Journal of immunological methods最新文献

{"title":"Construction, expression, and immunogenicity analysis of a fusion gene based on the PE/PPE family in combination with adjuvants.","authors":"Xiaochun Wang, Mingming Zhou, Lifa Xu, Zian Zhang, Sun Qishan, Shi Zilun, Yun Xu, Qiangsen Zhong, Runlin Wang, Xinxin Pi, Yang Huini, Zhang Jiaqi, Wang Bingxin, Jiagao Wu","doi":"10.1016/j.jim.2026.114131","DOIUrl":"https://doi.org/10.1016/j.jim.2026.114131","url":null,"abstract":"<p><p>Tuberculosis (TB) remains the leading global cause of death from a single infectious agent and a major fatal infectious disease worldwide. However, the Bacillus Calmette-Guérin (BCG) vaccine-currently the only licensed TB vaccine in clinical practice-has critical limitations in preventing latent infection and disease reactivation, failing to deliver comprehensive protective immunity. Given these gaps, targeting Mycobacterium tuberculosis (M.tb) antigens with strong immunogenic potential is a key strategy for next-generation TB vaccine development. The pathogenic M.tb harbors the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motif gene families, named for their N-terminal conserved proline (P) and glutamic acid (E) residues, which are increasingly recognized as promising vaccine targets due to their roles in host-pathogen interaction and immune modulation. To address BCG's limitations, this study selected the PE/PPE family members Rv0832 and Rv3653 for full-length gene synthesis to construct a fusion antigen designated ZMM6, which was expressed using the pET-28a vector. The immunogenicity of the ZMM6 subunit vaccine was then evaluated in M.tb-infected human cohorts and animal models. The results showed that the fusion protein ZMM6 induced peripheral blood mononuclear cells (PBMCs) from M. tb-infected individuals to secrete high levels of IFN-γ, IL-2, and IL-6 cytokines. Moreover, the levels of cytokine secretion induced by the fusion protein were higher than those induced by the individual subunit proteins, suggesting that the fusion protein possesses higher immunogenicity. In mice immunized with the protein vaccine (adjuvant formulation) ZMM6/MnJ, high titers of various IgG subclass antibodies were elicited, with the immune response skewed toward a Th1-type profile, accompanied by robust induction of multiple Th1-type cytokines. Flow cytometry (FCM) analysis further revealed a significant increase in CD4⁺IFN-γ⁺ single-positive T cells in ZMM6/MnJ-immunized mice. Collectively, these findings demonstrate that the protein vaccine (adjuvant formulation) ZMM6/MnJ exhibits strong immunogenicity.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"114131"},"PeriodicalIF":1.7,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149182504","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":"Efficient detection of human antigen-specific CD4+ T cells with peptide-exchangeable HLA-DP4 tetramers and multiplexed magnetic enrichment.","authors":"Shawn A Mahmud, Grace D Frechette, Lisa M Burzenski, Sandhya Appiah, Leonard D Shultz, Marc K Jenkins, Thamotharampillai Dileepan","doi":"10.1016/j.jim.2026.114129","DOIUrl":"10.1016/j.jim.2026.114129","url":null,"abstract":"<p><p>The tremendous variation in human MHCII alleles is a barrier to using fluorophore-labeled peptide:MHCII tetramers to study antigen-specific CD4+ T cells. This is because matching of the MHCII molecule in the tetramer with each human subject is required. We show that fluorophore-labeled tetramers of peptides complexed to the most common human MHCII molecule-encoded by DPA1*01:03 and DPB1*04:01-detect antigen-specific cells in most humans. New peptides can be successfully exchanged into these tetramers. Combining these novel exchangeable DP4 tetramers with a multiplexed magnetic enrichment step allows the detection of antigen-specific CD4+ T cell responses in ≥60% of humans without forehand knowledge of HLA type making pilot experiments with new tetramers more practical and efficient. This system could be adopted by the field to study CD4+ T cell responses to multiple independent antigens or multiple epitopes within an antigen (i.e., as a system for epitope discovery).