Journal of immunological methods最新文献

{"title":"Development of an ELISA strategy for the serological diagnosis of farmer's lung disease","authors":"Adeline Rouzet ,&nbsp;Eliane Devillers ,&nbsp;Coralie Barrera ,&nbsp;Emeline Scherer ,&nbsp;Laurence Millon ,&nbsp;Anne-Pauline Bellanger","doi":"10.1016/j.jim.2025.113902","DOIUrl":"10.1016/j.jim.2025.113902","url":null,"abstract":"<div><h3>Context</h3><div>Farmer's lung disease (FLD) is a form of hypersensitivity pneumonitis due to regular exposure to microbial antigens present in moldy hay. Serological diagnosis is usually based on immunoprecipitation techniques using 1 to 12 in-house antigens. To improve standardization, recombinant antigens (r-Ags) have been produced from three etiological agents involved in FLD.</div></div><div><h3>Aim</h3><div>The aim of this study was to develop a strategy using an ELISA test for the serological diagnosis of FLD.</div></div><div><h3>Methods</h3><div>The ELISA strategy was developed on an initial group of patients and then validated using sera from 29 patients with FLD and 54 exposed but non-FLD patients. Using sera from the second group of patients, the ELISA was simplified by using purified antigens from <em>Saccharopolyspora rectivirgula</em>, <em>Lichtheimia corymbifera</em>, and <em>Eurotium amstelodami</em>, each mixed with a corresponding r-Ag. The performance of the ELISA was evaluated according to the antigens used and also associated with an immunoprecipitation technique.</div></div><div><h3>Results</h3><div>The ELISA test using three purified antigens (PAgs) mixed with 3 r-Ags showed a sensitivity of 97 % and a specificity of 83 % (AUC: 0.84). The use of ELISA as a screening technique and double diffusion (using 6 somatic antigens) as a confirmatory technique in cases of moderate or positive ELISAs showed a sensitivity of 93 % and a specificity of 84 % (AUC: 0.86).</div></div><div><h3>Conclusion</h3><div>The use of a mixture of purified protein antigens and highly standardized recombinant antigens from three etiological agents involved in FLD offers an effective and innovative strategy for the serological diagnosis of FLD cases. This strategy has been successfully applied in the routine workflow of our laboratory.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113902"},"PeriodicalIF":1.6,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144491070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An unexpected bioanalytical challenge caused by positive control antibodies in a clinical immunogenicity assay - A simple solution and broadly applicable recommendations.","authors":"John Hok Nin Lowe, Jenny Yanhong Li, Mauricio Maia","doi":"10.1016/j.jim.2025.113901","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113901","url":null,"abstract":"<p><p>Anti-drug antibody (ADA) assays are an important element in the suite of bioanalytical methods required for assessment of the safety and efficacy of recombinant-protein therapeutics. As such, and following extensive optimization, there is an expectation that clinical ADA assays be fully validated for multiple performance parameters, including sensitivity, specificity, reagent stability, and robustness. Among critical reagents used in ADA assays, ADA positive controls (PCs) play a crucial role in multiple stages of assay development and validation, including selection of assay format, establishing assay cut-points, estimating assay relative sensitivity, assessing assay precision, as well as ensuring acceptable assay performance during sample testing. This manuscript highlights an unexpected and highly-impactful PC performance inconsistency attribute we encountered prior to validation of a clinical ADA assay. We also describe the investigation that identified the root cause of this problem: the immune complexation between the murine monoclonal antibody used as the surrogate PC and human anti-mouse antibody present in the serum used to prepare the PC. We conclude that murine monoclonal antibodies are not fully appropriate reagents for use as PCs in clinical ADA assays. Finally, potential approaches to circumvent or mitigate this specific problem in clinical ADA assays are discussed.