{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T16:39:34Z","timestamp":1766421574537,"version":"3.48.0"},"reference-count":39,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T00:00:00Z","timestamp":1766361600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"scientific and technological programme \u201cDevelopment of new diagnostic test systems for particularly dangerous viral infections\u201d","award":["BR24992948"],"award-info":[{"award-number":["BR24992948"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>The emergence and spread of coronavirus infections have created a necessity to develop serological methods for assessing population immunity. The enzyme-linked immunosorbent assay (ELISA) remains one of the most accessible and informative approaches for these purposes. The choice of recombinant proteins plays an important role in the sensitivity and specificity of the test system, and in this regard, the creation of a domestic ELISA based on the chimeric SM protein to the SARS-CoV-2 virus is relevant. In this work, a recombinant chimeric SM protein expressed in the E. coli system and purified using metal-affinity chromatography on Ni-NTA agarose was constructed and presented for the first time. An ELISA test system was developed and tested using panels of positive and negative sera, including samples obtained before the COVID-19 pandemic. The obtained sensitivity (90.48%) and specificity (93.65%) indicators with a ROC curve AUC = 0.9623 (OD450 = 0.213) indicate the diagnostic accuracy of the test system. The positive diagnostic ratio (LR+) = 14.25.0 indicates the reliability of a positive result. The domestically developed ELISA test system can be used for serological monitoring and assessment of the immune status of the population.<\/jats:p>","DOI":"10.3390\/mps9010004","type":"journal-article","created":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T16:19:26Z","timestamp":1766420366000},"page":"4","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of an ELISA Using Recombinant Chimeric SM Protein for Serological Detection of SARS-CoV-2 Antibodies"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5500-9913","authenticated-orcid":false,"given":"Gulnur","family":"Nakhanova","sequence":"first","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7954-6246","authenticated-orcid":false,"given":"Olga","family":"Chervyakova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kamshat","family":"Shorayeva","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5623-7571","authenticated-orcid":false,"given":"Aisha","family":"Issabek","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8353-1629","authenticated-orcid":false,"given":"Sabina","family":"Moldagulova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Asankadyr","family":"Zhunushov","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, National Academy of Sciences of the Kyrgyz Republic, Bishkek 720071, Kyrgyzstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aknur","family":"Ulankyzy","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aigerim","family":"Zhakypbek","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alisher","family":"Omurtay","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6593-0271","authenticated-orcid":false,"given":"Aziz","family":"Nakhanov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zharkinay","family":"Absatova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yeraly","family":"Shayakhmetov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kuanysh","family":"Jekebekov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Temirlan","family":"Baiseit","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aslan","family":"Kerimbayev","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems LLP, National Holding QazBioPharm JSC, Almaty 080409, Kazakhstan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,22]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2025, April 15). Timeline of WHO\u2019s Response to COVID-19. World Health Organization. Available online: https:\/\/www.who.int\/emergencies\/diseases\/novel-coronavirus-2019\/interactive-timeline."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1007\/s00125-022-05670-0","article-title":"Incidence of newly diagnosed diabetes after COVID-19","volume":"65","author":"Rathmann","year":"2022","journal-title":"Diabetologia"},{"key":"ref_3","unstructured":"World Health Organization (2022). Therapeutics and COVID-19: Living Guideline, World Health Organization. Available online: https:\/\/iris.who.int\/handle\/10665\/340374."},{"key":"ref_4","unstructured":"Crist, C. (2022, June 06). Paxlovid Reduces Risk of COVID Death by 79% in Older Adults: Study. Medscape. Available online: https:\/\/www.medscape.com\/viewarticle\/975949."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1016\/j.amepre.2022.10.007","article-title":"Associations of physical inactivity and COVID-19 outcomes among subgroups","volume":"64","author":"Young","year":"2023","journal-title":"Am. J. Prev. Med."