Quantitative analysis of microplate bioassays, such as ELISA, requires robust nonlinear regression tools. These are typically available either as expensive commercial software or as simple web-based alternatives with insufficient analytical capabilities. In this work, we present Labreador-a freely available web platform built on a client-side architecture that eliminates the need to transfer data to external servers. The system integrates automated selection from five regression models (including 4PL and 5PL) with the Levenberg-Marquardt optimization algorithm, weighted regression for heteroscedasticity correction, interactive outlier management, and a three-level quality control system with automatic blocking of interpolation when the calibration curve quality is insufficient (R2 < 0.95). The platform's accuracy was verified on a synthetic reference dataset. Following the application of 1/Y2 weighted regression and interactive outlier management, the algorithm recovered an excellent fit (R2 ≥ 0.98) and the true EC₅₀ reference value of 150.0 fell within the recovered 95% confidence interval, confirming the statistical validity of the parameter estimate in the presence of 10% CV biological noise. Labreador provides robust computational accuracy sufficient for rigorous laboratory practice in a free, privacy-oriented environment accessible without any technical barriers.

As new SARS-CoV-2 subvariants emerge alongside continuous reports of post-acute long-term sequelae after COVID-19, there is an urgent need to mitigate the harmful effects associated with SARS-CoV-2 infection. While antiviral protection is commonly assessed by measuring neutralizing antibodies, specific antiviral CD8[+] T cell responses are also relevant for long-term immunity. In this context, tools that improve our knowledge regarding anti-SARS-CoV-2 CD8[+] T cell immunity are important for monitoring antiviral responses, supporting the development of new approaches to induce long-term immunity. Here, we sought to establish a cost-effective platform that enables the direct identification of SARS-CoV-2 peptide-reactive CD8[+] T cells restricted to HLA-A*02, a highly frequent allele family in the Brazilian population. For this, we produced an HLA-A*02 dimeric molecule (HLA-A*02:01-IgG) in-house, coupled with peptides derived from SARS-CoV-2, and used in flow cytometry assays to identify antigen-specific CD8[+] T cell populations. Using low concentrations of HLA-A*02:IgG-peptide complex (0.25 μg/mL), we were able to detect anti-SARS-CoV-2-specific CD8[+] T cells in HLA-A*02-positive individuals, but not in HLA-A*02-negative individuals, as expected. Furthermore, ROC curve analysis showed maximum sensitivity and specificity (100%, AUC = 1.0), highlighting the effectiveness of our method Overall, our assay identified virus-specific CD8[+] T cell populations in HLA-A*02 individuals using low concentrations of HLA-A*02:IgG-peptide complex, supporting its use to evaluate the CD8[+] T cell responses restricted to SARS-CoV-2. This method may provide insights into cellular immunity in the context of the current vaccination and help guide new vaccine and/or therapeutic platform development, including the analysis of memory CD8[+] T cell subsets.

Each T cell typically carries a specific T-cell receptor (TCR) that determines its specificity against an epitope presented by the HLA complex on a target cell. Antigenic challenge triggers the expansion of reactive cells within a diverse pool of T cells with randomly generated receptors, a process that results in epitope-driven shifts of TCR frequencies over time. Here, we analyze the effects of SARS-CoV-2 vaccination on the TCR populations in peripheral blood drawn from seven COVID-naive individuals during the early phase of vaccine rollout. To identify SARS-CoV-2 vaccine-associated TCR sequences among the ∼10[5]-10[6] TCR sequences sampled before and after vaccination, we develop statistical criteria to detect significant increases in abundance of positive TCR clones. Application of our statistical methods shows a robust identification of TCR sequences that respond to SARS-CoV-2 vaccination in vivo, illustrating the feasibility of quantifying the clone-specific dynamics of T-cell abundance changes following immunological perturbations.

Extractable nuclear antigen (ENA) antibody testing is universal to the diagnosis of systemic autoimmune rheumatic diseases (SARDs). However, current commercial multiplex platforms require expensive instruments and are often cost-prohibitive for routine testing in low- and middle-income settings. Pictor PictHealth® Immunoscreen ENA assay was developed as an affordable, scalable alternative for the simultaneous detection of ENA antibodies. The assay performance was compared with the Euroimmun ENA Line immunoassay and BioPlex 2200 ENA assays using clinical sera from patients with autoimmune diseases. Concordance among assays was evaluated using three-way and pairwise analyses, expressed as positive percent agreement (PPA), negative percent agreement (NPA), and overall concordance. Analytical precision was assessed within and between manufacturing lots. The Pictor multiplex assay showed high concordance with established commercial platforms. Three-way agreement across all assays ranged from 67.3% for TRIM21/Ro52 to 100% for Jo-1, with Sm, CENP-B showing concordance of 95%. Pairwise comparisons demonstrated PPA and NPA values exceeding 85% for most autoantibodies. The Pictor PictHealth® Immunoscreen ENA assay provides diagnostic performance comparable to the Euroimmun and BioPlex platforms while maintaining the ease of implementation and scalability of a traditional ELISA format. These results support its potential as a cost-effective and accessible tool for ENA antibody testing in laboratories with varying resource levels.