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113901"},"PeriodicalIF":1.6,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of microfluidic ELISA for measuring humoral responses to clostridial antigens in vaccinated cattle.","authors":"Joo Youn Park, Amelia Woolums, Robert Wills, Rhonda Vann, Keun Seok Seo","doi":"10.1016/j.jim.2025.113900","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113900","url":null,"abstract":"<p><p>Clostridial diseases significantly threaten livestock health, particularly in cattle, underscoring the need for effective vaccination strategies. This study develops and optimizes a microfluidic-based enzyme-linked immunosorbent assay (ELISA) for the rapid (< 1 h) and cost-effective measurement of IgG antibody levels against various clostridial toxins in cattle vaccinated with a multivalent clostridial vaccine. This assay requires only 5 μL of sample and reagent volume, demonstrating high repeatability and reproducibility with coefficient of variation (CV) values ranging from 0.1 % to 8.5 % across all tested clostridial antigens. The Limit of Detection (LOD) of the assay ranged from 1:150 to 1:800, allowing for sensitive detection of antibody levels. For Clostridium perfringens ε toxin, antibody titers were measured using a commercial kit, while microfluidic ELISA was applied to assess titers against tetanus toxoid, Clostridium septicum α toxin, Clostridium novyi type B toxins, and Clostridium sordellii toxins. Significant increases in IgG antibody levels were observed for C. perfringens ε toxin and tetanus toxoid following both primary and booster vaccine doses, peaking by day 42. Antibody titers against C. septicum α toxins and C. novyi type B toxins increased after the primary dose, peaking at day 42, while no booster effect was seen for C. sordellii. These findings highlight the utility of microfluidic ELISAs as a practical and efficient tool for assessing humoral immune responses to clostridial toxins in vaccinated cattle, with potential application for the herd level disease surveillance and vaccine efficacy assessment.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113900"},"PeriodicalIF":1.6,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144497233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of vectors for reformatting scFv fragments derived from phage display libraries into native IgG1 structures for in vivo imaging and therapeutic applications","authors":"Marton Fogarasi ,&nbsp;Corina Roman ,&nbsp;Simona Dima","doi":"10.1016/j.jim.2025.113899","DOIUrl":"10.1016/j.jim.2025.113899","url":null,"abstract":"<div><div>Monoclonal antibodies are a crucial class of therapeutic agents utilized for the treatment of various disorders. Typically, fully human therapeutic antibodies are created using the antibody fragment phage display from naive libraries and are selected against the antigen in a format known as the single-chain Fv (scFv) fragment. To be employed in immunotherapy, the scFv fragment needs to be reformatted into the complete IgG structure with a native-like conformation and expressed in mammalian cells. In this context, we have established an expression system for reformatting scFv fragments into complete IgG1 molecules, enabling both antibody chains to be cloned within the same vector. These constructs are based on the IgGγ1 heavy chain, with the light chain belonging to either the lambda or kappa isotype. For <em>in vivo</em> imaging, we have designed vectors with specific mutations in the IgG1 lower hinge region and C_H2 domain to impede the antibody's effector function, ensuring that immune cells are not activated. The efficiency of this expression system was evaluated by producing two antibodies: one with the lambda light chain and the other with the kappa light chain. Their expression levels were assessed in HEK-293 and CHO-K1 cell lines. Structurally, the resulting antibodies are properly assembled and folded into their quaternary structure, while functionally, these immunoglobulins specifically recognize the antigens and inhibit cancer cell invasion.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113899"},"PeriodicalIF":1.6,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144366071","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of an enteric pathogen multiplex immunoassay to measure antibody responses in blood and saliva for integrated serology applications","authors":"Lindsay N. Avolio ,&nbsp;Nora Pisanic ,&nbsp;Kate L. Kruczynski ,&nbsp;William J. Moss ,&nbsp;Kawsar R. Talaat ,&nbsp;Robert W. Kaminski ,&nbsp;Kristen A. Clarkson ,&nbsp;Sarah Elwood ,&nbsp;Restituta Mosha ,&nbsp;Eric R. Houpt ,&nbsp;Estomih R. Mduma ,&nbsp;James A. Platts-Mills ,&nbsp;Christopher D. Heaney","doi":"10.1016/j.jim.2025.113898","DOIUrl":"10.1016/j.jim.2025.113898","url":null,"abstract":"<div><h3>Background</h3><div>Enteric infections are a leading cause of global morbidity and mortality, with the highest burden of disease in young children in low- and middle-income countries. Comprehensive profiling of enteric infections, which requires intensive sampling and molecular and culture-based detection methods, may miss recent and historical infections. High-throughput antibody-based testing of blood or non-invasive specimens like saliva can fill this gap, especially in vulnerable populations.