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Mladenovic Stokanic, M., Simovic, A., Jovanovic, V., Radomirovic, M., Udovicki, B., Krstic Ristivojevic, M., Djukic, T., Vasovic, T., Acimovic, J., and Sabljic, L. (2024). Sandwich ELISA for the quantification of nucleocapsid protein of SARS-CoV-2 based on polyclonal antibodies from two different species. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25010333"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1111\/1348-0421.13173","article-title":"Development of a double-antibody sandwich ELISA for detection of SARS-CoV-2 variants based on nucleocapsid protein-specific antibodies","volume":"68","author":"Lv","year":"2024","journal-title":"Microbiol. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1877","DOI":"10.1007\/s00705-021-05072-4","article-title":"Establishment of an indirect ELISA-based method involving the use of a multiepitope recombinant S protein to detect antibodies against canine coronavirus","volume":"166","author":"Hao","year":"2021","journal-title":"Arch. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Ramos, F.F., Pereira, I.A.G., Cardoso, M.M., Bandeira, R.S., Lage, D.P., Scussel, R., Anastacio, R.S., Freire, V.G., Melo, M.F.N., and Oliveira-da-Silva, J.A. (2023). 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. Viruses, 15.","DOI":"10.3390\/v15091877"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Malik, Y.S., Kumar, P., Ansari, M.I., Hemida, M.G., El Zowalaty, M.E., Abdel-Moneim, A.S., Ganesh, B., Salajegheh, S., Natesan, S., and Sircar, S. (2021). SARS-CoV-2 spike protein extrapolation for COVID diagnosis and vaccine development. Front. Mol. Biosci., 8.","DOI":"10.3389\/fmolb.2021.607886"},{"key":"ref_11","first-page":"363","article-title":"SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity","volume":"18","author":"Liang","year":"2005","journal-title":"Biomed. Environ. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Herrscher, C., Eymieux, S., Gaborit, C., Blasco, H., Marlet, J., Stefic, K., Roingeard, P., Grammatico-Guillon, L., and Hourioux, C. (2022). ELISA-based analysis reveals an anti-SARS-CoV-2 protein immune response profile associated with disease severity. J. Clin. Med., 11.","DOI":"10.3390\/jcm11020405"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Martin, S., Heslan, C., J\u00e9gou, G., Eriksson, L.A., Le Gallo, M., Thibault, V., Chevet, E., Godey, F., and Avril, T. (2021). SARS-CoV-2 integral membrane proteins shape the serological responses of patients with COVID-19. PLoS Pathog., 17.","DOI":"10.1101\/2021.02.15.431237"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"J\u00f6rri\u00dfen, P., Sch\u00fctz, P., Weiand, M., Vollenberg, R., Schrempf, I.M., Ochs, K., Fr\u00f6mmel, C., Tepasse, P.R., Schmidt, H., and Zibert, A. (2021). Antibody response to SARS-CoV-2 membrane protein in patients of the acute and convalescent phase of COVID-19. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.679841"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"113489","DOI":"10.1016\/j.jim.2023.113489","article-title":"Development and validity assessment of ELISA test with recombinant chimeric protein of SARS-CoV-2","volume":"519","author":"Fernandez","year":"2023","journal-title":"J. Immunol. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Freitas, N.E.M., Santos, E.F., Leony, L.M., Silva, \u00c2.A.O., Daltro, R.T., Vasconcelos, L.C.M., Duarte, G.A., Oliveira da Mota, C., Silva, E.D., and Celedon, P.A.F. (2022). Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera. PLoS Neglected Trop. Dis., 16.","DOI":"10.1371\/journal.pntd.0010290"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Santos, F.L., Celedon, P.A., Zanchin, N.I., Brasil Tde, A., Foti, L., Souza, W.V., Silva, E.D., Gomes Yde, M., and Krieger, M.A. (2016). Performance assessment of four chimeric Trypanosoma cruzi antigens based on antigen-antibody detection for diagnosis of chronic Chagas disease. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0161100"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Ferra, B.T., Chyb, M., Skwarecka, M., and Gatkowska, J. (2025). The trivalent recombinant chimeric proteins containing immunodominant fragments of Toxoplasma gondii SAG1 and SAG2 antigens in their core: A good diagnostic tool for detecting IgG antibodies in human serum samples. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26125621"},{"key":"ref_19","unstructured":"Kasenov, M.M., Orynbayev, M.B., Zakarya, K., Kerimbayev, A.A., Zhugunissov, K.D., Sultankulova, K.T., Myrzakhmetova, B.S., Nakhanov, A.K., Kassenov, M.M., and Abduraimov, Y.O. (2020). Strain \u201cSARS-CoV-2\/KZ_Almaty\/04.2020\u201d of Coronavirus Infection COVID-19 Used for Preparation of Means of Specific Prophylaxis, Laboratory Diagnostics, and Evaluation of Vaccine Biological Protection Effectiveness. (34762), Patent, Republic of Kazakhstan."},{"key":"ref_20","unstructured":"NCBI GenBank (2025, January 24). SARS-CoV-2 Isolate hCoV-19\/Kazakhstan\/MZ379258.1, Complete Genome (Accession No. MZ379258.1). National Center for Biotechnology Information, Available online: https:\/\/www.ncbi.nlm.nih.gov\/nuccore\/MZ379258.1."