Open Peer Review Any reports and responses or comments on the article can be found at the end of the article. RESEARCH ARTICLE 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 [version 3; peer review: 2 approved] Gholib Gholib , Sri Wahyuni , Muslim Akmal , Muhammad Hasan , Muhammad Agil , Bambang Purwantara 5 Physiology Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia Anatomy Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia Histology Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia Clinical Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia Department of Veterinary Clinic Reproduction and Pathology, Bogor Agricultural University, Bogor, Jawa Barat, 16680, Indonesia Abstract To obtain accurate measurements of cortisol (C) and Background: 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. Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) Methods: 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 C (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups). Parallelism, accuracy, and precision tests showed that both C Results: 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). 1 2 3 4 5 5 1 2 3 4 5 Reviewer Status Invited Reviewers version 3 (revision) 06 Mar 2020 version 2 (revision) 04 Feb 2020 version 1 30 Jul 2019 1 2 report report report , Gadjah Mada University (UGM), Pudji Astuti Yogyakarta, Indonesia 1 , Autonomous University of Annaïs Carbajal Barcelona, Barcelona, Spain 2 30 Jul 2019, :1220 ( First published: 8 ) https://doi.org/10.12688/f1000research.19804.1 04 Feb 2020, :1220 ( Second version: 8 ) https://doi.org/10.12688/f1000research.19804.2 06 Mar 2020, :1220 ( Latest published: 8 ) https://doi.org/10.12688/f1000research.19804.3 v3 O Page 1 of 19 F1000Research 2020, 8:1220 Last updated: 06 MAR 2020