Antibody responses to individual antigens vary widely in their relative contributions of the four human IgG subclasses. Detection of IgG subclasses is usually done using specific monoclonal antibodies. Ideally, such antibodies recognize a subclass irrespective of polymorphic structural variation (also called allotypes), while not recognizing any of the other subclasses. For IgG2 this is especially challenging. There are many monoclonal antibodies commercially available, but whether they meet the abovementioned requirements is unclear. In this study, we examined twelve commercially available monoclonal antibodies against human IgG2 (HP6002/AB4-DH4, HP6014/AC3-AA11/MH162.1, HP6200/HG2-56F, KT138, 985028/MAB9794, 31-7-4, MTG211E, 52G1, EPR4418, 2348B/MAB97941, and RM118) for their ability to selectively bind to human IgG2, and not to the other three subclasses. We used a panel of recombinant monoclonal antibodies comprising different human IgG subclasses and allotypes, and tested binding in ELISA. Of the twelve anti-human IgG2 clones tested, only three were found to be highly specific for IgG2 (>1000-fold). Two further clones demonstrated good selectivity albeit with measurable cross-reactivity to IgG4 of up to ca. 1-2%. Three clones were cross-reactive to certain IgG4 and IgG3 allotypes. These clones may possess G4m(b) specificity. The remaining clones showed only low selectivity for IgG2. In conclusion, specificity determinations of anti-IgG subclass reagents should take into account the genetic/allotypic variation across IgG subclasses. Antibodies with proclaimed IgG2 specificity do not always meet requirements for subclass-specific antibody detection.

Sepsis is a life-threatening condition that requires reliable biomarkers for appropriate clinical assessment. Histidine-rich glycoprotein (HRG) has emerged as a promising biomarker candidate associated with sepsis severity. We previously developed an ELISA method using monoclonal antibodies, 69-1 A and 75-2D for sensitive and specific quantification of plasma HRG. However, the assay required extensive sample dilution and further optimization to improve analytical robustness. In the present study, we developed an optimized ELISA by reducing sample dilution ratio and extending the measurable range toward higher HRG concentrations through a competitive reaction system incorporating a small amount of free 75-2D antibody. The optimized assay covered a plasma HRG range of 16-250 μg/mL and showed lower coefficients of variation than the previous method. The optimized method also showed good correlation with the previous ELISA. In addition, we evaluated recombinant single-isoform HRG protein as candidate calibrator material for this assay system. Because both isoforms showed comparable reactivity and dilution linearity, recombinant Form-1 HRG (an isoform bearing Pro at 204-position, 75 kDa) was selected as a practical candidate calibrator based on its simpler glycosylation-related characteristics and quantitative assignment by amino acid analysis. Our new findings support the optimization of ELISA system and a single-isoform recombinant calibrator for more reproducible HRG measurement. It is expected that analytical standardization and clinical validation will subsequently be performed to establish in vitro diagnostic application in actual clinical use.

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.

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.

Following the initial infection, herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) establish latency in sensory nerve ganglia. The viruses can later reactivate, producing recurrent lesions such as painful vesicles or ulcers. HSV-1 is most often associated with oral herpes (cold sores), whereas HSV-2 is more commonly linked to genital herpes. Serologic IgG tests can be used to determine if a person has antibodies to HSV-1 or HSV-2 from past infection. In this paper, we describe the development and performance evaluation of an antibody based lateral flow immunochromatographic assay (LFA) device for the detection and differentiation between HSV-1&2 IgG antibody in fingerstick blood. Analytical and clinical analyses were performed to compare the performance characteristics of the HSV-1&2 IgG LFA (VIZIHSV™) with Western Blot. VIZIHSV™ results from 373 subjects seeking STI care and 267 pregnant women at four clinical sites were compared with results obtained by testing serum samples obtained from the same study participants with Western Blot. When compared to Western Blot, the Positive Predictive Agreement (PPA) and Negative Predictive Agreement (NPA) in individuals seeking care for STIs was 94.6% (95% CI: 91.4-96.6%) and 94.9% (95% CI: 87.5-98.0%), respectively. PPA for HSV-2 in this group was 99.0%, 95% CI (96.3%,99.7%) and the NPA was 91.6%, 95% CI (86.6%, 94.8%). Performance in pregnant women was similar to that in the STI-care-seeking group with an HSV-1 PPA of 90.4% (95% CI: 85.0-94.0%) and NPA of 96.0% (95% CI: 90.2-98.4%). PPA for HSV-2 in pregnant women was 91.3% (95% CI: 79.7-96.6%) and NPA was 91.7% (95% CI: 87.3-94.7). Results from the clinical study also showed that 29% of the 373 subjects seeking STI care and 66.3% of pregnant women did not indicate a past history of HSV-1 or HSV-2 infection, indicating that HSV-1&2 IgG LFA type-specific serology will enhance the ability to detect past infection and make the diagnosis and guide clinical management in select patients. VIZIHSV™ has been demonstrated to be simple and sufficiently robust for use in CLIA-waived point-of-care (POC) facilities by untrained healthcare professionals and to detect and differentiate between HSV1&2 IgG antibody from fingerstick blood in 30 min. Therefore, this test provides a fast, reliable, and highly sensitive method to detect IgG antibody to HSV-1 and/or HSV-2 viral infection.

