Journal of Immunological Methods

Tacrolimus is an immunosuppressant drug commonly used in transplantation. Although multiple studies have demonstrated that polymorphisms in the CYP3A5 gene impact the metabolism of tacrolimus, routine pre-transplant testing for these markers is still not broadly implemented. TacroType - a new laboratory developed test implemented by One Lambda Laboratories - utilizes a qPCR-based six-plex assay for CYP3A5 genotyping and detects the three most common genetic variants (*3, *6 and *7) associated with loss of CYP3A5 protein function and reduced tacrolimus metabolism. TacroType was optimized to address known sources of protocol, technical or sample variability to achieve accurate and reproduceable genotyping results. An analytical performance study was completed following CLSI guidelines. Accuracy was confirmed for each possible CYP3A5 genotype involving 6 target alleles using 32 well-characterized reference samples. TacroType exhibited accurate performance within a broad range of DNA concentrations and quality. Precision studies indicated consistent genotyping results across 4 operators, 2 instrument types and 5 lots of reagents. Accurate and reproducible assay performance was demonstrated using whole blood from 100 and buccal swabs from 70 donors. The analytical performance of TacroType was evaluated in 4014 total qPCR reactions, with a report rate of 99.8% and genotyping accuracy of 100% (95% confidence interval of 99.9%).

Antibody Fc glycosylation is modulated in a variety of disease and immune response contexts, altering downstream functional responses including antibody-dependent cellular cytotoxicity through modified immune cell Fc receptor binding. Accessible, high-throughput glycosylation assays such as enzyme-linked lectin assays (ELLAs) are essential to advance understanding of glycosylation regulation and function. However, current ELLA protocols lack standardization and optimization, and results are reported out in arbitrary absorbance units, limiting reproducibility and cross-study comparability. We developed an optimized multi-lectin parallel ELLA with three specific improvements: systematic optimization of incubation times and reagent concentrations; incorporation of Protein A for IgG specificity; and use of commercially available bovine fetuin B as a quantitative surrogate standard for cross-study reproducibility. Our panel of 8 lectins, SNA, RCA, LCA, PHA-E, PHA-L, MAL-I, WGA, and DSL, cover the major IgG glycoforms. We demonstrate that our ELLA panel can reveal biologically relevant cytokine-induced plasticity of IgG glycosylation profiles in immortalized B cells.

Abstract/SummaryWe present a streamlined protocol that enables the characterization of the metabolic state of immune cell populations through their distinct NADH/FAD autofluorescence fingerprints using a FACSymphony A5 spectral cytometer. We demonstrate the utility of this approach by profiling the metabolic status of diverse splenic B-cell subsets and assessing metabolic changes associated with their activation state.

Flow cytometry can establish T cell clonality by detecting a restricted expression pattern of the T cell receptor (TCR) {beta} constant region (TRBC), expressed in association with CD3. However, T cell neoplasms frequently lose surface expression of the CD3/TCR complex, posing a challenge to demonstrating T cell lineage and clonality. To address this challenge, here we present a 12-color flow cytometry panel, called cytoTCR, to characterize cytoplasmic expression of CD3/TCR complex components. We apply cytoTCR to 38 patient specimens with immunophenotypically abnormal T cell populations, demonstrating this approach can efficiently establish T cell lineage and clonality in challenging T cell neoplasms that have lost surface CD3 expression. While we show that natural killer (NK)-lineage neoplasms can express cytoplasmic CD3 at similar levels to T cells, we show that absent expression of cytoplasmic TCR components by mature lymphocytes can help confirm NK cell lineage. We demonstrate that cytoTCR can detect cytoplasmic TRBC-restriction in challenging cases of null-phenotype anaplastic large cell lymphoma, which lack surface expression of pan-T cell antigens. In cases of T-lymphoblastic leukemia, cytoTCR shows that cytoplasmic TRBC expression matches the expected developmental stage of the leukemia. Finally, we use cytoTCR to characterize atypical cCD3-CD7- T cells in a patient with a history of T-lymphoblastic leukemia as well as recent CAR-T therapy, showing that this atypical population is polytypic and represents CAR-T product rather than residual disease. Our study presents a broadly applicable flow cytometric approach to simultaneously assess T cell lineage and clonality in suspected T lineage populations with absent surface CD3 expression.

We demonstrate the use of fluorescence correlation spectroscopy (FCS) to characterize fluorescently-labeled protein production. We use cell-free protein synthesis to express the protein YFP-CopB, a fusion of Chlamydia Outer Protein (Cop) B and Yellow Fluorescent Protein (YFP). CopB is a [~]50 kDa protein believed to have a critical role in chlamydial infection.1 After adding a plasmid encoding YFP-CopB to an E. coli cell-free lysate, protein expression begins. We track the cell-free reaction over several hours using the EI-FLEX, a commercial instrument with FCS capability. As protein is expressed over time, YFP-CopB increases in concentration, and the EI-FLEX detects an increase in fluorescent signal above the background of the cell-free lysate. The FCS data collected gives information about the size, aggregation tendencies, rates of production and fluorescent protein maturation, and concentration of the YFP-CopB produced. The use of FCS concurrent with cell-free synthesis presents a simple method to characterize proteins of interest as they are produced without the need for purification.

