Journal of immunological methods最新文献

{"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}

{"title":"Multiplex ACE2-RBD binding inhibition assay: An integrated tool for assessing neutralizing antibodies to SARS-CoV-2 variants and protection against breakthrough infections","authors":"Emma Bloch ,&nbsp;Laura Garcia ,&nbsp;Françoise Donnadieu ,&nbsp;Jason Rosado ,&nbsp;Delphine Planas ,&nbsp;Timothée Bruel ,&nbsp;Laurent Hocqueloux ,&nbsp;Thierry Prazuck ,&nbsp;Olivier Schwartz ,&nbsp;Laura Tondeur ,&nbsp;Laurie Pinaud ,&nbsp;Arnaud Fontanet ,&nbsp;Stéphane Pelleau ,&nbsp;Michael White","doi":"10.1016/j.jim.2025.113886","DOIUrl":"10.1016/j.jim.2025.113886","url":null,"abstract":"<div><div>SARS-CoV-2 remains a significant health threat due to its high infection and mutation rates. The emergence of new variants of concern poses challenges as they can lead to immune escape mutations, potentially reducing the efficacy of vaccines and antibody therapeutics. The receptor binding domain (RBD) of SARS-CoV-2 is particularly noteworthy as it is both the most rapidly evolving domain and the principal target of neutralizing antibodies. As an alternative to time-consuming and expensive neutralization assays, we have developed a bead-based multiplex surrogate virus neutralization test based on ACE2-RBD binding inhibition. We demonstrated how our high-throughput assay allows us to simultaneously assess anti-RBD neutralizing antibodies levels against multiple SARS-CoV-2 variants, providing data that is consistent with the gold-standard live virus neutralization assay. The utility of this assay was demonstrated by applying it to a large French population cohort to demonstrate that hybrid immunity (generated by a combination of vaccination and infection) is associated with protection against infection with the Omicron BA.1 and BA.2 lineages of SARS-COV-2.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113886"},"PeriodicalIF":1.6,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144168562","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 performance evaluation of a novel, fully automated, high-throughput GAD65 autoantibody assay on the Phadia platform","authors":"Daniel Filchtinski ,&nbsp;José Kayser ,&nbsp;Virginie Ettner ,&nbsp;Sanja Holz ,&nbsp;Bjarne Kristensen ,&nbsp;Johannes Schulte-Pelkum ,&nbsp;Isabel Wilhelm ,&nbsp;Raimund Fiedler","doi":"10.1016/j.jim.2025.113885","DOIUrl":"10.1016/j.jim.2025.113885","url":null,"abstract":"<div><h3>Objectives</h3><div>In clinics, diabetes is determined by blood glucose levels or by glycosylated hemoglobin (HbA1c). However, to distinguish between Type 1 and Type 2 diabetes (T1D and T2D) for adequate treatment, patients are tested for the presence of Type 1 diabetes-relevant autoantibodies in the second tier.</div><div>Anti-GAD65 autoantibody is one of the main markers for T1D in both pediatric and adult T1D patients. If the anti-GAD65 autoantibody is detectable, the patients have type 1 diabetes and need to be treated with insulin.</div><div>In the clinical routine diagnostics of type 1 diabetes, GAD65-serology is performed using a special assay architecture known as bridging ELISA. Adapting these assays onto automated ELISA systems has shown to be a constant challenge, so they can be hardly applied for high throughput testing in a routine clinical laboratory.</div></div><div><h3>Methods</h3><div>We developed the first fully automated, high-throughput GAD65 autoantibody fluorescence enzyme immunoassay based on the bridging immunoassay format on Phadia instrument platform, termed EliA GAD65 and compared its performance with the bridging ELISA from RSR™ (GADAb ELISA) and the fully automated MAGLUMI® GAD65 chemiluminescence assay (CLIA) from Snibe Diagnostics.</div></div><div><h3>Results</h3><div>When tested for clinical performance, ROC Analysis of EliA GAD65 showed an Area under the curve (AUC) of 0.906 (95 % CI 0.864 to 0.949) compared to the AUC of 0.923 (95 % CI 0.884to 0.963) of GADAb ELISA with no significant difference between the two assays (<em>p</em> = 0.384). When adjusted to a specificity of 96 %, EliA GAD65 showed a sensitivity of 76 % (CI 95 % 66.4 % to 84.0 %) compared to a sensitivity of 74 % (95 % CI 64.6 % to 81.6 %) of GADAb ELISA. When the fully automated MAGLUMI® GAD65 CLIA was compared to GADAb ELISA, it showed a total agreement of 92.9 % (95 % CI 86.6 % to 96.4 %) with less positive samples detected in contrast to the total agreement of 100 % (95 % CI 96.7 % to 100 %) of EliA GAD65 to GADAb ELISA.</div></div><div><h3>Conclusions</h3><div>Fully automated EliA GAD65 assay displays similar clinical characteristics as the well-established bridging GADAb ELISA and a higher agreement with the GADAb ELISA than the automated MAGLUMI® GAD65 CLIA.