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.

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. IMPORTANCE: 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.

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.

The diagnosis of leprosy remains challenging because available diagnostic tests show variable sensitivity and typically require blood sampling. In this study, we assessed the diagnostic performance of the chimeric M2 protein using ELISA assay based on both serum and urine samples from paired specimens of 315 participants. The cohort included paucibacillary (PB; n = 50) and multibacillary (MB; n = 60) leprosy patients, individuals diagnosed with leishmaniasis, Chagas disease, tuberculosis, or HIV infection, as well as household contacts and healthy controls. In serum-based ELISA, mean optical density (OD) values were significantly higher in PB and MB patients (0.544 ± 0.070 and 0.795 ± 0.085, respectively) compared with healthy controls (0.192 ± 0.025). Household contacts exhibited lower OD means (0.130 ± 0.017 for PB contacts and 0.147 ± 0.024 for MB contacts), while samples from patients with other infectious diseases remained below the established cut-off. Receiver operating characteristic curve (ROC) demonstrated sensitivity and specificity exceeding 96.0%, indicating strong discriminatory capacity of the M2 antigen in serum. Urine-based ELISA similarly revealed elevated OD values among PB and MB patients (0.336 ± 0.069 and 0.547 ± 0.076, respectively). In contrast, controls showed a mean OD of 0.170 ± 0.027, and PB and MB household contacts presented lower values (0.108 ± 0.017 and 0.121 ± 0.018, respectively). All other disease groups remained below the cut-off threshold. ROC curve analysis yielded sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) greater than 97.8%, confirming high diagnostic accuracy. Furthermore, M2-specific IgG levels declined substantially following treatment, with reductions of 70.0%-84.0% in MB patients and 55.0%-64.0% in PB patients. Correspondingly, serum and urine OD values decreased from 0.774/0.547 to 0.365/0.243, respectively. Collectively, these findings indicate that the M2 protein represents a promising biomarker for leprosy, demonstrating strong diagnostic performance in both serum and urine assays and supporting its potential utility for diagnosis and treatment monitoring.

Reliable assessment of vaccine induced antibody responses depends on the specificity and validation of immunological reagents, however, many commercially available antibodies remain insufficiently validated and may exhibit substantial off target binding. This limitation is particularly relevant for isotype specific analyses, where cross reactivity can profoundly affect data interpretation. In this study, we examined serum IgA responses following vaccination in pigs using two different commercially available polyclonal anti-porcine IgA antibodies and evaluated their specificity against monoclonal antibody-based assays and recombinant immunoglobulin isotypes. Initial analyses using a widely applied polyclonal antibody showed IgA response kinetics closely resembling IgG responses, though at lower titers, consistent with numerous previous reports. The unexpectedly similar half-lives of serum IgA and IgG prompted us to test an alternative polyclonal antibody, which revealed only transient IgA responses detectable one week after immunization. This short-lived IgA response was independently confirmed using a biotinylated monoclonal antibody ELISA specific for porcine IgA. Correlation analyses across all time points, together with direct binding assays employing isotype-switched monoclonal antibodies and recombinantly produced porcine immunoglobin isotypes, demonstrated pronounced cross reactivity between one commercial anti-IgA polyclonal antibody and porcine IgG, and a cross reactivity of both commercial anti-IgG polyclonal antibodies, which showed comparable levels of detection of IgA as the IgA-specific polyclonal antibodies. Collectively, these findings demonstrate a critical lack of specificity in commonly used polyclonal secondary antibodies for porcine IgA and IgG detection. Importantly, antibody choice fundamentally alters the interpretation of antibody isotype responses, highlighting the need for rigorous validation of immunological reagents in both preclinical vaccine research and infectious disease immunopathology studies.