</div></div><div><h3>Methods</h3><div>We developed a bead-based multiplex immunoassay (MIA) that includes enteric pathogen targets from bacteria (<em>Shigella</em>, Enterotoxigenic <em>Escherichia coli</em> [ETEC], <em>Campylobacter</em>, typhoidal <em>Salmonella</em>, non-typhoidal <em>Salmonella</em>); viruses (norovirus, rotavirus, hepatitis E virus, adenovirus, astrovirus); and protozoa (<em>Cryptosporidium</em>, <em>Giardia</em>), as well as select targets for respiratory viruses and vaccine preventable diseases (VPDs). Coupling was optimized for peptide and lipopolysaccharide (LPS) antigens separately and assay performance characterized for both serum and salivary applications.</div></div><div><h3>Results</h3><div>The ability to assess IgG and IgA antibody responses in serum ranging over 4 orders of magnitude was demonstrated, allowing for assessment of various phases of infection. The mean inter-operator assay precision was excellent (6.7 % CV in serum IgG and 10.6 % CV in saliva IgG). The MIA was highly correlated (<em>r</em> = 0.72–0.96, <em>p</em> &lt; 0.0001) with reference ELISA antibody titers in selected antigens.</div></div><div><h3>Conclusions</h3><div>Improved scalability of enteric pathogen detection through an MIA may be combined with existing serosurveillance for VPDs and respiratory viruses and complement broad enteric pathogen burden and vaccine studies. This framework for integrated serology provides methods for assessing disease burden to inform prevention and treatment guidelines and guide targeted interventions.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113898"},"PeriodicalIF":1.6,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144368942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Indirect ELISA diagnostic based on recombinant 3AB3 nonstructural protein of foot-and-mouth disease virus for use in porcine serology","authors":"M. Rout ,&nbsp;L.K. Pandey ,&nbsp;B.R. Prusty ,&nbsp;J.K. Mohapatra ,&nbsp;R.P. Singh","doi":"10.1016/j.jim.2025.113897","DOIUrl":"10.1016/j.jim.2025.113897","url":null,"abstract":"<div><div>Foot-and-mouth disease (FMD), a disease of risk group 4 with transboundary importance, is a highly contagious devastating menace bearing serious concern that affects all susceptible cloven-hoofed animals eventuating striking economic losses to the livestock world. The elementary objective of this study was to optimize and validate the working parameters of an indirect ELISA based on prokaryotically expressed recombinant 3AB3 nonstructural protein (NSP) to estimate the 3AB3 NSP antibodies emanating from FMD virus (FMDV) infection and evaluate its performance highlighting the potential utility as a serological implement for surveillance of the disease in pig. Diversified categories of enzyme-linked immunosorbent assays (ELISAs) have been developed with wider use for evaluation of herd immunity in animals. However, recombinant antigen-based ELISAs are contemplated to be the preferred alternatives to tedious virus neutralization test, while antibody to 3AB3 NSP has been explored to be the most dependable and trust-worthy indicator for FMD. The 3AB3 NSP indirect ELISA, already in use in the country as a test for screening FMDV infection-specific antibodies in the bovine species was modified using anti-porcine IgG-horse radish peroxidase (HRP) conjugate and the test was successfully applied for pig. The assay performance was compared with an internationally accepted PrioCHECK® FMDV NS blocking ELISA kit. The overall diagnostic sensitivity (DSn) of the indirect ELISA was estimated to be 96.20 % (228/237), while the diagnostic specificity (DSp) on naïve and vaccinated animals varied at 96.63 % (316/327) and 96.94 % (286/295), respectively. In India, where FMD is prevalent and the pig population is high in some states, this ‘in-house’ optimized test system can be of immense utility paving the way for sero-epidemiological study in pigs, essentially critical for effective implementation of disease control strategies.