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113885"},"PeriodicalIF":1.6,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187131","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":"Reference intervals for functional lymphocyte proliferation studies using 3H thymidine uptake in adults","authors":"Rohan Ameratunga ,&nbsp;Danny Lim ,&nbsp;Hilary Longhurst ,&nbsp;James Mehrtens ,&nbsp;Euphemia Leung ,&nbsp;Klaus Lehnert ,&nbsp;Richard Steele ,&nbsp;See-Tarn Woon","doi":"10.1016/j.jim.2025.113884","DOIUrl":"10.1016/j.jim.2025.113884","url":null,"abstract":"<div><h3>Aims</h3><div>This study aimed to establish the reference intervals for the ³H thymidine uptake assays of functional cellular immunity. Functional tests of cellular immunity play a critical role in several clinical scenarios. An excellent T cell response to vaccine antigens is arguably the best in vitro test of normal cellular immunity.</div></div><div><h3>Methods</h3><div>In this article the reference intervals for over 250 healthy adults were calculated for lectins, anti-CD3 (OKT3) and antigens comprising Candida, tetanus and diphtheria toxoids. These samples were provided by volunteer blood donors as controls to be run in parallel with diagnostic tests of in vitro cellular immunity in patients with suspected immunodeficiency.</div></div><div><h3>Results</h3><div>The control samples uniformly responded to PHA. The response to other lectins, OKT3 and antigens was more heterogeneous. The variable responses to diphtheria and tetanus toxoids likely reflected the immunization status of these blood donors to these vaccine antigens.</div></div><div><h3>Conclusions</h3><div>Although there has been a recent move to implement non-radioactive methods for functional cellular immunity, ³H thymidine uptake continues to be utilized in many diagnostic laboratories because of excellent sensitivity. Robust vaccine-induced cellular responses in immunodeficient patients may inform clinical decisions such as using live attenuated viral vaccines. Similarly, markedly impaired responses to phytohemagglutinin A (PHA), in the appropriate clinical context, supports a diagnosis of Severe Combined Immunodeficiency (SCID) and the need for urgent hematopoietic stem cell transplantation (HSCT). The reference intervals established by this audit will assist other diagnostic laboratories using this platform.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113884"},"PeriodicalIF":1.6,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144181180","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 a one-step diagnostic and differential immunochromatographic method for mpox and chickenpox","authors":"Laiqing Li ,&nbsp;Qiang Jia ,&nbsp;Hongrui Lai ,&nbsp;Guoliang Luo ,&nbsp;Yanhong Lu ,&nbsp;Huankun Liang ,&nbsp;Wenqi Dong ,&nbsp;Cuicui Chen","doi":"10.1016/j.jim.2025.113881","DOIUrl":"10.1016/j.jim.2025.113881","url":null,"abstract":"<div><h3>Objective</h3><div>Mpox and chickenpox are highly contagious diseases with similar clinical symptoms and co-infection, and diagnostic tests are extremely useful in their differentiation. Mpox virus and chickenpox virus are the main causative pathogens of mpox and chickenpox respectively. This study aims to develop a rapid diagnostic immunochromatographic trip for differential diagnosis of mpox and chickenpox viruses.</div></div><div><h3>Methods</h3><div>An immunochromatographic assay (ICA) based on the double-antibody sandwich format was developed. First, the paired monoclonal antibodies (MAbs) were prepared: for mpox, the detecting MAb1 and capture MAb2; for chickenpox, the detecting MAb3 and capture MAb4. These MAbs were against the recombinant antigens (AR/gE) of mpox and chickenpox viruses. Then, the detecting MAbs were conjugated to gold nanoparticles and coated onto the conjugate pads. The capture MAbs were immobilized onto a nitrocellulose membrane at two detection zones to form the Test 1 (T1, to test AR) line and Test 2 (T2, to test gE) line, respectively. Goat anti-mouse IgG antibodies were immobilized onto the nitrocellulose membrane to form the control (C, to verify the effectiveness of the strip) line as the control. Finally, it was assembled into a one-step double T-line colloidal gold immunochromatographic test strip for the differential diagnosis of mpox and chickenpox viruses. The sensitivity, specificity, precision and methodology comparison were evaluated using mpox pseudovirus and clinical samples.</div></div><div><h3>Results</h3><div>A dual-index ICA strips for the simultaneous detection of mpox and chickenpox were successfully prepared. The sensitivity of the dual-index ICA strips to AR antigen was 0.125 ng/mL, to gE antigen was 0.25 ng/mL, with high specificity, precision and reproducibility. The results can be judged within 10–15 min. Methodology comparison study (PCR vs. ICA strips) of 56 clinical samples found there was no statistically significant difference between the ICA strips and PCR method.</div></div><div><h3>Conclusion</h3><div>The dual-index ICA strips had good sensitivity, specificity, simple procedure and rapid detection. This protocol is very suitable for early diagnosis and differentiation of mpox and chickenpox in medical institutions and individuals.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113881"},"PeriodicalIF":1.6,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144134524","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}