</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"114129"},"PeriodicalIF":1.7,"publicationDate":"2026-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149109878","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":"EYRAplex platform for quantification of viral antigen-specific IgG responses","authors":"David Wullimann,&nbsp;John Tyler Sandberg,&nbsp;Steve Angeloni,&nbsp;Bartek Makower,&nbsp;Hans-Gustaf Ljunggren","doi":"10.1016/j.jim.2026.114107","DOIUrl":"10.1016/j.jim.2026.114107","url":null,"abstract":"<div><div>Quantification of antigen-specific antibodies is fundamental to studies of infection, vaccination, and humoral immunity. Conventional serological assays typically measure responses to a single antigen and require relatively large sample volumes, whereas multiplex platforms enable simultaneous analysis of multiple analytes while conserving sample material. Here, we describe the use of EYRAplex as a bead-based multiplex platform to quantify antigen-specific IgG responses against multiple viral antigens. The major structural envelope proteins from three flaviviruses, tick-borne encephalitis virus, Japanese encephalitis virus, and yellow fever virus, were coupled to color-coded magnetic beads and used to measure IgG responses in longitudinal serum samples from vaccinated individuals. Assay performance was optimized by evaluating antigen coupling densities and serum dilutions, and analytical sensitivity was assessed using flavivirus-specific monoclonal antibodies. The assay reliably quantified the magnitude and kinetics of vaccine-induced antibody responses and distinguished type-specific from cross-reactive antibodies across flavivirus antigens. These findings demonstrate that EYRAplex provides a robust, scalable, and versatile multiplex platform for quantifying antigen-specific IgG responses in human samples.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"550 ","pages":"Article 114107"},"PeriodicalIF":1.7,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148818414","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dynamic effects of short-term storage temperature and duration on the transcriptome and functional pathways of umbilical cord blood-derived NK cells","authors":"Jing Xu,&nbsp;Yanqin Wang,&nbsp;Dongmei Yang,&nbsp;Zehua Zhang,&nbsp;Li Zhang,&nbsp;Shanshan Xu,&nbsp;Jing Dong,&nbsp;Xuesong Gao,&nbsp;Chenyu Liu","doi":"10.1016/j.jim.2026.114110","DOIUrl":"10.1016/j.jim.2026.114110","url":null,"abstract":"<div><h3>Objective</h3><div>This study aims to elucidate the dynamic impact of different storage conditions (temperature and time) on the function of natural killer (NK) cells derived from umbilical cord blood (UCB) at the transcriptome level, providing a theoretical basis for optimizing the standardized post-collection processing protocol of UCB in clinical settings.</div></div><div><h3>Methods</h3><div>Four healthy full-term UCB samples were collected and assigned to a fresh control group (0H) and experimental groups stored at 4 °C (4C) or 25 °C (RT) for 24 h (24H) and 72 h (72H). Umbilical cord blood mononuclear cells (CBMCs) were isolated and expanded in vitro to derive NK cells. Using RNA sequencing (RNA-seq) technology, combined with principal component analysis (PCA), screening of differentially expressed genes (DEGs), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the effects of storage conditions on the transcriptome of NK cells were comprehensively analyzed.</div></div><div><h3>Results</h3><div>The results indicated that storage time was the primary factor causing transcriptomic differences. Compared to the 0H group, storage for 24 h induced a limited number of Differentially Expressed Genes (DEGs) (only 11 in the 24H-RT group), indicating overall transcriptome stability. In contrast, storage for 72 h triggered profound transcriptomic reprogramming, with DEGs significantly enriched in immune activation-related pathways. Specifically, at the 72-h time point, storage at 4 °C compared to 25 °C prevented the downregulation of key metabolic pathways such as ‘response to decreased oxygen levels’ and ‘regulation of leukocyte cell-cell adhesion’.</div></div><div><h3>Conclusion</h3><div>CBMC-derived NK cells can tolerate up to 24 h of ex vivo storage while maintaining transcriptome and core functional stability. However, extending the ex vivo storage period to 72 h leads to significant reprogramming in the expression of immune and metabolism-related genes. Notably, the degree of metabolic suppression under refrigerated conditions at 4 °C was less pronounced than that at 25 °C room temperature storage. This study recommends that the ex vivo storage time of UCB should ideally be limited to within 24 h, with room temperature storage being a viable option. If prolonged storage is necessary, refrigeration at 4 °C is advised.