Neutrophil extracellular traps (NETs) play a crucial role in host defense, yet their dysregulation is associated with inflammation and tissue damage. Consequently, researching NETs in vitro is essential for understanding their role in both health and disease. However, accurate quantification of NETs in vitro remains a challenge due to limitations in manual methods and semi-automated tools, which often require extensive user input and/or are time-consuming. Moreover, some methods identify neutrophils and NETs based on a single fixed parameter, such as size, which may lead to inaccuracies in recognizing neutrophils due to the cells' biological complexity. In this study, we developed a user-friendly framework for NET quantification using LABKIT, an ImageJ plugin, together with a custom Jython-based script that streamlines image processing and NET classification. By combining fluorescence microscopy with a machine learning tool, this approach enables rapid, scalable analysis of large multi-dimensional datasets with improved flexibility, reliability, and accessibility. Leveraging LABKIT for quantification ensures objective and consistent segmentation of NETs and neutrophils, reducing user bias and processing time while supporting reproducible data curation. Overall, EasyQuant provides a foundation for a freely accessible pipeline that can be integrated into diverse research workflows, herein specifically applied to improve the accuracy and throughput of neutrophil quantification.

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).

Respiratory syncytial virus (RSV) represents a substantial public health burden worldwide, causing lower respiratory infections primarily in children under 5 and adults over 65. In recent years vaccines and monoclonal antibodies (mAbs) have demonstrated promising prophylaxis against RSV in vulnerable populations, benefitting individuals, families, and healthcare systems. However, this protection is incomplete, highlighting the importance of continued research and development towards novel and improved therapeutics. Clinically qualified endpoint assays are a crucial tool in these efforts, giving researchers insight into the magnitude, composition, and neutralization efficacies of the humoral immune response to RSV antigens. Here we describe the methodologies and clinical qualification of two correlated RSV assays, an Enzyme Linked Immunosorbent Assay (ELISA) and a Plaque Reduction Neutralization Test (PRNT). We demonstrate each assay is precise, linear, and translational. Publicly available controls increase reproducibility and transferability to other laboratories.

Public single-nucleus transcriptomes are increasingly reused to count gamma delta T cells in the human small intestine, but how far that count depends on analytical choices has not been quantified. Using a public 3-prime single-nucleus dataset of 33,600 nuclei from six small-intestinal biopsies, we benchmarked three calling strategies across three quality-control levels, tested six module-score cutoff rules, and applied two specificity checks that need no additional assay. The recovered fraction spanned 3.38% to 12.49% on the deposited matrix and 5.46% to 21.62% under strict filtering, a 3.70-fold to 4.08-fold range depending on the filter. Tightening the module-score cutoff corrected the count but not the membership: at a cutoff matched to the gated call, agreement with it reached only 0.408 by Jaccard index. Within one quality-control stratum the call rate rose from 0.51% to 12.17% across deciles of genes per nucleus, so detection sensitivity rather than abundance dominates. Constant-region genes were near-absent from non-T epithelial nuclei (TRGC2 0.25% versus 36.19% of CD3-positive nuclei), indicating that broad TRGC2 detection reflects expression that is not lineage restricted rather than ambient RNA. However, 96.3% of gated calls also carried TRAC or TRBC counts, which is hard to reconcile with excision of the TRD locus during TRA rearrangement and points to unquantified alpha-beta admixture. Variable-region genes were near-undetectable, so subtype assignment was infeasible. We conclude that absolute gamma delta counts from 3-prime nuclear data are not interpretable on their own, and we supply a reporting checklist and open code.

Mutations in the Iduronate 2-sulfatase (IDS) gene cause Hunter syndrome (also known as mucopolysaccharidosis type II, or MPS II), a rare genetic condition that causes glycosaminoglycans (GAGs) to accumulate. Although enzyme replacement therapy (ERT) is the usual course of treatment, it can cause immunological reactions, such as the generation of IgE antibodies specific to the drug, which can result in hypersensitivity reactions. In this study, an ELISA-based test for characterizing anti-idursulfase beta IgE antibodies in human serum was developed and its analytical feasibility was validated. The complex challenges of generating antibodies against the IgE isotype using conventional methods make it difficult to generate an appropriate positive control antibody for IgE-specific tests. In order to overcome this difficulty, Anti-IDS IgG and Human IgE antibodies were chemically linked to generate IgG-IgE conjugate antibodies. Using this conjugated IgG-IgE positive control antibody, we developed the assay and validated it following immunogenicity guidelines, assessing validation parameters such as cut point, sensitivity, specificity, selectivity, precision, drug tolerance, and stability. The results demonstrate the methodological suitability of this assay for IgE isotype characterization.

