{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T01:17:41Z","timestamp":1777943861082,"version":"3.51.4"},"reference-count":52,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T00:00:00Z","timestamp":1761177600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>Toxoplasma gondii is a globally distributed protozoan parasite, and reliable serodiagnosis is essential for effective management of toxoplasmosis. Conventional assays rely on tachyzoite lysate antigen (TLA), which suffers from limited standardization and reproducibility. In this study, immunodominant fragments of six dense granule proteins\u2014GRA29, GRA35, GRA36, GRA45, GRA54, and GRA64\u2014were expressed in Escherichia coli, purified, and evaluated as candidate antigens in IgG ELISAs using human sera. This study represents the first assessment of their diagnostic utility. Initial screening identified GRA29, GRA45, and GRA54 as promising candidates, with AUC values of 0.9983, 0.8507, and 0.9323, respectively, while GRA35-, GRA36-, and GRA64-based ELISA showed poor discrimination between seropositive and seronegative samples. Extended evaluation of GRA29-based assay with a larger serum panel (n = 286) confirmed excellent diagnostic performance, yielding an AUC of 0.9942 and higher sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) compared with TLA-ELISA. A comparative analysis revealed that GRA29 produced stronger reactivity in positive sera and lower background in negatives. These findings highlight GRA29 as a promising recombinant antigen for the serodiagnosis of human toxoplasmosis and a potential standardized alternative to TLA.<\/jats:p>","DOI":"10.3390\/cimb47110879","type":"journal-article","created":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T08:14:04Z","timestamp":1761207244000},"page":"879","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development and Evaluation of Six Novel Recombinant GRA Proteins in Serodiagnosis of Human Toxoplasmosis"],"prefix":"10.3390","volume":"47","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0127-4244","authenticated-orcid":false,"given":"Karolina","family":"So\u0142owi\u0144ska","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Microbiology, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 Narutowicza Str., 80-233 Gda\u0144sk, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5421-6905","authenticated-orcid":false,"given":"Lucyna","family":"Holec-G\u0105sior","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Microbiology, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 Narutowicza Str., 80-233 Gda\u0144sk, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1965","DOI":"10.1016\/S0140-6736(04)16412-X","article-title":"Toxoplasmosis","volume":"363","author":"Montoya","year":"2004","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.12703\/r\/12-1","article-title":"Advances towards the Complete in Vitro Life Cycle of Toxoplasma gondii","volume":"12","author":"Warschkau","year":"2023","journal-title":"Fac. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1128\/CMR.05013-11","article-title":"Epidemiology of and Diagnostic Strategies for Toxoplasmosis","volume":"25","author":"Florence","year":"2012","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Akins, G.K.H., Furtado, J.M., and Smith, J.R. (2024). Diseases Caused by and Behaviors Associated with Toxoplasma gondii Infection. Pathogens, 13.","DOI":"10.3390\/pathogens13110968"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"101203","DOI":"10.1016\/j.spen.2025.101203","article-title":"Congenital Toxoplasmosis","volume":"54","author":"Cerisola","year":"2025","journal-title":"Semin. Pediatr. Neurol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Bollani, L., Auriti, C., Achille, C., Garofoli, F., De Rose, D.U., Meroni, V., Salvatori, G., and Tzialla, C. (2022). Congenital Toxoplasmosis: The State of the Art. Front. Pediatr., 10.","DOI":"10.3389\/fped.2022.894573"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Garweg, J.G., Kieffer, F., Mandelbrot, L., Peyron, F., and Wallon, M. (2022). Long-Term Outcomes in Children with Congenital Toxoplasmosis-A Systematic Review. Pathogens, 11.","DOI":"10.3390\/pathogens11101187"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1186\/s40001-024-02055-4","article-title":"Trend in Serological and Molecular Diagnostic Methods for Toxoplasma gondii Infection","volume":"29","author":"Kim","year":"2024","journal-title":"Eur. J. Med. