The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, the reliability of C and T concentration measurements in the serum of Aceh cattle, was tested using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum was evaluated. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C secretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20 (O)C (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups). Results: Parallelism, accuracy, and precision tests showed that both C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.01). Concentrations of T in bulls were significantly higher than in cows (p<0.01). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.