concentration in equine plasma samples. Plasma IL-6 concentration was high (> 10,000 pg/mL) in 9.1% of the Standardbred racehorses, which warrants further investigation.