Journal of immunological methods最新文献

{"title":"Development and clinical application of a highly sensitive serological assay for detecting thrombospondin type-1 domain-containing 7A antibodies in idiopathic membranous nephropathy","authors":"Tianyu Zheng ,&nbsp;Xuanli Tang ,&nbsp;Shangbin Kao ,&nbsp;Nannan Tian ,&nbsp;Yufeng Yan ,&nbsp;Dian Li ,&nbsp;Canlong Yan ,&nbsp;Yuan Qin ,&nbsp;Xiumei Zhou ,&nbsp;Yigang Wang ,&nbsp;Guoqin Wang ,&nbsp;Biao Huang","doi":"10.1016/j.jim.2026.114065","DOIUrl":"10.1016/j.jim.2026.114065","url":null,"abstract":"<div><h3>Objective</h3><div>To establish a highly sensitive serological assay for detecting serum thrombospondin type-1 domain-containing 7 A antibodies (THSD7A-Ab) in patients with idiopathic membranous nephropathy (IMN), thereby addressing the current gap in available diagnostic biomarkers.</div></div><div><h3>Methods</h3><div>A time-resolved fluorescence immunoassay (TRFIA) was established by immobilizing THSD7A protein on the solid phase, which captured THSD7A-Ab present in serum samples. Detection was performed using a europium (Eu³⁺)-labeled secondary antibody. Serum THSD7A-Ab levels were measured in both THSD7A-positive IMN patients and M-type phospholipase A2 receptor (PLA2R)-negative IMN patients, and the corresponding seropositivity rates were determined.</div></div><div><h3>Results</h3><div>The TRFIA-THSD7A-Ab assay demonstrated a sensitivity of 0.13 RU/mL, with a linear range from 0.13 to 600 RU/mL. The intra-assay precision ranged from 3.35% to 4.63%, while the inter-assay precision ranged from 5.21% to 9.07%. The recovery rate was 93.78% to 107.20%. Serum THSD7A-Ab levels in THSD7A-associated IMN patients (176.54 ± 224.77 RU/mL) were significantly higher PLA2R-associated IMN patients (11.38 ± 4.76 RU/mL, <em>P</em> &lt; 0.0001) and healthy controls (7.86 ± 2.92 RU/Ml, P &lt; 0.0001). The cut-off value for distinguishing THSD7A-associated IMN was determined to be 13.70 RU/mL. Among PLA2R-negative IMN patients, the positive rate of THSD7A-Ab was 1.6%.</div></div><div><h3>Conclusion</h3><div>A highly sensitive TRFIA method for detecting serum THSD7A-Ab was successfully established for the first time, demonstrating excellent discriminative capability for identifying THSD7 A-positive patients.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114065"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147982001","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":"Integrated workflow for production and in vitro characterization of equine anti-SARS-CoV-2 hyperimmune serum with epitope mapping","authors":"Karla de Santana Evangelista ,&nbsp;Emerson de Castro Barbosa ,&nbsp;Adriana de Souza Andrade ,&nbsp;Liz Ardisson ,&nbsp;Aline Andrade Martins ,&nbsp;Patrícia Ferreira Boasquivis ,&nbsp;Myrian Morato Duarte ,&nbsp;Bárbara de Oliveira Ribeiro ,&nbsp;Job Alves de Souza Filho ,&nbsp;Márcia Helena Borges ,&nbsp;Gladstony de Oliveira Souza ,&nbsp;Carlos Chávez Olórtegui ,&nbsp;Alisson Bruno Luzia ,&nbsp;Sérgio Caldas","doi":"10.1016/j.jim.2026.114059","DOIUrl":"10.1016/j.jim.2026.114059","url":null,"abstract":"<div><div>This study establishes an integrated workflow linking antibody production, neutralization assessment, and epitope mapping, supporting the identification of antigenic regions recognized by polyclonal antibodies generated in vivo. The viral suspension was inactivated with β-propiolactone under biosafety level 3 (BSL-3) conditions, with no cytopathic effect or plaque formation observed, confirming loss of viral replicative capacity. The produced immunogen retained antigenic activity, as demonstrated by reactivity with IgG antibodies from vaccinated and previously infected individuals. Protein analysis by Western blot revealed the presence of major structural proteins, spike and nucleocapsid, in different molecular forms, consistent with previous reports. Sequential immunizations in horses induced robust humoral responses, with antibody titers increasing up to 16-fold after the booster cycle. The purified F(ab')₂ fraction showed 80.4% purity, as estimated by densitometry, and retained antigen binding activity in ELISA assays, supporting the effectiveness of the purification process. The preparation achieved PRNT₅₀ values of 1:97,000 against