BACKGROUND:Luminex bead-based solid-phase immunoassays (SFIA) are the gold standard for anti-human leukocyte antigen (HLA) antibody detection in transplantation immunology. However, preanalytical interferences may compromise assay validity. Although automated hemolysis, icterus, and lipemia (HIL) indices are routinely used to assess sample quality, their relevance for predicting interference in Luminex-based HLA antibody testing remains unclear. METHODS:A total of 1308 patient samples were retrospectively evaluated using Luminex SFIA. Internal control bead mean fluorescence intensity (MFI) values were assessed according to manufacturer-defined criteria. Samples with control bead deviations were analyzed in relation to automated HIL indices and categorized into four interference profiles based on control bead behavior. Control bead normalization ratios and correlation analyses were performed. RESULTS:Control bead MFI values outside acceptable ranges were observed in 125 samples (9.7%). After exclusion of 16 samples, 109 were included in the final analysis. Elevated lipemia indices were associated with assay interference in two groups, manifesting as signal amplification or signal quenching. Notably, more than half of the deviated samples showed marked signal suppression despite normal HIL indices, consistent with silent interference. Another interference profile demonstrated pronounced control bead signal elevation in the absence of abnormal HIL indices, indicating an index-independent matrix effect. CONCLUSION:Lipemia represents an underrecognized source of interference in Luminex-based HLA antibody testing. Automated HIL indices alone did not capture all analytically compromised samples in this cohort. Systematic monitoring of internal control bead values may improve detection of silent and index-independent interferences and enhance analytical accuracy.
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.
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 (R2 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.
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.
OBJECTIVE: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. METHODS: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. RESULTS: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'. CONCLUSION: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.
The prevailing chromium contamination in water resources sets an exigency in developing a rapid detection tool with high sensitivity and target-specificity for impactful environmental monitoring. The present study deals with the development and validation of a highly specific immunosensor based chromium detection platform. Primarily, chromium-protein conjugate was prepared, using EDTA (Ethylenediaminetetraacetic acid) as chelating agent and BSA (Bovine serum albumin) as carrier protein. The optimized ratio of chromium, EDTA, and BSA was 1:1:1 at 0.33 mg/ml concentration each. The successful conjugation of Cr-EDTA-BSA was confirmed through SDS-PAGE analysis. The prepared conjugate was immunized into Balb/c mice for the development of polyclonal and monoclonal antibodies. These antibodies were screened and optimized using sandwich ELISA. The optimized experimental concentration was 50 ng per well antigen (Cr-EDTA-BSA) with an antibody dilution of 1:1500 at OD value of 0.75 (450 nm). The assay showed high chromium specificity and less cross-reactivity (<0.6%) with other metal ions, except Fe(II) (1.7%). ELISA depicted 1.38 ng/ml as the limit of detection. ELISA and ICP-MS showed recovery percentage ranging from 93.3%-111.2% and 96%-115.6%, respectively. The coefficient of variation (CV %) ranged from 10.9% to 14.6% and 11.6% to 14.6% in between the above assays respectively. Significant correlation (R2 = 0.995) between both the methods were also observed in terms of chromium concentrations detection. Further, antibody pair was integrated to develop an immunochromatographic test (ICT) sensor, which later processed for lab validation using 0-40 ng/ml concentration of chromium in water samples. The visual limit of detection (vLOD) of the developed ICT assay was 10 ng/ml. The integration of targeted monoclonal-polyclonal antibody pairs for the development of immunosensor, is a novel approach adapted so far. Antibody-pair based immunochromatographic sensor offers rapid, onsite and accurate detection and monitoring of chromium in water sources, promoting a sustainable-healthy environmental practice.