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"550 ","pages":"Article 114110"},"PeriodicalIF":1.7,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148857366","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":"Intracellular CD3 and surface CD8β distinguish activated human T cells from natural killer cells after αCD3/αCD28 stimulation","authors":"Cade M. McDonald ,&nbsp;Sofia C. Alzugaray Orellana ,&nbsp;Susan E. Murray","doi":"10.1016/j.jim.2026.114079","DOIUrl":"10.1016/j.jim.2026.114079","url":null,"abstract":"<div><div>Many studies of human T cell function involve activating primary T cells in vitro to assess functional outcomes. A common model of human T cell activation is polyclonal stimulation with αCD3/αCD28 antibodies. However, subsequent identification of these stimulated T cells in a mixed population is complicated by surface CD3 loss. In particular, both cytotoxic T cells and natural killer (NK) cells can appear CD3⁻CD8α⁺, leading to misidentification of NK cells as cytotoxic T cells. To address this limitation, we investigated incorporating intracellular CD3 and surface CD8β staining following αCD3/αCD28 stimulation to improve discrimination between cytotoxic T cells and NK cells. We found that intracellular CD3 staining completely recovers lost CD3 expression. When intracellular staining is not amenable to downstream applications in which live cells are required, we found that CD8β more accurately identifies cytotoxic T cells while excluding NK cells than does the more common CD8α staining. We validated CD8β as a more faithful marker of cytotoxic T cells by comparing cytokine profiles of CD8α or CD8β gated PBMC to those of purified T cells, showing that CD8β most closely mimics the cytokine profile of cytotoxic T cells within a purified T cell population. We conclude that i) intracellular CD3 staining is ideal when cell fixation and permeabilization is possible and ii) surface CD8β staining is best when intracellular CD3 staining is not possible, e.g., when recovery of live cells from fluorescence-activated cell sorting is necessary.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114079"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148225964","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 chimeric DGP-IgG antibodies as quality control for celiac disease diagnosis","authors":"Liang Zheng ,&nbsp;Xiaotao Lin ,&nbsp;Xufu Xiang ,&nbsp;Chungen Qian ,&nbsp;Fangming Cheng ,&nbsp;Gang Wang ,&nbsp;Yuxi He ,&nbsp;Hui Tan","doi":"10.1016/j.jim.2026.114077","DOIUrl":"10.1016/j.jim.2026.114077","url":null,"abstract":"<div><h3>Background</h3><div>Celiac disease (CD) is an autoimmune disorder triggered by an immune response to dietary gluten in the small intestine, which is frequently undiagnosed or misdiagnosed owing to its non-specific symptoms. The detection of specific serum antibodies, including anti-tissue transglutaminase (tTG) IgA and anti-deamidated gliadin peptide (DGP) IgG, is essential for diagnosing celiac disease. However, the limited availability of DGP IgG positive human sera for use as quality control materials, particularly in countries with low celiac disease prevalence, poses a significant challenge.</div></div><div><h3>Objective</h3><div>To overcome the limited supply of positive serum for DGP IgG, chimeric human DGP IgG antibodies were developed as alternative quality control materials (first time in China). Additionally, several in-house DGP IgG-specific assays (ELISA and CLIA) targeting human and mouse antibodies were developed to monitor each step during chimeric antibodies preparation as quality control materials.</div></div><div><h3>Methods</h3><div>Mouse monoclonal antibodies against DGP IgG were screened from hybridoma cells derived from mice immunized with recombinant DGP. Chimeric antibodies of DGP IgG comprising murine variable regions and human IgG constant regions were produced in mammalian expression system using HEK293F cells. Four in-house DGP IgG specific assays were developed to monitor each step of chimeric antibody preparation, i.e. DGP IgG ELISA (mouse), DGP IgG CLIA (mouse), DGP IgG ELISA (human) and DGP IgG CLIA (human). Final chimeric antibodies were prepared as quality control materials and tested for homogeneity and open-vial stability. SPR (Surface Plasmon Resonance) was used to measure binding affinity for DGP chimeric antibodies and DGP antigen for DGP IgG assays mentioned above.