SARS-CoV-2 under the conditions tested. The use of chemically inactivated whole virus as the immunogen provides a robust approach for generating antibodies recognizing a broad repertoire of viral epitopes while preserving structural complexity. This workflow, which can be safely implemented in BSL-3 laboratories, represents an alternative to recombinant protein-based approaches and may be adaptable to emerging infectious threats. Moreover, the identification of multiple linear immunogenic peptides recognized by the generated antibodies provides experimentally derived information on antibody-binding profiles, supporting potential applications in diagnostics and immunobiotechnology.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114059"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147873913","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":"Specific and quantitative assay for duck variant orthoreovirus utilizing the loop-mediated isothermal amplification technique","authors":"Shuai Zhang ,&nbsp;Xinhui Wei ,&nbsp;Min Han ,&nbsp;Guannan Li ,&nbsp;Xiaomeng Wang ,&nbsp;Youxiang Diao ,&nbsp;Dalin He ,&nbsp;Yi Tang","doi":"10.1016/j.jim.2026.114063","DOIUrl":"10.1016/j.jim.2026.114063","url":null,"abstract":"<div><div>Since 2018 and up to the present, newly discovered avian orthoreovirus (NDRV) types have been a prominent cause of illness concerns in ducks in China. Infected birds with the NDRV frequently had significant splenic necrosis. For effective avian reovirus (ARV) infection management and prevention, it is essential to have access to improved diagnostic techniques for quickly diagnosing recently discovered ARV variations. To identify NDRVs from clinical samples of various avian origins, a unique quantitative loop-mediated isothermal amplification assay (qLAMP) was created in this work. By utilizing 10-fold serial dilutions of the standard substance, excellent low detection limits (1.0 copies/μL) were observed. The test had no cross-reaction with avian viruses other than NDRV, and the generated qLAMP could successfully identify NDRV reference strains. Intra- and interassay tests with variability ranging from 0.08% to 2.78% were used to demonstrate the assay's reproducibility. The qLAMP results demonstrated a greater detection efficiency when compared to those from the examination of 90 clinical samples using traditional PCR and RT-qPCR. Overall, this assay was sensitive and specific for the identification of NDRV clinical samples from diverse bird species and should be used as a standard diagnostic laboratory test.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114063"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147987845","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":"Novel methods for neural IgG characterization and validation in autoimmune neurologic disorders","authors":"Michael Gilligan ,&nbsp;Andrew McKeon","doi":"10.1016/j.jim.2026.114062","DOIUrl":"10.1016/j.jim.2026.114062","url":null,"abstract":"<div><div>Neural autoantibody testing is standard for patients with suspected autoimmune neurologic disorders. The number of neural antibodies has increased dramatically in the past 40 years, with over 50 disease-specific neural antibodies defined to date. The advent of immune-profiling assays such as protein array and phage immunoprecipitation sequencing (PhIP-Seq) has further expedited the rate of antibody discovery, with many recent targets identified using these methods. In this review, we discuss traditional and novel methods of antibody discovery, with an emphasis on the incorporation of protein array and PhIP-Seq into our antibody discovery approach. Advantages, limitations, and potential future uses of these platforms within the field of autoimmune neurology are also considered.