Accurate detection of antigen-specific antibodies by ELISA requires minimizing nonspecific background, yet blocking conditions are rarely optimized for antibody isotype. Here, we systematically evaluated the impact of blocking reagents on isotype-specific detection of antibodies against influenza A/PR8 hemagglutinin (HA) in mice using an antigen-specific indirect ELISA with isotype-specific detection. Sera from naïve and infected animals were analyzed for HA-specific IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, and IgM across multiple blocking conditions. No single reagent was universally optimal: 1.5% bovine serum albumin (BSA) yielded the highest signal-to-noise ratios for IgG1, IgG2a, IgG2b, IgG2c, and IgA, whereas IgG3 required 20% fetal bovine serum (FBS) to reduce background. In contrast, IgM detection exhibited persistently high background with all conventional blocking reagents tested, including BSA, FBS, and non-fat dry milk. Screening of alternative protein sources identified soy milk as uniquely effective for IgM detection. Soy milk markedly reduced background in naïve serum while preserving strong antigen-specific signal in immune samples, resulting in a substantial improvement in signal-to-noise ratio. These findings demonstrate that ELISA blocking conditions are strongly isotype-dependent and highlight the use of unconventional protein blockers as an effective strategy for improving detection of challenging antibody classes.

Health monitoring of laboratory rodents is essential for ensuring animal welfare, meeting regulatory requirements for zoonosis control, and maintaining the integrity and reproducibility of scientific studies. Routine serological screening, conducted in accordance with FELASA guidelines, plays a critical role in the detection of infectious agents. While conventional serological assays typically target individual pathogens, bead-based multiplex platforms require costly instrumentation and an additional bead functionalization step, which limits their routine implementation in animal facilities. In this study, we evaluated the performance and usability of a multiplex ELISA-like assay (MIRIAD®, GD Biotech, France), designed for in-house serological surveillance of laboratory rodents. A total of 68 mouse and 27 rat serum samples were screened using the recommended quarterly serological monitoring panel. A subset of 22 mouse and 5 rat serum samples was further evaluated using the recommended annual monitoring panel. The results were compared with two commercially available single-target ELISAs, one for mice (Charles River, Wilmington, MA, USA) and one for rats (Biotech Trading Partners, Encinitas, CA, USA). Concordance rates ranging from 94% to 100% were observed across all 26 target pathogens tested. Compatibility with dried blood spot (DBS) cards was also evaluated, demonstrating reliable antibody detection after storage and elution, and supporting the potential for reduced sample volumes and simplified sample handling. These findings highlight the multiplex ELISA-like assay potential as a practical and efficient tool for routine serological health monitoring in laboratory animal facilities. Its ease of use, minimal sample requirements, and compatibility with DBS technology can facilitate more frequent testing, support early detection of pathogens, and promote adherence to the 3Rs (Replacement, Reduction, and Refinement) principles through refinement of health-monitoring practices.

