STABILITY OF AN ELISA TEST SYSTEM BASED ON A RECOMBINANT CHIMERIC SM PROTEIN FOR THE DETECTION OF ANTIBODIES TO SARS-COV-2 UNDER DIFFERENT TEMPERATURE CONDITIONS
Main Article Content
Authors
G.D. Nakhanova
Research Institute for Biological Safety Problems, QazBioPharm National Holding JSC, 080409, Zhambyl Region, Republic of Kazakhstan
Institute of Biotechnology, National Academy of Sciences of the Kyrgyz Republic, 720071, Bishkek, Kyrgyz Republic
O.V. Chervyakova
Research Institute for Biological Safety Problems, QazBioPharm National Holding JSC, 080409, Zhambyl Region, Republic of Kazakhstan
A.T. Zhunushov
Institute of Biotechnology, National Academy of Sciences of the Kyrgyz Republic, 720071, Bishkek, Kyrgyz Republic
A.K. Nakhanov
Research Institute for Biological Safety Problems, QazBioPharm National Holding JSC, 080409, Zhambyl Region, Republic of Kazakhstan
A. Ulankyzy
Research Institute for Biological Safety Problems, QazBioPharm National Holding JSC, 080409, Zhambyl Region, Republic of Kazakhstan
A.S. Zhakypbek
Research Institute for Biological Safety Problems, QazBioPharm National Holding JSC, 080409, Zhambyl Region, Republic of Kazakhstan
A. Tileukhanova
Research Institute for Biological Safety Problems, QazBioPharm National Holding JSC, 080409, Zhambyl Region, Republic of Kazakhstan
K.A. Shorayeva
Research Institute for Biological Safety Problems, QazBioPharm National Holding JSC, 080409, Zhambyl Region, Republic of Kazakhstan
Abstract
Assessment of the stability of kit components is a critical step in the development of enzyme-linked immunosorbent assay (ELISA) systems, as it directly determines the accuracy and reproducibility of diagnostic results during storage and routine use. In the present study, the stability and reproducibility of a newly developed ELISA test system based on the recombinant chimeric SM protein were evaluated under different temperature conditions. The stability of the kit components was assessed by monitoring changes in the analytical signal, expressed as optical density (OD), and the variability of replicate measurements, expressed as the coefficient of variation (CV, %), over a controlled period at -20ºC, 4ºC, 22ºC, and 37ºC. The results demonstrated that the stability of the developed ELISA test system was strongly temperature-dependent. Standard refrigerated storage at 4ºC was identified as the optimal condition, providing the highest preservation of the analytical signal, with 81.3% of the initial signal retained (OD₄₅₀ = 1.933), while maintaining high reproducibility (CV = 1.74%), consistently below the predefined acceptance threshold of ≤5%. Storage at -20ºC showed notable limitations. Despite maintaining a relatively high analytical signal (OD₄₅₀ = 1.623) and 68.2% residual activity, freezing and thawing of the reagents resulted in increased analytical variability (CV = 5.56%), exceeding the established acceptance criterion. Exposure to elevated temperatures of 22ºC and 37ºC had a pronounced detrimental effect on test-system stability, resulting in degradation of protein components and a loss of more than 65% of the initial activity. These findings demonstrate that, to ensure long-term preservation of the sensitivity, specificity, and reproducibility of the developed ELISA diagnostic kit, storage at 4ºC under properly maintained cold-chain conditions is an essential technological requirement.
Keywords
ELISA test system, stability, reagents, optical density, recombinant protein
Article Details
References
Coronavirus disease (COVID-19) [Electronic resource] / World Health Organization. - URL: URL
COVID-19 epidemiological update - 14 March 2025. Edition 177 [Electronic resource] / World Health Organization. - Geneva : WHO, 2025. - URL: URL.
Expression and characterization of SARS-CoV-2 spike proteins / J.M. Schaub, C.W. Chou, H.C. Kuo [et al.] // Nature Protocols. - 2021. - Vol. 16, No. 11. - P. 5339–5356. - DOI: 10.1038/s41596-021-00623-0.
Strategy for the construction of SARS-CoV-2 S and N recombinant proteins and their immunogenicity evaluation / P.H.G. Borges, B. Gregio, H.T. Suzukawa [et al.] // BioTech. - 2025. - Vol. 14, No. 2. - Art. 38. - DOI: 10.3390/biotech14020038.
Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications / A. Bhatwa, W. Wang, Y.I. Hassan [et al.] // Frontiers in Bioengineering and Biotechnology. - 2021. - Vol. 9. - Art. 630551. - DOI: 10.3389/fbioe.2021.630551.
Escherichia coli recombinant expression of SARS-CoV-2 protein fragments / B.E. McGuire, J.E. Mela, V.C. Thompson [et al.] // Microbial Cell Factories. - 2022. - Vol. 21. - Art. 21. - DOI: 10.1186/s12934-022-01753-0.
Antisera produced using an E. coli-expressed SARS-CoV-2 RBD and complemented with a minimal dose of mammalian cell-expressed S1 subunit of the spike protein exhibits improved neutralization / T. Yoshizue, S. Brindha, R. Wongnak [et al.] // International Journal of Molecular Sciences. - 2023. - Vol. 24, No. 13. - Art. 10583. - DOI: 10.3390/ijms241310583.
Wingfield P.T. Overview of the purification of recombinant proteins // Current Protocols in Protein Science. - 2015. - Vol. 80. - P. 6.1.1–6.1.35. - DOI: 10.1002/0471140864.ps0601s80.
B-cell epitopes-based chimeric protein from SARS-CoV-2 N and S proteins is recognized by specific antibodies in serum and urine samples from patients / F.F. Ramos, I.A.G. Pereira, M.M. Cardoso [et al.] // Viruses. - 2023. - Vol. 15, No. 9. - Art. 1877. - DOI: 10.3390/v15091877.
Development and validity assessment of ELISA test with recombinant chimeric protein of SARS-CoV-2 / Z. Fernandez, R. de Arruda Rodrigues, J.M. Torres [et al.] // Journal of Immunological Methods. - 2023. - Vol. 519. - Art. 113489. - DOI: 10.1016/j.jim.2023.113489.
Validation of a chimeric proteins-based ELISA for the diagnosis of brucellosis in Kazakhstan / A. Bulashev, S. Eskendirova, B. Ingirbay [et al.] // Turkish Journal of Veterinary and Animal Sciences. - 2023. - Vol. 47, No. 6. - Art. 10. - DOI: 10.55730/1300-0128.4329.
Designing and expression of recombinant chimeric spike protein from SARS-CoV-2 in Escherichia coli and its immunogenicity assessment / S. Karimi, S. Nazarian, F. Sotoodehnejadnematalahi [et al.] // Iranian Journal of Pharmaceutical Research. - 2023. - Vol. 22, No. 1. - Art. e137751. - DOI: 10.5812/ijpr-137751.
Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity / M. Cano-Muñoz, D. Polo-Megías, A. Cámara-Artigas [et al.] // International Journal of Biological Macromolecules. - 2022. - Vol. 222, Pt. B. - P. 2467–2478. - DOI: 10.1016/j.ijbiomac.2022.10.031.
Enhancing the stability of COVID-19 serological assay through metal-organic framework encapsulation / Y. Wang, Z. Wang, P. Gupta [et al.] // Advanced Healthcare Materials. - 2021. - Vol. 10, No. 18. - Art. e2100410. - DOI: 10.1002/adhm.202100410.
Development of a new indirect ELISA test for the detection of anti-feline coronavirus antibodies in cats / I. Ferrero, S. Dewilde, P. Poletti [et al.] // Veterinary Sciences. - 2025. - Vol. 12, No. 3. - Art. 245. - DOI: 10.3390/vetsci12030245.
Development and validation of ELISA for in vitro diagnosis of SARS-CoV-2 infection / L.C.M. Vasconcelos, L. Maia Leony, Â.A.O. Silva [et al.] // COVID. - 2025. - Vol. 5. - Art. 108. - DOI: 10.3390/covid5070108.
Nakhanova, G.; Chervyakova, O.; Shorayeva, K.; Issabek, A.; Moldagulova, S.; Zhunushov, A.; Ulankyzy, A.; Zhakypbek, A.; Omurtay, A.; Nakhanov, A.; et al. Development of an ELISA Using Recombinant Chimeric SM Protein for Serological Detection of SARS-CoV-2 Antibodies. Methods Protoc. 2026, 9, 4. Crossref.
ISO 23640:2015. In vitro diagnostic medical devices - Evaluation of stability of in vitro diagnostic reagents. - Geneva : International Organization for Standardization, 2015.