OBJECTIVE:This study aimed to establish and verify reference intervals (RIs) for coagulation factor XII (FXII) and antithrombin III (ATIII) in an adult population from the Jiangsu region of China and to evaluate the influence of analytical platform on RI interpretation. METHODS:We retrospectively analyzed data from 301 apparently healthy adults attending the Health Examination Center of Nanjing Drum Tower Hospital. Plasma FXII and ATIII activities were measured in parallel using two automated coagulation analyzers, the Sysmex CS-5100 and CS-6500. Statistical analysis followed CLSI EP28-A3 guidelines. Distribution normality was assessed to determine the appropriate method for RI establishment: the non-parametric percentile method for non-normally distributed data and the parametric method for normally distributed data. Analyzer-related agreement was further assessed using Bland-Altman analysis. The derived RIs were verified using an independent sample set of 20 apparently healthy adults. RESULTS:FXII showed a non-Gaussian distribution, whereas ATIII showed an approximately Gaussian distribution. For the CS-5100 analyzer, the RI was 32.2%-109.1% for FXII and 84.1%-104.7% for ATIII. For the CS-6500 analyzer, the corresponding RIs were 37.1%-111.1% for FXII and 86.1%-106.7% for ATIII. The newly established RIs differed from the manufacturer-provided ranges currently used in routine practice. No clinically meaningful sex-based partitioning was identified. Although analyzer-related differences were statistically significant, Bland-Altman analysis showed modest mean biases between the CS-6500 and CS-5100 platforms, with mean differences of +5.09% for FXII and + 1.17% for ATIII. All verification samples fell within the proposed RIs. CONCLUSION:This study established and verified regional RIs for FXII and ATIII in healthy adults from Jiangsu, China. Although analyzer-related differences between the CS-5100 and CS-6500 platforms were modest, the findings highlight the importance of local RI verification when applying manufacturer-derived intervals or changing analytical platforms. These locally derived RIs may improve the interpretation of FXII and ATIII results and reduce unnecessary follow-up testing caused by inappropriate generic intervals.
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.
Following the incidence of life-threatening cytokine release syndrome (CRS) in healthy volunteers in the 2006 first-time-in-human (FTIH) trial of a CD28 mAb (TGN1412), there is now a regulatory expectation to investigate immuno-stimulatory mAbs for CRS hazard using in vitro cytokine release assays (CRAs) prior to FTIH trials, and CRAs incorporating more than one test article may be used prior to combination clinical trials. To this end, many organisations stimulate healthy donor human peripheral blood mononuclear cells (PBMCs) using immobilised (plate-bound) mAbs, however methodology is not standardized, and differences in methods used throughout the pharmaceutical / biotechnology industry could influence assay outcomes. This study investigated the impact of micro-titre plate type on mAb immobilisation and cytokine release. We observed superior assay performance when stimulating PBMCs with OKT3 anti-CD3 mAb immobilised onto polystyrene plates of the Costar type. In contrast, Falcon polystyrene plates and polypropylene plates elicited reduced cytokine induction, and lower apparent potency, reflecting slower association of mAbs onto Falcon polystyrene, and higher background stimulation of PBMCs by polypropylene plates. We observed essentially no difference between Costar polystyrene plates with flat- and round-bottom wells. Costar polystyrene plates were saturated by OKT3 mAb solutions of 10 μg/mL (2 μg/well) or higher. We therefore recommend using Costar polystyrene plates for CRAs supporting FTIH clinical studies for immuno-stimulatory mAbs, similar biologics, and their combinations. These observations should aid the design and optimisation of CRAs, hence helping to inform clinical development.