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/j.pt.2020.09.006","article-title":"Serology for Toxoplasma in Immunocompromised Patients: Still Useful?","volume":"37","author":"Dupont","year":"2021","journal-title":"Trends Parasitol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"de Oliveira Azevedo, C.T., do Brasil, P.E.A.A., Guida, L., and Lopes Moreira, M.E. (2016). Performance of Polymerase Chain Reaction Analysis of the Amniotic Fluid of Pregnant Women for Diagnosis of Congenital Toxoplasmosis: A Systematic Review and Meta-Analysis. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0149938"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Yba\u00f1ez, R.H.D., Yba\u00f1ez, A.P., and Nishikawa, Y. (2020). Review on the Current Trends of Toxoplasmosis Serodiagnosis in Humans. Front. Cell. Infect. Microbiol., 10.","DOI":"10.3389\/fcimb.2020.00204"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1186\/s13071-015-0902-6","article-title":"Diagnosis of Toxoplasmosis and Typing of Toxoplasma gondii","volume":"8","author":"Liu","year":"2015","journal-title":"Parasit. Vectors"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1343","DOI":"10.1128\/CVI.00117-13","article-title":"Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies","volume":"20","year":"2013","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1111\/j.1365-3024.1988.tb00242.x","article-title":"Induction of a Protective Antibody-Dependent Response against Toxoplasmosis by in Vitro Excreted\/Secreted Antigens from Tachyzoites of Toxoplasma gondii","volume":"10","author":"Darcy","year":"1988","journal-title":"Parasite Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/S0923-2494(05)80096-3","article-title":"Excreted\/Secreted Antigens of Toxoplasma gondii\u2014Their Origin and Role in the Host-Parasite Interaction","volume":"144","author":"Capron","year":"1993","journal-title":"Res. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Rezaei, F., Sharif, M., Sarvi, S., Hejazi, S.H., Aghayan, S., Pagheh, A.S., Dodangeh, S., and Daryani, A. (2019). A Systematic Review on the Role of GRA Proteins of Toxoplasma gondii in Host Immunization. J. Microbiol. Methods, 165.","DOI":"10.1016\/j.mimet.2019.105696"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1073\/pnas.91.2.509","article-title":"The Parasitophorous Vacuole Membrane Surrounding Intracellular Toxoplasma gondii Functions as a Molecular Sieve","volume":"91","author":"Schwab","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Griffith, M.B., Pearce, C.S., and Heaslip, A.T. (2022). Dense Granule Biogenesis, Secretion, and Function in Toxoplasma gondii. J. Eukaryot. Microbiol., 69.","DOI":"10.1111\/jeu.12904"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1038\/s41541-022-00563-0","article-title":"Toxoplasmosis Vaccines: What We Have and Where to Go?","volume":"7","author":"Zhang","year":"2022","journal-title":"npj Vaccines"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1007\/s11845-022-02998-9","article-title":"Toxoplasma gondii Vaccine Candidates: A Concise Review","volume":"192","author":"Mamaghani","year":"2023","journal-title":"Ir. J. Med. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.exppara.2007.12.002","article-title":"Toxoplasma gondii: Enzyme-Linked Immunosorbent Assay Using Different Fragments of Recombinant Microneme Protein 1 (MIC1) for Detection of Immunoglobulin G Antibodies","volume":"119","author":"Holec","year":"2008","journal-title":"Exp. Parasitol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1006\/prep.1999.1044","article-title":"Cloning, Overexpression, and Purification of the Recombinant His-Tagged SSB Protein of Escherichia coli and Use in Polymerase Chain Reaction Amplification","volume":"16","author":"Dabrowski","year":"1999","journal-title":"Protein Expr. Purif."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.exppara.2009.10.010","article-title":"Toxoplasma gondii: Recombinant GRA5 Antigen for Detection of Immunoglobulin G Antibodies Using Enzyme-Linked Immunosorbent Assay","volume":"124","author":"Kur","year":"2010","journal-title":"Exp. Parasitol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.csda.2013.07.015","article-title":"Finding the Optimal Cut-Point for Gaussian and Gamma Distributed Biomarkers","volume":"69","author":"Rota","year":"2014","journal-title":"Comput. Stat. Data Anal."