</div></div><div><h3>Results</h3><div>The chimeric antibodies against DGP demonstrated high affinity to DGP antigen by using SPR analysis. The chimeric DGP IgG as quality materials exhibited satisfactory homogeneity (CV &lt; 10%) and open-vial stability (for at least 5 days at 2–8 °C and at least 90 days at −20 °C).</div></div><div><h3>Conclusion</h3><div>This study represents the first development of chimeric DGP IgG as quality control materials and four in-house DGP IgG assays in China. These DGP IgG quality control materials and in-house DGP IgG assays can serve as valuable tools for future DGP IgG assay development and standardization.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114077"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148258286","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":"Stepwise single-cell-resolved deep immunophenotyping pipeline to characterise immune heterogeneity and functionality in health and disease","authors":"Aristeidis Roubanis,&nbsp;Zi Mei,&nbsp;Fanny Hedin,&nbsp;Wim Ammerlaan,&nbsp;Markus Ollert,&nbsp;Antonio Cosma,&nbsp;Feng Q. HeFeng","doi":"10.1016/j.jim.2026.114076","DOIUrl":"10.1016/j.jim.2026.114076","url":null,"abstract":"<div><div>Providing comprehensive insights into both immune cellular compositions and molecular profiles at single-cell resolution remains challenging. Here we proposed a stepwise single-cell-resolved deep immunophenotyping (SCRIPT) pipeline to enable not only a high-dimensional characterization of the immune compositions and functionalities, but also an unbiased genome-scale molecular profiling of specific immune subsets. Our pipeline integrated rigorous pre-analytical measures, including consideration of circadian timing during sample collection, multiple quality control steps, and critical parameters at different stages of the workflow. Our pipeline leveraged the first-step cytometry analysis to rationally guide subsequent efficient and unbiased single-cell RNA sequencing characterization on specific cell subsets, which otherwise has to be achieved by trial and error and at a much higher cost. We exampled our pipeline with specifically affected CD8 T-cell subsets and a guided genome-scale molecular analysis into those CD8 subsets in a clinical study. Together, our pipeline could be generally applicable to various disease contexts for a comprehensive analysis at both molecular and cellular levels.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114076"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148252023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isosulfan blue sentinel lymph node biopsy enables reliable lymph node harvest and multicolor flow cytometry in mice","authors":"Sydney Jeffs,&nbsp;April Espinoza,&nbsp;Samantha Owusu-Antwi,&nbsp;Alejandra Suarez-Arnedo,&nbsp;Tatiana Segura,&nbsp;Holly Lewis","doi":"10.1016/j.jim.2026.114082","DOIUrl":"10.1016/j.jim.2026.114082","url":null,"abstract":"<div><div>Accurate and reproducible collection of the draining lymph nodes (DLNs) is essential for studying immune responses in preclinical models, yet technical variability and the small size of murine lymph nodes make consistent harvest challenging, particularly across users with differing surgical experience. To address this limitation, we adapted the clinical technique of isosulfan blue sentinel lymph node biopsy (IB-SLNB) for use in mice. Despite its widespread application in surgical oncology, IB has not been systematically evaluated for compatibility with fluorescence-based immunologic assays. Here, we show that IB-SLNB enables rapid and reliable identification of axillary DLNs, improving reproducibility of lymph node harvest. Importantly, IB labeling does not interfere with multicolor flow cytometry or downstream colorimetric analyses, preserving analytic fidelity. This facile, low-cost method enhances consistency in lymphatic tissue collection and supports high-quality immune profiling across diverse experimental contexts, including models of infection, cancer, and transplantation.