Application of human-specific ELISA kits for the analysis of laboratory animal biomaterial

   Background. Enzyme-linked immunosorbent assay (ELISA) is widely used in the study of pharmacological safety, pharmacokinetics, pharmacodynamics of both original and reproduced (biosimilar) drugs; it is the method of choice for the evaluation of various biomarkers in clinical and preclinical studies. A feature limiting the use of this method is its species specificity.

   Objective. The article discusses the use and adaptation of ELISA sets specific for humans for the analysis of biomaterial from laboratory animals.

   Design and methods. The materials of several studies performed in the organization using rats as one of the most common test systems in preclinical studies, and including the use of ELISA kits for the determination of steroid hormones in the blood (testosterone, estradiol), stress hormones (cortisol, corticosterone) and cardiac biomarkers (myoglobin, troponin).

   Results. Examples of using reagent kits without any changes are shown (cortisol), after various optimization options (corticosterone, testosterone, estradiol). On the myoglobin cardiomarker example an algorithm for biomaterial type choosing is presented, which allows expanding the initial field of reagent kit use and unifying the biomaterial selection in an experimental study, and troponin I turned out to be an example of the unsuitability of the kit for analyzing rat biomaterial.

   Conclusion. Based on experimental experience, an algorithm and decision-making scheme for approbation and adaptation of ELISA reagent kits specific for humans in the analysis of laboratory animal biomaterial are formulated.

1. Cox BM, Alsawah F, McNeill PC, et al. Neurochemical, hormonal, and behavioral effects of chronic unpredictable stress in the rat. Behavioural Brain Research. 2011;220(1):106–111. doi: 10.1016/j.bbr.2011.01.038

2. Pereira VH, Marques F, Lages V, et al. Glucose intolerance after chronic stress is related with downregulated PPAR-γ in adipose tissue. Cardiovascular Diabetology. 2016;15(1):114. doi: 10.1186/s12933-016-0433-2

3. López AL, Villanueva ME, Padilla MB, et al. Chronic unpredictable mild stress progressively disturbs glucose metabolism and appetite hormones in rats. Acta endocrinologica. 2018;14(1):16–23. doi: 10.4183/aeb.2018.16

4. Oosterholt BG, Maes JHR, Van der Linden D, et al. Burnout and cortisol: Evidence for a lower cortisol awakening response in both clinical and non-clinical burnout. Journal of Psychosomatic Research. 2015;78(5):445–451. doi: 10.1016/j.jpsychores.2014.11.003

5. Merkulov VM, Klimova NV, Merkulova TI. The ultradian rhythm of glucocorticoid secretion and the time course of target gene regulation. Vavilov Journal of Genetics and selection. 2015;19(2):214–221. (In Russ.) doi: 10.18699/VJ15.027

6. Morris CJ, Aeschbach D, Scheer FA. Circadian system, sleep and endocrinology. Mo.l Cell Endocrinol. 2012; 349(1):91–104. doi: 10.1016/j.mce.2011.09.003

7. Lightman SL, Wiles CC, Atkinson HC et al. The significance of glucocorticoid pulsatility. European journal of pharmacology. 2008;583(2–3):255–262. doi: 10.1016/j.ejphar.2007.11.073

8. Nunes KP, de Oliveira AA, Szasz T et al. Blockade of toll-like receptor 4 attenuates erectile dysfunction in diabetic rats. J. Sex. Med. 2018;15(9):1235–1245. doi: 10.1016/j.jsxm.2018.07.005

9. Al-Oanzi ZH. Erectile dysfunction attenuation by naringenin in streptozotocin-induced diabetic rats. J. Food Biochem. 2019;43(7):e12885. doi: 10.1111/jfbc.12885

10. Yang BB, Hong ZW, Zhang Z, et al. Epalrestat, an aldose reductase inhibitor, restores erectile function in streptozocin-induced diabetic rats. Int. J. Impot. Res. 2019;31(2):97–104. doi: 10.1038/s41443-018-0075-x

11. Yang R, Wang J, Chen Y, et al. Effect of caffeine on erectile function via up-regulating cavernous cyclic guanosine monophosphate in diabetic rats. J. Androl. 2008;29(5):586–591. doi: 10.2164/jandrol.107.004721

12. Luo L, Dai DZ, Cheng YS, et al. Sildenafil improves diabetic vascular activity through suppressing endothelin receptor A, iNOS and NADPH oxidase which is comparable with the endothelin receptor antagonist CPU0213 in STZ-injected rats. J. Pharm. Pharmacol. 2011;63(7):943–951. doi: 10.1111/j.2042-7158.2011.01268.x

13. Zenkevich IG, Klimova IO. Use of standard addition method in quantitative chromatographic analysis. J. of Analyt. Chem. 2006; 61(10):967–972. (In Russ.) doi: 10.1134/S1061934806100042

14. Zenkevich IG, Morozova TE. Features of the method of standard addition for quantitation of analytes in complex mixtures with sorption properties. Analytics and Control. 2010;14(3):164–171. (In Russ.)