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e43","DOI":"10.1093\/nar\/30.10.e43","article-title":"DNAWorks: An automated method for designing oligonucleotides for PCR-based gene synthesis","volume":"30","author":"Hoover","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein measurement with the Folin phenol reagent","volume":"193","author":"Lowry","year":"1951","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_24","first-page":"530","article-title":"Assessment of the humoral immune response of experimental animals to the administration of recombinant ectodomain of SARS-CoV-2 S-glycoprotein with ISCOM-adjuvant","volume":"23","author":"Evseenko","year":"2023","journal-title":"Bioprep. Profil. Diagn. Lechenie"},{"key":"ref_25","unstructured":"Molecular Devices (2025, September 05). Better Metrics for Comparing Instruments and Assays [Application Note]. Available online: https:\/\/www.moleculardevices.com\/en\/assets\/app-note\/br\/better-metrics-for-comparing-instruments-and-assays."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/oxfordjournals.aje.a118408","article-title":"A simple method of estimating fifty percent endpoints","volume":"27","author":"Reed","year":"1938","journal-title":"Am. J. Epidemiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A simple statistical parameter for use in evaluation and validation of high throughput screening assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. Biomol. Screen."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"108915","DOI":"10.1016\/j.celrep.2021.108915","article-title":"Linear epitope landscape of the SARS-CoV-2 spike protein constructed from 1051 COVID-19 patients","volume":"34","author":"Li","year":"2021","journal-title":"Cell Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1988","DOI":"10.1080\/22221751.2020.1815591","article-title":"Functional mapping of B-cell linear epitopes of SARS-CoV-2 in COVID-19 convalescent population","volume":"9","author":"Yi","year":"2020","journal-title":"Emerg. Microbes Infect."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"eabd4250","DOI":"10.1126\/science.abd4250","article-title":"Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity","volume":"370","author":"Shrock","year":"2020","journal-title":"Science"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1007\/s13721-021-00347-x","article-title":"In-silico design of a multi-epitope for developing sero-diagnosis detection of SARS-CoV-2 using spike glycoprotein and nucleocapsid antigens","volume":"10","author":"Mamaghani","year":"2021","journal-title":"Netw. Model. Anal. Health Inform. Bioinform."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4290","DOI":"10.1038\/s41598-021-83730-y","article-title":"In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19","volume":"11","author":"Phan","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2806","DOI":"10.1038\/s41467-020-16638-2","article-title":"Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients","volume":"11","author":"Poh","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Farrera-Soler, L., Daguer, J.P., Barluenga, S., Vadas, O., Cohen, P., Pagano, S., Yerly, S., Kaiser, L., Vuilleumier, N., and Winssinger, N. (2020). Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma. PLoS ONE, 15.","DOI":"10.1101\/2020.06.15.20131391"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1006\/prep.1999.1162","article-title":"Purification of his-tagged proteins by immobilized chelate affinity chromatography: The benefits from the use of organic solvent","volume":"18","author":"Franken","year":"2000","journal-title":"Protein Expr. Purif."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1186\/s12985-022-01768-4","article-title":"Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections","volume":"19","author":"Luo","year":"2022","journal-title":"Virol. J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e03228-23","DOI":"10.1128\/spectrum.03228-23","article-title":"Comprehensive, comparative evaluation of 25 automated SARS-CoV-2 serology assays","volume":"12","author":"Dimech","year":"2024","journal-title":"Microbiol. Spectr."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Souris, M., Tshilolo, L., Parzy, D., Lobaloba Ingoba, L., Ntoumi, F., Kamgaing, R., Ndour, M., Mbongi, D., Phoba, B., and Tshilolo, M.A. (2022). Pre-pandemic cross-reactive immunity against SARS-CoV-2 among Central and West African populations. Viruses, 14.","DOI":"10.3390\/v14102259"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Kharlamova, N., Dunn, N., Bedri, S.K., Jerling, S., Almgren, M., Faustini, F., Gunnarsson, I., R\u00f6nnelid, J., Pullerits, R., and Gjertsson, I. (2021). False positive results in SARS-CoV-2 serological tests for samples from patients with chronic inflammatory diseases. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.666114"}],"container-title":["Methods and Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/1\/4\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T16:37:28Z","timestamp":1766421448000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/1\/4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,22]]},"references-count":39,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["mps9010004"],"URL":"https:\/\/doi.org\/10.3390\/mps9010004","relation":{},"ISSN":["2409-9279"],"issn-type":[{"value":"2409-9279","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,22]]}}}