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114082"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148338846","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":"Optimized intracellular flow cytometry panel enables CD4 and CD8 T cell cytokine profiling in Syrian hamsters","authors":"Tolunay Ataç,&nbsp;Julia Sid Hansen,&nbsp;Louise Krag Isling,&nbsp;Gabriel Kristian Pedersen,&nbsp;Katharina Wørzner","doi":"10.1016/j.jim.2026.114084","DOIUrl":"10.1016/j.jim.2026.114084","url":null,"abstract":"<div><div>The Syrian golden hamster has emerged as a valuable animal model in challenge experiments and vaccine efficacy studies, particularly for infectious diseases that are poorly represented in mice. In recent years, research has demonstrated that hamsters can better recapitulate certain human infectious diseases compared to mice. Syrian hamsters are used in studies with Yellow fever virus, Helicobacter spp., Hantavirus pulmonary syndrome, Japanese encephalitis, Nipah virus, Adenovirus, Influenza A/B, and SARS-CoV-2, among others. However, the model has been limited by a lack of immunological tools, forcing researchers to rely on clinical outcomes and histopathology rather than detailed immunoprofiling. While some studies have identified cross-reactive antibodies, a comprehensive and complete intracellular flow cytometry panel for measuring cytokine responses in CD4 and CD8 T cell subsets has not yet been established. This paper introduces such an optimized panel, enabling deeper insights into cell-mediated immunity and supporting the broader use of hamsters in vaccine development, therapeutic evaluation, and immune correlate discovery.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114084"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148345376","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":"Performance evaluation of Meso Scale Discovery (MSD) quantitative serological assays for detection of binding (IgG, IgA, IgM) and ACE2 inhibitory antibody levels for SARS-CoV-2","authors":"Camila Macedo Cincotta,&nbsp;Dante Coleman,&nbsp;Steven Padilla,&nbsp;Jennifer Enoch,&nbsp;Ines Lakhal-Naouar,&nbsp;Matthew Bell,&nbsp;Jason Ouellette,&nbsp;Sheida Mansouri,&nbsp;Oussama M'hamdi,&nbsp;Janice Darden,&nbsp;Jennifer Malia,&nbsp;Sheila A. Peel,&nbsp;Kristina K. Peachman","doi":"10.1016/j.jim.2026.114081","DOIUrl":"10.1016/j.jim.2026.114081","url":null,"abstract":"<div><div>A performance evaluation of a quantitative research use only (RUO) immunological assay utilizing the Meso Scale Discovery (MSD) platform for assessing IgM, IgA, and IgG binding and SARS-CoV-2 pseudo-neutralization was performed. Comparative analyses of antibody quantification of IgA, IgG, and IgM isotypes against SARS-CoV-2 nucleocapsid, spike, Receptor Binding Domain (RBD) and N-Terminal Domain (NTD) antigens were performed using MSD multi-spot electro-chemiluminescent V-PLEX COVID-19 test kits, and two well characterized serological panels. Cross reactivity was assessed against COVID-19 negative/Influenza samples. Precision was assessed by comparing variation between duplicate values within run; accuracy was assessed by comparing values obtained across runs against expected values. The MSD IgG, IgA, and IgM binding, and ACE2 inhibitory antibody assays demonstrated acceptable precision and accuracy within the expected 20%/30% variability, respectively per manufacturer's protocol; no cross reactivity with influenza samples was observed. Different MSD plate configurations, multiple (two) MSD analysis instruments, and multiple operators did not impact antibody quantification; thus, confirming the robustness of both assays. Comparative analysis between MSD and commercial Euroimmun, Siemens ADVIA, and Beckman Coulter DXLTM ACCESS assays demonstrated better performance overall for the MSD assay.</div></div><div><h3>Importance</h3><div>This work describes and validates the MSD platform, as a highly flexible, customizable, operator independent, and reliable platform for antibody assessment of COVID-19 response which is highly adaptable to immune assessment of emerging or re-emerging pathogens.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114081"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148239459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}