The accurate detection of intrathecal antibodies against the rabies virus (RABV) is critical for antemortem diagnosis and disease monitoring, particularly when the virus-neutralizing activity is low or delayed. In this study, indirect ELISA assays were developed to detect IgM, IgG, and polyvalent (IgA/IgM/IgG) antibodies against the RABV glycoprotein (RABV G) in cerebrospinal fluid. The RABV G antigen, purified by Lens culinaris lectin affinity chromatography, retained its physicochemical and immunoreactive properties, as confirmed by SDS-PAGE, monoclonal antibody recognition and lectin-binding assays. The analytical validation demonstrated low limits of detection (0.056-0.063), high precision and reproducibility (intra-assay CV ≤ 1.84%), acceptable recovery (101.1-115.6%), linear response (R[2] up to 0.989), and minimal matrix effects. Diagnostic performance using a composite reference standard showed moderate agreement for IgM (AUC 0.8150) and IgG (AUC 0.7917), and excellent performance for the polyvalent format (AUC 0.9695), which presented the highest sensitivity (88.6%) and overall agreement (84.8%). RFFIT analysis of ELISA-positive samples demonstrated neutralizing activity in a subset of IgM-positive cases, and a higher proportion of IgG- and polyvalent-positive samples. All RFFIT-positive samples were positive in the polyvalent ELISA format. Serial CSF sampling from one patient indicated that ELISA-detected binding antibodies preceded measurable neutralizing activity. Overall, indirect ELISA based on the native RABV G provides sensitive detection of intrathecal antibody responses, but it does not consistently predict virus-neutralizing activity. These findings support the complementary use of binding and functional assays with molecular diagnostics for a comprehensive antemortem evaluation of rabies infection in the central nervous system. Future studies in larger cohorts are necessary to define the clinical applicability.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), remains the leading cause of death from a single infectious agent. The only approved vaccine for TB, Bacille Calmette-Guerin (BCG), provides variable and incomplete protection from disease, underscoring the need to improve vaccine development. Preclinical murine models are widely used to triage TB vaccine candidates, but immunological endpoints in mice frequently rely on splenocytes or infected tissues, whereas human clinical studies commonly evaluate peripheral blood mononuclear cells or whole blood. This difference in sample type creates a methodological gap that can limit cross-species comparison of antigen-specific T cell responses. Here, we describe the development of a 96-well, small-volume murine whole blood intracellular cytokine staining assay designed to better align preclinical immune monitoring with blood-based clinical endpoints. Iterative optimization identified blood handling and red blood cell processing as key determinants of assay performance. Heparinized collection combined with red blood cell lysis before culture enabled detection of mitogen-induced cytokine responses in murine blood, while stimulation with a broad M. tb epitope pool enabled detection of low-frequency infection-driven antigen-specific CD4[+] and CD8[+] T cell responses. In contrast, low-frequency ID93-specific recall responses following subunit vaccination were readily detected in splenocytes but remained difficult to detect reproducibly in blood. These results define the utility and sensitivity limits of murine whole blood ICS for TB vaccine studies and provide a practical workflow for infection-driven immune monitoring that more closely parallels clinical assay formats.

Thymic epithelial cells (TECs) play a vital role in the generation of immunocompetent and immunotolerant T cells. The understanding of TEC biology in model animals such as mice has advanced thanks to the cellular and molecular analyses of purified TEC populations, including cortical TECs (cTECs) and medullary TECs (mTECs). On the other hand, studies of human TECs have fallen behind because of the lack of a well-established method for purifying TEC populations. Here, we show that using 0.5 U/mL liberase to digest thymus tissue into single cells is quicker than using collagenase D or 0.1 U/mL liberase. Thymus digestion with 0.5 U/ml liberase preserves CD205 expressed by cTECs. RNA sequencing analysis of TEC populations isolated from 0.5 U/mL liberase-digested human thymic tissue revealed the expression of molecules known as cTEC- and mTEC-specific genes in mouse thymus in each human TEC population. Our study is expected to fuel further cellular and molecular studies of human TECs, including those related to the collapse of immune tolerance in humans.

Although most available therapeutic antibodies are immunoglobulin (Ig) G, their limited access at mucosal sites has prompted increasing interest in alternative isotypes, such as IgM. Therapeutic IgM antibodies have primarily been obtained from patient sera; however, methods for generating IgM antibodies that specifically target antigenic regions remain poorly established. In the present study, we aimed to generate IgM antibodies recognising the 160-loop region of haemagglutinin (HA) of influenza B virus using peptide immunisation. BALB/c mice were subcutaneously immunised with a synthetic peptide corresponding to the HA 160-loop region. As a result, 666 hybridomas were established, from which 20 peptide-reactive and 8 HA-reactive IgM antibodies were obtained. The most effective clone, BHA177, exhibited a concentration-dependent neutralising activity against the parental IBV strain, achieving 40% neutralisation at 50 μg/mL. These findings indicate that peptide immunisation can elicit functional IgM antibodies recognising the HA 160-loop region and provide a proof-of-concept for further optimisation of peptide-based IgM generation strategies.