Analytical performance of canine acylated and unacylated ghrelin enzyme-linked immunosorbent assays

Ghrelin is a major appetite-stimulating hormone. It circulates as acylated ghrelin (AG) and unacylated ghrelin (UAG), which could have different metabolic actions in obesity. Our objective was to study the analytical performance of two new canine AG and UAG ELISAs using blood samples from healthy, normal-weight dogs. Additionally, the effect of a protease inhibitor (PI) on short-term sample storage was analyzed.

The intra- and inter-assay precision for low, intermediate, and high AG and UAG concentrations, accuracy, limits of quantification (LQ), and detection limit (DL) of a blank sample were determined in ten healthy dogs. To study the effects of a PI on ghrelin concentrations, and AG and UAG concentrations were compared in five canine plasma samples stored for 1 month with (PI+), without (PI-), and with PI added at sample thawing (PI+th).

The intra- and inter-assay coefficients of variation were 1.8%-5.7% and 2.9%-6.4% for the AG assay, and 0.8%-7.5% and 2.8%-13.4% for the UAG assay, respectively. Accuracy analyses showed nonsignificant deviation from linearity for the AG (R²  = .99; Runs test: P = .37) and UAG (R²  = .99; Runs test: P = .42) assays. For the AG assay, the upper LQ was >1261 pg/mL, the lower LQ was 6.2 pg/mL, and the DL was 0.3 pg/mL. For the UAG assay, the upper LQ was >1785 pg/mL, the lower LQ was 16.3 pg/mL, and the DL was 1.8 pg/mL. No differences in the AG (P = .54) and UAG (P = .95) concentrations were detected in the plasma samples subjected to PI+, PI-, and PI+th.

The AG and UAG ELISA assays had acceptable precision, accuracy, lower LQ, and DLs, but upper LQ could not be established. An influence of the PI on short-term storage was not detectable. Long-term storage ± PI was not evaluated and should be investigated further.