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113897"},"PeriodicalIF":1.6,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144336612","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of antigen B subunit 2 (AgB2) gene polymorphism in sheep isolates of Echinococcus granulosus sensu lato and effect on serologic response","authors":"Harun Kaya Kesik ,&nbsp;Figen Celik ,&nbsp;Seyma Gunyakti Kilinc ,&nbsp;Muhammet Uslug ,&nbsp;Sami Simsek","doi":"10.1016/j.jim.2025.113896","DOIUrl":"10.1016/j.jim.2025.113896","url":null,"abstract":"<div><div>Cystic Echinococcosis (CE) is one of the most common helminth infections in many parts of the world. Antigen B (AgB) is a key molecule secreted by both the germinal membrane and protoscoleces during the larval stages of <em>Echinococcus granulosus</em>. Characterizing polymorphisms in the genes encoding AgB can improve the interpretation of serological diagnostic tests. This study aimed to determine the polymorphism in the AgB subunit 2 (AgB2) gene in sheep isolates of <em>E. granulosus</em> sensu lato and to investigate its effect on the serological response. Germinal membranes from 41 sheep hydatid cysts and corresponding blood serum samples were collected from slaughterhouses in Elazig and Bingol provinces of Türkiye. Total genomic DNA was isolated, and PCR amplification of the AgB2 gene region was followed by DNA sequencing to evaluate genetic diversity. Western Blot (WB) analysis was performed using partially purified cyst fluid antigen rich in AgB. Sequence analysis revealed that the 663 bp AgB2 gene region exhibited high polymorphism. A total of 33 polymorphic sequences were identified and classified into 10 different haplotypes (AgB2.Hap_01 to AgB2.Hap_10). Among these, 31 (93.9 %) samples were WB-positive, while 2 (6.1 %) were negative. The WB test demonstrated a sensitivity of 80.4 % and a specificity of 100 %. These results suggest a relationship between the polymorphism in the AgB2 gene and variations in the serological response.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113896"},"PeriodicalIF":1.6,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144254802","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of multiplexed flow based assay for simultaneous identification and estimation of meningococcal capsular polysaccharide serogroups A, C, W, Y and X for conjugate vaccine manufacturing","authors":"Sushil Patni,&nbsp;Reena Verma,&nbsp;Mahavir Desarda,&nbsp;Sandip Naikwade,&nbsp;Hemant Deorukhakar,&nbsp;Rajeev Dhere,&nbsp;Asha Mallya","doi":"10.1016/j.jim.2025.113895","DOIUrl":"10.1016/j.jim.2025.113895","url":null,"abstract":"<div><div>The development of a multivalent meningococcal polysaccharide vaccine requires the analysis of active ingredient, i.e., capsular polysaccharides, at various stages of its development. Furthermore, in accordance with WHO guidelines for the release of any batch of purified polysaccharides, it is imperative to determine whether any heterologous polysaccharides, with a threshold of less than 1 % of their dry weight, are present in the sample. We have developed a flow-based multiplexed bead-based competitive inhibition assay (BBCIA), which identifies the meningococcal polysaccharide A, C, Y, W, and X at purified polysaccharides, conjugate bulk, and final vaccines development, and quantified the 1 % heterologous polysaccharides as contaminant in purified polysaccharides. Polysaccharides are coupled to the carboxylated microsphere and incubated with a serogroup-specific antibody-antigen mixture. Unneutralized antibodies bind to respective antigens coupled onto the beads and are expressed in terms of median fluorescence intensity (MFI). The BBCIA identified meningococcal polysaccharides with ≥90 % inhibition during identity, and estimated the heterologous polysaccharides as contaminants with 70–130 % recovery of spiked polysaccharides with a percentage CV &lt; 20 %. Results were validated as per ICH guidelines, such as limit of detection and quantification, linearity, precision, robustness, specificity, and accuracy in accordance to international standards. We are reporting an assay tool that simultaneously identifies and quantifies polysaccharides of the multivalent meningococcal vaccine with the advantages of less time, labour and sample volume in analysis with higher sensitivity and accuracy. With high throughput capabilities and increased sensitivity, BBCIA can be readily adopted to identify and estimate various serogroup-specific antigens for vaccine development.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113895"},"PeriodicalIF":1.6,"publicationDate":"2025-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144266422","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative flow cytometry using quantitative streptavidin-protein G-biotin beads (qBeads)","authors":"Maitreyi Bharath ,&nbsp;Anh Le ,&nbsp;Vaishnavi Konda ,&nbsp;Shivika Srivastava ,&nbsp;Mark Beliaev ,&nbsp;Mengbing Chen ,&nbsp;Anand George ,&nbsp;Andy Zhang ,&nbsp;Sophie Tan ,&nbsp;Eben Ginto ,&nbsp;Aaditya Awatiger ,&nbsp;Kushagra Bahuguna ,&nbsp;Vienna Li ,&nbsp;Suditi Kedambadi ,&nbsp;Xiaolin Cao ,&nbsp;Hao Zeng ,&nbsp;Guofei Xiong ,&nbsp;Mihika Prabhu ,&nbsp;Ayana Modi ,&nbsp;Yanlan Wang ,&nbsp;John