15. Zenkevich IG, Barkhatova DD, Belysheva MN, et al. Comparative characterization of different kinds of chromatographic quantification using the double standard addition method. Analytics and Control. 2021;25(2):146–154. (In Russ.) doi: 10.15826/analitika.2021.25.2.010

16. Matichin AA, Faustova NM, Kargopolceva DR, et al. Circadian fluctuation in plasma testosterone levels in adult male rats. Laboratory Animals for Science. 2020;2:36–42. (In Russ.) doi: 10.29296/2618723X-2020-02-04

17. Begemann K, Rawashdeh O, Olejniczak I, et al. Endocrine regulation of circadian rhythms. Biol Timing Sleep. 2025;2:10. doi: 10.1038/s44323-025-00024-6

18. Dymova OV. Modern biomarkers in cardiology. Medicinskij sovet. 2018;(16):118–123. (In Russ.) doi: 10.21518/2079-701X-2018-16-118-123

19. Vittorini S, Clerico A. Cardiovascular biomarcers: increasing, impact of laboratory medicine in cardiology practice. Clinical Chemistry and Laboratory Medicine. 2008;46(6):748–763. doi: 10.1515/CCLM.2008.188

20. Saprygin DB. Cardiospecific troponins: their significance in the diagnosis, risk stratification, and prognosis of acute coronary syndrome. 1. Diagnostic value of traditional and modern markers of myocardial damage. International Journal of Interventional Cardioangiology. 2003;2:65–70. (In Russ.)

21. Vanek T, Kohli A. Biochemistry, myoglobin. StatPearls Treasure Island: StatPearls Publishing; 2025.

22. Ordway GA, Garry DJ. Myoglobin: an essential hemoprotein in striated muscle. J. Exp. Biol. 2004;207(Pt20):3441–3446. doi: 10.1242/jeb.01172.

23. Katruha IA. Human cardiac troponin complex. Structure and functions. Uspekhi Biologicheskoi Khimii. 2013; 53:149–194. (In Russ.)

24. Morrou D, Kennon KP, Dzhess RL, et al. Guidelines of the National Academy of Clinical Biochemistry for laboratory medicine practice: Clinical characteristics and utilization of biochemical markers in acute coronary syndromes. Laboratory Diagnostics. 2008.1(17):16–24. (In Russ.)

25. Kolmanová E, Bartošová L, Khazneh E, et al. Comparison of the specificity of cardiac troponin I and creatine kinase MB in isoproterenol-induced cardiotoxicity model in rats. Acta Veterinaria Brno. 2015;84(4):343–350. doi: 10.2754/avb201584040343

26. Clements P, Brady S, York M, et al. Time course characterization of serum cardiac troponins, heart fatty acid-binding protein, and morphologic findings with isoproterenol-induced myocardial injury in the rat. Toxicologic Pathology. 2010;38:703–714. doi: 10.1177/0192623310374969

27. Berezkin VA, Bondareva ED, Dobryanskaya SS, et al. Technological processes in preclinical studies. A risk-based approach. Consultant GLP-Planet 2022. Pharmaceutical industry opinion. Saint Petersburg: Research-and-manufacturing company «HOME OF PHARMACY» Joint Stock Company; 2022. P. 152–173. (In Russ.) doi: 10.57034/978-5-6048955-0-4-s7

28. Bolsunovskaya YuR, Engalycheva GN, Ivkin DYu, et al. Design of a pharmacological experiment. Implementation of the ARRIVE principles in research center operations. Consultant GLP-Planet 2022. Pharmaceutical industry opinion. Saint Petersburg: Research-and-manufacturing company «HOME OF PHARMACY» Joint Stock Company, 2022. P. 52–71. (In Russ.) doi: 10.57034/978-5-6048955-0-4-s3

29. Piccoli SP. Points to consider document: scientific and regulatory considerations for the analytical validation of assays used in the qualification of biomarkers in biological matrices [Internet]. Biomarker Assay Collaborative Evidentiary Considerations Writing Group; 2019 [cited 2025 Apr 30]. Available from: https://media.c-path.org/wp-content/uploads/20240427170639/evidconsid-whitepaper-analyticalsectionv2019.pdf

30. Kosman VM, Karlina MV, Faustova NM, et al. Validating bioanalytical methods for biomarker quantitation : a regulatory document review. Regulatory Research and Medicine Evaluation. 2025;15(5):550–564. doi: 10.30895/1991-2919-2025-766