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1128\/CDLI.7.4.607-611.2000","article-title":"Enzyme-Linked Immunosorbent Assays Using the Recombinant Dense Granule Antigens GRA6 and GRA1 of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies","volume":"7","author":"Lecordier","year":"2000","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1779","DOI":"10.1128\/JCM.42.4.1779-1781.2004","article-title":"Usefulness of Toxoplasma gondii-Specific Recombinant Antigens in Serodiagnosis of Human Toxoplasmosis","volume":"42","author":"Pietkiewicz","year":"2004","journal-title":"J. Clin. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.1128\/JCM.38.3.1144-1150.2000","article-title":"Recombinant Antigens to Detect Toxoplasma gondii-Specific Immunoglobulin G and Immunoglobulin M in Human Sera by Enzyme Immunoassay","volume":"38","author":"Aubert","year":"2000","journal-title":"J. Clin. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"107722","DOI":"10.1016\/j.exppara.2019.107722","article-title":"Identification of Novel Antigens for Serum IgG Diagnosis of Human Toxoplasmosis","volume":"204","author":"Luo","year":"2019","journal-title":"Exp. Parasitol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1128\/CVI.00341-08","article-title":"GRA2 and ROP1 Recombinant Antigens as Potential Markers for Detection of Toxoplasma gondii-Specific Immunoglobulin G in Humans with Acute Toxoplasmosis","volume":"16","author":"Kur","year":"2009","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1016\/j.diagmicrobio.2007.03.003","article-title":"The Dense Granule Protein GRA2, a New Marker for the Serodiagnosis of Acute Toxoplasma Infection: Comparison of Sera Collected in Both France and Iran from Pregnant Women","volume":"58","author":"Golkar","year":"2007","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1016\/S0732-8893(03)00156-1","article-title":"Evaluation of Toxoplasma gondii Recombinant Proteins for the Diagnosis of Recently Acquired Toxoplasmosis by an Immunoglobulin G Analysis","volume":"47","author":"Nigro","year":"2003","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.diagmicrobio.2007.09.003","article-title":"Serodiagnosis of Recently Acquired Toxoplasma gondii Infection in Pregnant Women Using Enzyme-Linked Immunosorbent Assays with a Recombinant Dense Granule GRA6 Protein","volume":"61","author":"Golkar","year":"2008","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"700","DOI":"10.1007\/s004360050473","article-title":"Serodiagnosis of Acute Toxoplasmosis Using a Recombinant Form of the Dense Granule Antigen GRA6 in an Enzyme-Linked Immunosorbent Assay","volume":"84","author":"Redlich","year":"1998","journal-title":"Parasitol. Res."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Yba\u00f1ez, R.H., and Nishikawa, Y. (2022). Comparative Performance of Recombinant GRA6, GRA7, and GRA14 for the Serodetection of T. gondii Infection and Analysis of IgG Subclasses in Human Sera from the Philippines. Pathogens, 11.","DOI":"10.3390\/pathogens11020277"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.micres.2015.11.006","article-title":"Immunodiagnosis and Molecular Validation of Toxoplasma gondii-Recombinant Dense Granular (GRA) 7 Protein for the Detection of Toxoplasmosis in Patients with Cancer","volume":"183","author":"Fallahi","year":"2016","journal-title":"Microbiol. Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2133","DOI":"10.1128\/JCM.00237-06","article-title":"Chimeric Antigens of Toxoplasma gondii: Toward Standardization of Toxoplasmosis Serodiagnosis Using Recombinant Products","volume":"44","author":"Beghetto","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Ferra, B.T., Holec-G\u0105sior, L., Gatkowska, J., Dziadek, B., Dzitko, K., Gr\u0105\u017alewska, W., and Lautenbach, D. (2019). The First Study on the Usefulness of Recombinant Tetravalent Chimeric Proteins Containing Fragments of SAG2, GRA1, ROP1 and AMA1 Antigens in the Detection of Specific Anti-Toxoplasma gondii Antibodies in Mouse and Human Sera. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0217866"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3291","DOI":"10.1007\/s00436-015-4552-6","article-title":"A New Toxoplasma gondii Chimeric Antigen Containing Fragments of SAG2, GRA1, and ROP1 Proteins\u2014Impact of Immunodominant Sequences Size on Its Diagnostic Usefulness","volume":"114","author":"Ferra","year":"2015","journal-title":"Parasitol. Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e00792-20","DOI":"10.1128\/mSphere.00792-20","article-title":"Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection","volume":"5","author":"Young","year":"2020","journal-title":"mSphere"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3963","DOI":"10.1038\/s41467-019-11855-w","article-title":"A CRISPR Platform for Targeted in Vivo Screens Identifies Toxoplasma gondii Virulence Factors in Mice","volume":"10","author":"Young","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e02388-18","DOI":"10.1128\/mBio.02388-18","article-title":"Three Toxoplasma gondii Dense Granule Proteins Are Required for Induction of Lewis Rat Macrophage Pyroptosis","volume":"10","author":"Wang","year":"2019","journal-title":"MBio"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Wang, Y., Hollingsworth, L.R., Sangar\u00e9, L.O., Paredes-Santos, T.C., Krishnamurthy, S., Penn, B.H., Wu, H., and Saeij, J.P.J. (2024). Host E3 Ubiquitin Ligase ITCH Mediates Toxoplasma gondii Effector GRA35-Triggered NLRP1 Inflammasome Activation and Cell-Autonomous Immunity. MBio, 15.","DOI":"10.1128\/mbio.03302-23"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1342","DOI":"10.1016\/j.vaccine.2024.01.050","article-title":"Protective Effect against Toxoplasmosis in BALB\/C Mice Vaccinated with Recombinant Toxoplasma gondii CDPK3, GRA35, and ROP46 Protein Cocktail Vaccine","volume":"42","author":"Li","year":"2024","journal-title":"Vaccine"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Shi, Y., Li, J., Yang, W., and Chen, J. (2023). Protective Immunity Induced by DNA Vaccine Containing TgGRA35, TgGRA42, and TgGRA43 against Toxoplasma gondii Infection in Kunming Mice. Front. Cell. Infect. Microbiol., 13.","DOI":"10.3389\/fcimb.2023.1236130"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"e00858-19","DOI":"10.1128\/mSphere.00858-19","article-title":"Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells","volume":"5","author":"Cygan","year":"2020","journal-title":"mSphere"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e01796-18","DOI":"10.1128\/mBio.01796-18","article-title":"Aspartyl Protease 5 Matures Dense Granule Proteins That Reside at the Host-Parasite Interface in Toxoplasma gondii","volume":"9","author":"Coffey","year":"2018","journal-title":"MBio"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"5258","DOI":"10.1038\/s41467-020-18991-8","article-title":"Genome-Wide Screens Identify Toxoplasma gondii Determinants of Parasite Fitness in IFN\u03b3-Activated Murine Macrophages","volume":"11","author":"Wang","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"112592","DOI":"10.1016\/j.celrep.2023.112592","article-title":"Host Genetics Highlights IFN-\u03b3-Dependent Toxoplasma Genes Encoding Secreted and Non-Secreted Virulence Factors in In Vivo CRISPR Screens","volume":"42","author":"Tachibana","year":"2023","journal-title":"Cell Rep."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e01442-22","DOI":"10.1128\/mbio.01442-22","article-title":"Dense Granule Protein GRA64 Interacts with Host Cell ESCRT Proteins during Toxoplasma gondii Infection","volume":"13","author":"Mayoral","year":"2022","journal-title":"MBio"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"So\u0142owi\u0144ska, K., and Holec-G\u0105sior, L. (2024). Single Cell Expression Systems for the Production of Recombinant Proteins for Immunodiagnosis and Immunoprophylaxis of Toxoplasmosis. Microorganisms, 12.","DOI":"10.3390\/microorganisms12081731"},{"key":"ref_51","first-page":"67","article-title":"Expression and Single-Step Purification of GRA8 Antigen of Toxoplasma gondii in Escherichia Coli","volume":"3","author":"Babaie","year":"2011","journal-title":"Avicenna J. Med. Biotechnol."},{"key":"ref_52","first-page":"235","article-title":"Multiple B-Cell Epitopes in a Recombinant GRA2 Secreted Antigen of Toxoplasma gondii","volume":"34","author":"Murray","year":"1993","journal-title":"Appl. Parasitol."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/11\/879\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T08:39:06Z","timestamp":1761208746000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/11\/879"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,23]]},"references-count":52,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["cimb47110879"],"URL":"https:\/\/doi.org\/10.3390\/cimb47110879","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,23]]}}}