Wang","doi":"10.1016/j.jim.2025.113883","DOIUrl":"10.1016/j.jim.2025.113883","url":null,"abstract":"<div><div>Quantitative flow cytometry (qFCM) is a powerful approach for the precise measurement of cellular and molecular characteristics, offering significant advantages in biomedical research, clinical diagnostics, and therapeutic applications. However, current qFCM beads, relying on chemical conjugation face several limitations. This study explored the use of streptavidin-coated beads with a defined quantity of Protein G-biotin for quantitative flow cytometry, demonstrating that these quantitative streptavidin-Protein G-biotin beads (qBeads) can be used for qFCM analysis through both direct and indirect methods, enabling the development of standardized assays for a wide range of applications.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113883"},"PeriodicalIF":1.6,"publicationDate":"2025-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144190358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and validation of dengue virus envelope protein domain III IgG antibody enzyme-linked immunosorbent assay","authors":"Juan Ignacio Marfía ,&nbsp;Ignacio Smith ,&nbsp;Alexandra Marisa Targovnik ,&nbsp;Federico Alejandro Di Lello ,&nbsp;Federico Javier Wolman ,&nbsp;Diego Martín Flichman ,&nbsp;María Victoria Miranda ,&nbsp;Silvina Noemí Valdez","doi":"10.1016/j.jim.2025.113887","DOIUrl":"10.1016/j.jim.2025.113887","url":null,"abstract":"&lt;div&gt;&lt;h3&gt;Introduction&lt;/h3&gt;&lt;div&gt;The global incidence of Dengue virus (DENV) infections has significantly increased in recent decades, becoming a public health emergency of global concern. Diagnostic tools for DENV infection include detection of the virus, viral RNA, or viral antigens, which are usually cumbersome or require sophisticated medical facilities and trained personnel. Serological tests are the most widely used methods to detect dengue infection due to low cost and operational simplicity. The detection of antibodies (IgM and IgG) in the blood of an infected individual is an indirect method of diagnosing DENV and IgG can be used as long-term detection. The aim of this work was to develop and validate an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-DENV IgG antibodies, and to determine its usefulness in the diagnosis and seroepidemiological survey of DENV.&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Materials and methods&lt;/h3&gt;&lt;div&gt;The production of the antigen was carried out using the tetravalent DENV E protein-based provided by Trebe Biotech. The recombinant baculovirus was obtained using the Bac to Bac® baculovirus expression system. The recombinant tetravalent dengue antigen was expressed in insect larvae, purified by IMAC and identified by SDS-PAGE and western blot analysis. The system used in this work has the advantage of using insect pests as biofactories. Recombinant protein production is high-yield and production times are short. Furthermore, it has no product size limit and is highly flexible to sequence changes or the emergence of new serotypes. An indirect ELISA was developed using purified tetravalent DENV antigen immobilized on polystyrene microplates; human sera samples from individuals with no history of DENV infection (&lt;em&gt;n&lt;/em&gt; = 22) and human sera samples from individuals with clinical history and diagnosed of DENV infection (&lt;em&gt;n&lt;/em&gt; = 23) were then incubated. Immune complexes were revealed with anti-human IgG-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. In order to establish DENV-IgG seroprevalence, the developed ELISA was applied to analyze samples from blood donor centers from different geographic regions of the country (&lt;em&gt;n&lt;/em&gt; = 504) and normal human sera (&lt;em&gt;n&lt;/em&gt; = 17).&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Results&lt;/h3&gt;&lt;div&gt;The efficiently produced recombinant dengue antigen was used for the development and validation of a high sensitivity (91.30 %) and specificity (90.91 %) indirect ELISA for the detection of anti-DENV IgG antibodies. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from normal control individuals and patients with DENV (AUC = 0.9901); the cut-off value was established at 2 SDs. In the assessment of seroprevalence levels of anti-DENV antibodies, 33 out of 504 analyzed samples (6.55 %) tested positive using our developed ELISA.&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Conclusions&lt;/h3&gt;&lt;div&gt;This technique is rap","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113887"},"PeriodicalIF":1.6,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187132","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}