Traditional immunohistochemistry techniques are limited in the number of fluorescent detection channels, markers, and staining resolution, which restricts the ability to fully evaluate bulk tissue samples and biopsies. Recent technical advancements, such as co-detection by indexing and spatial proteomics, have enabled in situ visualization of tissues by combining multiplex assays, repetitive staining, and quantitative image analysis. These technologies can identify cellular co-expression, cellular spatial relationships, tissue heterogeneity, and detect low-abundance molecules, which are critical for basic immunology, disease evaluation, and therapeutic evaluation studies. However, these methods require specially conjugated antibodies and protocols to analyze highly multiplexed tissue imaging (HMTI) readouts. Here, we utilize imaging cytometry as a viable alternative that also enables individual cellular cytometry analyses in two-dimensional formats. This technique has been used to investigate human and mouse tissues but is underexplored in the translationally relevant rhesus macaque (RM) model. Here, we demonstrate the use of this platform to image RM placenta, jejunum, kidney, and liver stored in OCT, using commercially available fluorophore-conjugated antibodies to identify structural markers (cytokeratin and vimentin), pan-immune cells (CD45), T cells (CD3 and CD8), natural killer cells (NKG2A/C), monocytes (CD16) and macrophages (CD68 and CD163), without any customization. We compared these preclinical samples to human samples to emphasize the potential for cross-species translational analyses using this platform. The flexibility to perform multiple rounds of photobleaching and fluorophore-based staining, combined with the ability to compensate for autofluorescence, makes this technology extremely valuable for deciphering tissue architecture and the spatial distribution of immune cells. Furthermore, we leveraged the platform's ability to export data in HMTI format and flow cytometry standard format, compatible with other quantitative downstream analysis pipelines, to simultaneously visualize the spatial distribution of various cell populations.
Activation-Induced Cytidine Deaminase (AID) induces DNA double-strand breaks (DSBs) at the switch (S) regions of the Immunoglobulin heavy chain (IgH) locus, which are essential for class switch recombination (CSR) and somatic hypermutation (SHM), key processes for effective antibody production. While AID activity is critical, its off-target effects, such as DSBs at the Myc locus, can cause chromosomal translocations like IgH-Myc fusions, contributing to B-cell lymphomas. The factors assembled on the IgH locus that help restrict AID-induced DSBs and subsequently CSR, remain unknown. To address this, we developed a method to isolate CSR-specific factors by inserting a 5×-GAL4-UAS sequence at the switch-mu (Sμ) region in CH12 cells. This engineered site enables recruitment of a 3-FLAG-GAL4 DNA-binding protein (3F-GAL4-DBD), allowing specific pulldown of proteins enriched at the Sμ region. Successful recovery of the known CSR regulator BRD2 from the Sμ region, along with enrichment of the DNA repair factors 53BP1 and gH2AX, validated this approach. Identification and characterization of IgH-enriched factors establish a validated methodological framework to facilitate future proteomic discovery of CSR regulators and highlight mechanisms that balance antibody diversification with genomic integrity in B cells.
Pseudorabies virus (PRV) remains a major threat to the global swine industry, and the development of rapid, sensitive, and reliable diagnostic methods is critical for effective disease control. In this study, we developed and validated a quantum dot-based fluorescent immunochromatographic strip assay (QDs-FICS) for the rapid detection of PRV glycoprotein B (gB). Recombinant PRV gB protein was expressed in HEK293F cells and used as the immunogen to generate monoclonal antibodies. An optimal antibody pair was screened using a double-antibody sandwich ELISA and subsequently employed to construct the fluorescent immunochromatographic assay. The performance of the assay was systematically evaluated in terms of specificity, sensitivity, and stability. Qualitative spike-in validation in PRV-negative porcine serum was further performed to verify its practical applicability in clinical samples. The results demonstrated that the QDs-FICS exhibited high specificity without cross-reactivity to other viral proteins, high sensitivity with a visual limit of detection of 102.5 TCID50/100 μL, and satisfactory stability during storage. Moreover, the assay could be completed within a short time and required minimal operational procedures. Collectively, the QDs-based fluorescent immunochromatographic assay established in this study provides a rapid, sensitive, and reliable tool for PRV antigen detection, and has strong potential for application in clinical diagnosis, epidemiological surveillance, and on-site detection of pseudorabies.