Evaluation of the humoral immune response in experimental animals to the administration of the recombinant SARS-CoV-2 S-glycoprotein ectodomain with an ISCOM adjuvant / V.A. Evseenko, A.V. Zaikovskaya, A.S. Gudymo [et al.] // Biopreparations. Prevention, Diagnosis, Treatment. - 2023. - Т. 23, № 4. - С. 530–543. - DOI: 10.30895/2221-996X-2023-23-4-530-543.
Reed L.J., Muench H. A simple method of estimating fifty percent endpoints // American Journal of Epidemiology. - 1938. - Vol. 27, No. 3. - P. 493–497. - DOI: 10.1093/oxfordjournals.aje.a118408.
Zhang J.-H., Chung T.D.Y., Oldenburg K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays // Journal of Biomolecular Screening. - 1999. - Vol. 4, No. 2. - P. 67–73. - DOI: 10.1177/108705719900400206.
Molecular and serological tests for SARS-CoV-2 detection in indeterminate serology: can we skip the second sample? / I.N. Sirakov, K. Shishkova, S. Shishkov, I. Alexiev // Current Issues in Molecular Biology. - 2025. - Vol. 47, No. 11. - Art. 919. - DOI: 10.3390/cimb47110919.
Richter S.M. [et al.]. The challenges of serology: towards reliable SARS-CoV-2 antibody assays [Electronic resource] // Biocompare. - 2020. - URL: URL.
El-Daly M.M. Advances and challenges in SARS-CoV-2 detection: a review of molecular and serological technologies // Diagnostics. - 2024. - Vol. 14, No. 5. - Art. 519. - DOI: 10.3390/diagnostics14050519.
Impact of high temperatures on enzyme-linked immunoassay (ELISA) performance for leptin measurements in human milk stored under varied freeze/thaw conditions / V. Bertacchi, M. Corley, G.P. Aronsen, R.G. Bribiescas // PLOS One. - 2025. - Vol. 20, No. 3. - Art. e0320366. - DOI: 10.1371/journal.pone.0320366.
Kang L., Smith S., Wang C. Stabilization of surface-bound antibodies for ELISA based on a reversible zeolitic imidazolate framework-8 coating // Journal of Colloid and Interface Science. - 2021. - Vol. 588. - P. 101–109. - DOI: 10.1016/j.jcis.2020.12.068.
El-Daly, M.M. Advances and Challenges in SARS-CoV-2 Detection: A Review of Molecular and Serological Technologies. Diagnostics 2024, 14, 519. Crossref.
Bertacchi V, Corley M, Aronsen GP, Bribiescas RG. Impact of high temperatures on enzyme-linked immunoassay (ELISA) performance for leptin measurements in human milk stored under varied freeze/thaw conditions. PLoS One. 2025 Mar 19;20(3):e0320366. doi: 10.1371/journal.pone.0320366. PMID: 40106448; PMCID: PMC11957766.
Lin Kang, Steve Smith, Congzhou Wang. Stabilization of surface-bound antibodies for ELISA based on a reversable zeolitic imidazolate framework-8 coating. / Journal of Colloid and Interface Science, Volume 588, 2021, Pages 101-109, ISSN 0021-9797, Crossref.
Khatkhatay MI, Desai MP, Sankolli GM, Pardhe DK, Joshi UM. Stability studies of the components of a prototype penicillinase (beta-lactamase)-linked ELISA kit. J Clin Lab Anal. 1993;7(2):95-9. - doi: 10.1002/jcla.1860070205.
Irene Ferrero, Paolo Poletti, Enrica Giachino, Joel Filipe, Alessia Libera Gazzonis, Carolina Allievi, Paola Dall’Ara. A newly developed rapid K39-based LeishmaCHECK Ab ELISA for high-throughput serological screening of canine leishmaniasis. Current Research in Parasitology & Vector-Borne Diseases. Volume 10, 2026, 100418, ISSN 2667-114X, Crossref.
Ferrero, I.; Dewilde, S.; Poletti, P.; Canepa, B.; Giachino, E.; Dall’Ara, P.; Filipe, J. Development of a New Indirect ELISA Test for the Detection of Anti-Feline Coronavirus Antibodies in Cats. Vet. Sci. 2025, 12, 245. Crossref.
M. I. Khatkhatay, M. P. Desai, G. M. Sankolli, D. K. Pardhe, U. M. Joshi. Stability studies of the components of a prototype penicillinase (β-lactamase)-linked ELISA kit. Journal of Clinical Laboratory Analysis, Volume 7, Issue 2 pp. 95-99.