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114062"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147973065","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":"Standardizing murine lamina propria leukocyte isolation: Peeling back the layers of intestinal immunology for experimental approaches","authors":"André Luiz Amorim da Costa ,&nbsp;João Victor Conceição Soares de Lima ,&nbsp;Juliana de Oliveira Ribeiro ,&nbsp;Lorraine de Oliveira da Cruz ,&nbsp;Juliana Guedes Nascimento ,&nbsp;Maria Clara Martins de Mattos Amorim De Sá ,&nbsp;Jesuíno Rafael Machado Ferreira ,&nbsp;Kamila Guimarães-Pinto ,&nbsp;Jefferson Antônio Leite ,&nbsp;Alessandra D'Almeida Filardy","doi":"10.1016/j.jim.2026.114060","DOIUrl":"10.1016/j.jim.2026.114060","url":null,"abstract":"<div><div>The intestinal lamina propria harbors a diverse repertoire of innate and adaptive immune cell populations, whose dynamic regulation is critical for maintaining mucosal immune homeostasis and for mounting effective inflammatory responses to infection or tissue injury. Accurate characterization of these populations in murine models requires reliable isolation of viable cells while preserving surface marker integrity.</div><div>However, the heterogeneous cellular composition of the lamina propria poses significant technical challenges, and variability in isolation protocols has contributed to reproducibility issues across studies in mouse experimental models. In particular, enzymatic digestion can markedly affect cell viability and surface molecule expression, with outcomes depending on the choice of enzyme and the inclusion of additives such as deoxyribonuclease I (DNase I) and/or dithiothreitol (DTT). Standardization of isolation methods in the immunology field is therefore essential to ensure consistent recovery of viable immune cells suitable for downstream analyses. To promote reproducibility across laboratories, we introduce a minimal yet comprehensive reporting checklist, together with a systematic overview of the key experimental variables that govern the yield, viability, and phenotypic integrity of lamina propria leukocytes (LPLs) in mice.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114060"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147868441","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":"ELISA method to assign weight-based antibody concentration to antigen-specific Ig in sera","authors":"Marta Benincasa,&nbsp;Salvatore Gemmellaro,&nbsp;Federica Boretto,&nbsp;Miren Iturriza,&nbsp;Rocio Canals,&nbsp;Simona Rondini,&nbsp;Omar Rossi,&nbsp;Francesca Mancini","doi":"10.1016/j.jim.2026.114037","DOIUrl":"10.1016/j.jim.2026.114037","url":null,"abstract":"<div><div>The ability to compare results obtained while assessing immune responses to different antigens using different assays is essential. Enzyme-linked Immunosorbent Assay (ELISA) is one of the most widely used assays to detect and quantify antibodies in human and animal samples. However, it carries the limitation of not giving absolute results but relative measures; specifically, ELISA titers are relative to the sample dilutions tested and ELISA Units/mL, assigned to samples, are dependent on the ELISA Units assigned to a standard serum used in the test, posing the question of how to compare results obtained across different ELISA assays. Some comparability limitations between immunoassays can be solved through calibration against International Standards, but they are not always available and they do not enable comparison across antigens. In an attempt to overcome this problem, we have developed a reliable ELISA-based assay that quantifies antigen-specific antibodies in sera with an absolute concentration of μg/mL, thus allowing comparison of results obtained running the assay with different coating antigens. The methodology is based on comparing the signal of the sera of interest against a curve made with quantified IgG. This method has been successfully applied to quantify total IgG in sera from different species: human, rabbit and mouse.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114037"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146035564","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":"Muscarinic 3 receptor antibodies in Sjögren's disease: Evaluating assay variability and detection methods","authors":"Martha Tsaliki ,&nbsp;Devavrat Dave ,&nbsp;Biji T. Kurien ,&nbsp;R. Hal Scofield","doi":"10.1016/j.jim.2026.114038","DOIUrl":"10.1016/j.jim.2026.114038","url":null,"abstract":"<div><div>The most common manifestations characterizing Sjögren's disease (SjD) are extensive ocular and oral dryness due to decreased lacrimal (keratoconjunctivitis sicca) and salivary gland function (xerostomia). Another hallmark of SjD is the presence of autoantibodies. Acinar cells in the exocrine glands express muscarinic-type-3-receptors (M3R), which act as regulators of saliva secretion. Studies have linked M3R-targeting autoantibodies to reduced saliva secretion in SjD. Earlier studies have proposed that anti-M3R antibodies can pose as potential SjD clinical markers and indicators of dryness. Although functional assays confirm that anti-M3R antibodies have inhibitory activity, other assays have shown a wide range of detection sensitivities for these antibodies (0–100%). Differences in assay type, reagents, or peptide conformation likely explain the wide variation in assay sensitivity and specificity. We searched PubMed/PubMed Central (up to 2024) to assess possible reasons for this discrepancy and identify the best assay with which to screen for these antibodies. This review highlights the importance of conformational assays and recommends standardization to improve diagnostic accuracy.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114038"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146125047","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":"Establishment of reference intervals for serum TNF-α, IFN-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17 and IFN-γ in healthy Chinese adults using flow cytometry","authors":"Hui Xie ,&nbsp;Guiting Zhang ,&nbsp;Yongquan Xia ,&nbsp;Jie Pan ,&nbsp;Han Shen ,&nbsp;Mao Xia","doi":"10.1016/j.jim.2026.114034","DOIUrl":"10.1016/j.jim.2026.114034","url":null,"abstract":"<div><h3>Background</h3><div>Cytokines, small protein molecules from immune and nonimmune cells, are crucial for immune regulation, hematopoiesis, cell growth, and tissue repair. This study employed flow cytometry to measure serum levels of tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interleukin-17 (IL-17) and interferon-γ (IFN-γ), establishing reference intervals to aid clinical diagnostics and treatment.</div></div><div><h3>Methods</h3><div>From January to March 2024, 299 participants were selected based on predetermined exclusion and inclusion criteria. The Kolmogorov-Smirnov test checked the distribution of cytokine levels, and reference intervals were set using the 95th percentile method following EP28-A3c and WS/T 402–2012 guidelines.</div></div><div><h3>Results</h3><div>The cytokines were not normally distributed, with notable sex differences in levels (<em>P</em> &lt; 0.05), necessitating separate reference intervals for each sex. For women, these were: TNF-α ≤2.94 pg/mL, IFN-α ≤7.69 pg/mL, IL-1β ≤2.40 pg/mL, IL-2 ≤ 2.40 pg/mL, IL-4 ≤ 3.93 pg/mL, IL-5 ≤ 2.42 pg/mL, IL-6 ≤ 15.96 pg/mL, IL-8 ≤ 23.42 pg/mL, IL-10 ≤ 7.10 pg/mL, IL-12p70 ≤ 7.36 pg/mL, IL-17 ≤ 4.14 pg/mL, IFN-γ ≤4.29 pg/mL. For men: TNF-α ≤ 2.65 pg/mL, IFN-α ≤ 7.50 pg/mL, IL-1β ≤2.40 pg/mL, IL-2 ≤ 2.52 pg/mL, IL-4 ≤ 4.58 pg/mL, IL-5 ≤ 2.40 pg/mL, IL-6 ≤ 14.61 pg/mL, IL-8 ≤ 15.88 pg/mL, IL-10 ≤ 3.94 pg/mL, IL-12p70 ≤ 7.18 pg/mL, IL-17 ≤ 3.63 pg/mL, IFN-γ ≤4.18 pg/mL.</div></div><div><h3>Conclusions</h3><div>We established reference intervals for serum TNF-α, IFN-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17 and IFN-γ in healthy adults from East China. These findings are crucial for assessing human immune status, diagnosing diseases, and advancing personalized medicine, laying the groundwork for studying inflammatory disease mechanisms.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114034"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145928989","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":"Optimization of a versatile cell ELISA for detection of antibodies against membrane protein BCMA","authors":"Petra Kern ,&nbsp;Ana Kolenc ,&nbsp;Valerija Kovač ,&nbsp;Vladka Čurin Šerbec","doi":"10.1016/j.jim.2025.114024","DOIUrl":"10.1016/j.jim.2025.114024","url":null,"abstract":"<div><div>Monoclonal antibodies against membrane proteins are commonly used for diagnostic and therapeutic applications. They are still mostly produced using hybridoma technology or phage display, generating large numbers of antibody candidates that need to be tested in a short period of time. As most membrane proteins do not retain their three-dimensional structure after their isolation from the cell membrane, cell-based high-throughput assays are needed for the primary screening of hundreds of antibody candidates.