PLXDC2 is a single-pass transmembrane glycoprotein expressed in multiple stem and immune-related cell types. In human induced pluripotent stem cells (hiPSCs), PLXDC2 mRNA and its corresponding protein have been confirmed by transcriptome analysis and mass spectrometry; however, surface detection by flow cytometry using the monoclonal antibody #4G3 cannot be achieved after routine TrypLE dissociation. This failure is attributable to degradation of protease-sensitive epitopes by TrypLE and membrane-associated proteases such as BACE-1 (β-site APP cleaving enzyme 1). To retain these epitopes, we evaluated a protease-free mechanical dissociation procedure in combination with BACE-1 inhibition during both cell dissociation and antibody staining. This approach enabled reproducible flow cytometric detection of PLXDC2 on 201B7 hiPSCs. Western blot analysis of membrane fractions similarly showed increased PLXDC2 signal intensity when samples were processed in the presence of a BACE-1 inhibitor. These results demonstrate a practical method for improving PLXDC2 detection in hiPSCs and provide a general strategy for analyzing cell-surface proteins that are susceptible to proteolytic cleavage.
Antivenoms are the only specific medications available for the treatment of snakebite envenomation, which represents an important global public health concern. The success of treatment depends, in part, on the ability of the antivenom used to bind to and neutralize the toxins present in the venom. To achieve this efficacy, it is important that the immunizing venom pool be representative of the snake fauna to be covered, which is challenging given the inter- and intraspecific variability among snake venoms. In Brazil, snakes of the genus Bothrops are responsible for most snakebite accidents, and the antivenom used in the country is produced from a pool of five different Bothrops venoms (B. jararaca, B. jararacussu, B. alternatus, B. moojeni, and B. neuwiedi). However, quality control of antivenom efficacy during the production process was carried out exclusively against B. jararaca venom. Thus, the aim of this study was to develop and standardize an avidity ELISA for the analysis of serum samples obtained from horses immunized with the Bothrops venom pool against the different venoms that comprise this pool, also including B. atrox venom, as an auxiliary tool for evaluating antivenom efficacy. The avidity ELISA revealed variations in avidity both among the four batches of equine serum samples analyzed (inter-batch variation) and among the six Bothrops venoms included in the study (intra-batch variation).The avidity ELISA showed good applicability for Bothrops venoms and good intra- and inter-plate repeatability, suggesting the potential of this assay for implementation in preliminary efficacy evaluations of Bothrops antivenom production.
Introduction Neutrophils are the most abundant circulating leukocytes and play a central role in innate immunity. As in vitro functional assays (NETosis, degranulation, phagocytosis, etc.) depend on cell viability, purity and basal activation, it is essential to validate neutrophil suspensions before experimental use. Objectives To characterize isolated neutrophils using an in-house flow cytometry workflow for assessing viability, purity, and activation, and to establish a reference interval (RI) for neutrophil basal activation. Methods Twenty healthy volunteers were recruited, and neutrophils were isolated from whole blood by negative selection. Cell viability was determined by propidium iodide staining. Leukocyte and neutrophil percentage were evaluated using conjugated antibodies against CD45 and CD15 respectively. Basal activation was assessed by measuring mean fluorescence intensity (MFI) using a conjugated antibody against CD66b. To validate the RI of basal activation, neutrophils were also stimulated with TNF. RI were calculated as mean ± 2 standard deviations (SD). Results Neutrophil suspensions displayed high quality across all criteria. Viability averaged 99.4% (SD =0.4). Leukocyte purity reached 99.3% (SD =0.4), and neutrophil purity was elevated, with a mean of 98.6% (SD = 0.5). Isolated neutrophils presented a mean CD66b MFI of 37.4 (SD =10.8), and CD66b expression increased significantly following TNF stimulation. The calculated RI was 37.4 ± 21.6. Conclusion This study demonstrated that neutrophils isolated by negative selection displayed high viability, purity, and minimal activation. It also allowed the definition of a RI for basal activation which could serve as objective criterion to validate neutrophil suspensions for functional assays.
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.
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.
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.