</div><div>We designed a simple cell-based ELISA by taking the protocols for indirect cell immunostaining and integrating them into a standard ELISA format. This cell ELISA is conducted in 96-well round bottom plates and consists of two immunostaining and washing steps, and a detection step. We designed the assay on B-cell maturation antigen as a model membrane protein antigen. We used several cell lines and a wide range of antibody concentrations. The assay was validated with imaging flow cytometry.</div><div>Although we obtained comparable results in specificity with cell ELISA and flow cytometry, screening multiple samples with cell ELISA is substantially faster than with flow cytometry, due to its ability to test several samples simultaneously. Using cell ELISA, a single operator can easily test several hundred antibodies per day. Key advantages of the protocol presented here are that it can be used for suspension or adherent cell lines and for cell lines that express the membrane protein in native or recombinant form. As such, it can potentially be translated to diverse antibody–antigen–cell line systems. This was confirmed by successfully detecting anti-CD3 and anti-CD19 antibodies binding to Jurkat and Raji cells, respectively.</div><div>Cell ELISA developed in our laboratory is a high-throughput, inexpensive, and easy-to-perform method for screening hybridoma that produce antibodies targeting membrane proteins. It does not require the use of soluble pure protein and it can be performed in any laboratory that specializes in antibody development and ELISA testing.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114024"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145833712","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":"PLGA-based nanovaccine delivering chimeric IpaD-StxB protein induces robust immune response against Shigella infection","authors":"Ayoub Fazeli ,&nbsp;Hosein Honari ,&nbsp;Davoud Sadeghi ,&nbsp;Hossein Samiei-Abianeh ,&nbsp;Rasoul Rafiei-Foroushani","doi":"10.1016/j.jim.2026.114036","DOIUrl":"10.1016/j.jim.2026.114036","url":null,"abstract":"<div><h3>Background and aim</h3><div>Shigellosis is a major cause of morbidity and mortality worldwide, especially among children under five in low- and middle-income countries. Despite its public health burden, there is no licensed vaccine. Recombinant and nanoparticle-based vaccine approaches offer promising alternatives to conventional methods. This study aimed to develop a chimeric nanovaccine based on the fusion of <em>Shigella flexneri</em> IpaD and StxB, encapsulated in PLGA nanoparticles, and to evaluate its immunogenic potential in a murine model.</div></div><div><h3>Methods</h3><div>The recombinant IpaD-StxB (IdSb) protein was expressed in <em>E. coli</em>, purified under denaturing conditions, and encapsulated into PLGA nanoparticles using a double emulsion solvent evaporation technique. Particle size, morphology, encapsulation efficiency, and in vitro release were characterized. BALB/c mice were immunized with either Nano-IdSb or free IdSb, and serum IgG levels were assessed via ELISA to evaluate the humoral immune response.</div></div><div><h3>Results</h3><div>The 24 kDa recombinant IdSb protein was successfully expressed in a prokaryotic system, yielding 10 mg/mL. PLGA nanoparticles encapsulating IdSb had an average diameter of 166 nm, high encapsulation efficiency (97%), and a loading capacity of 3.5%. They enabled sustained protein release over 40 days. Mice immunized with Nano-IdSb exhibited significantly stronger IgG responses than those receiving the free protein, indicating enhanced immunogenicity and antigen stability.</div></div><div><h3>Conclusion</h3><div>PLGA-encapsulated IdSb nanovaccine elicited a robust humoral immune response and demonstrated favorable physicochemical properties, supporting its potential as a cost-effective and broadly protective vaccine candidate against <em>Shigella</em> spp.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114036"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145952200","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}