Antibody- and cell-mediated immune responses against biologics, should they occur, can impact treatment efficacy and potentially pose severe risks to patient safety. Therefore, developers have focused on advancing strategies to mitigate such unwanted immunogenicity. Opportunities to address immunogenicity early in the development process, particularly during the drug design phase, have been identified. In vitro and in silico tools that facilitate the identification and removal of sequence liabilities have been established. For example, human cell-based in vitro T cell assays can be used to identify and remove CD4+ T cell epitopes, which are known to play a critical role in the development of anti-drug antibodies against recombinant proteins products as well as the transgenes of gene and therapy. Despite their widespread use in the industry, most of these assays lack thorough characterization, which undermines confidence in the results and comparability across laboratories. In this study, concepts of immunogenicity bioanalytical assay validation for study design and analysis were applied to characterize an internal CD4+ T cell proliferation assay as fit-for-purpose. A statistical path was applied to establish data acceptance criteria for handling of replicates, positivity and negativity of a signal, and donor cohort size. A Bayesian analysis was also performed and is proposed as an approach for sequence de-risking decision making. The in-depth characterization of the CD4+ T cell proliferation assay described here allows for accurate interpretation of the assay outcomes, thereby enhancing confidence in using this approach for mitigating the immunogenicity of biologics by design.

Lipoarabinomannan (LAM) is a useful biomarker for detection of M. tuberculosis infection and disease. Related antigens can be detected in urine samples of TB patients by combinations of monoclonal antibodies (mAbs) directed against specific epitopes expressed in LAM. While sensitive for samples from patients with active TB disease who have HIV-1 co-infections, these assays are less effective for other populations, and there is therefore a need for more sensitive antibodies that can improve the sensitivity of these assays. Here we characterize the antigen and epitope specificities, sequence diversity and isotype dependencies of eight LAM-specific human mAbs that target five distinct arabinose- and mannose-dependent epitopes present in LAM and lipoarabinomannan (LM). Whereas all of the mAbs recognized ManLAM, only a few, including A194-01, consistently detected antigens in TB+ urine samples. Converting A194-01 from the IgG1 to the IgM isotype resulted in broader recognition of poly-Ara glycan epitopes, and increased sensitivity for clinical antigens when combined with several capture reagents, including RU95-C1, a novel antibody targeting the mannan domain of LAM. These results define novel epitopes that are differentially expressed in bacterial and urinary forms of LAM, and identify novel antibody combinations which possess enhanced diagnostic utility for clinical forms of LAM.

BackgroundComplement-mediated hemolysis assays are essential for assessing immune function and diagnosing complement-related disorders. Conventional human erythrocyte derivatization with 2,4,6-trinitrobenzene sulfonic acid (TNBS) can induce nonspecific hemolysis and optical interference, complicating interpretation. Identifying a more biocompatible electrophile could improve assay specificity and reliability. MethodsA panel of aldehydes was screened for electrophilicity using a nucleophile consumption assay with glycine as a model nucleophile. Glyoxylic acid was selected based on reactivity, solubility, and visual neutrality, then neutralized with sodium bicarbonate to minimize baseline hemolysis. Human erythrocytes were sequentially treated with pancreatin and glyoxylic acid to generate glyoxylic acid-pancreatin-treated erythrocytes (GxPEs). Complement-mediated hemolysis was assessed using normal human serum, heat-inactivated serum, and pathway-specific conditions, with CH50 values calculated for total, alternative, and non-alternative pathways. ResultsGxPEs exhibited robust complement-specific hemolysis (maximum 93.56%) with negligible background activity in heat-inactivated serum. CH50 analysis confirmed activation via both alternative (0.9514 L) and non-alternative (1.963 L) pathways. Reconstitution experiments with factor B-depleted cryoprecipitate and cryosupernatant fractions demonstrated dependence on small complement components such as C2 and C4. ConclusionsGlyoxylic acid derivatization yields a reproducible, optically quiet, and complement-specific erythrocyte substrate suitable for functional hemolysis assays. This method offers a practical platform for complement diagnostics, research applications, and therapeutic evaluation.

Celiac disease (CD) is strongly associated with specific HLA DQ heterodimers, formed by HLA DQA1 and HLA DQB1 proteins. In particular DQ2.5 (DQB1*02 associated to DQA1*05) and DQ8 (DQB1*03:02 with DQA1*03) are present in virtually all celiac patients. HLA DQB1*02 is considered the main single genetic susceptibility marker and has been reported in 90 to 95% of CD patients. However, the distribution of these alleles may vary across populations, potentially impacting the performance of genetic screening strategies. In this study, we evaluated the prevalence of HLA DQ2.5 and DQ8 genotypes in celiac patients (n = 41) and an unbiased general population cohort (n = 60) from San Luis, Argentina, using a PCR-based genotyping approach. In addition, we assessed the feasibility of a simplified saliva direct PCR protocol for large scale testing. Overall, 95.1% of CD patients carried DQ2.5 and/or DQ8. Notably, 41.5% of patients were DQ8(+)/DQ2.5(-), and 36.6% lacked the DQB1*02 allele, indicating that DQB1*02 based screening alone would have reduced sensitivity in this population. In the general population, 53.3% of individuals carried CD associated genotypes, with a markedly higher prevalence of DQ8 compared to European cohorts. Genotype distributions deviated from Hardy Weinberg equilibrium in CD patients but not in the general population. We show that DQB1*03:02 is a reliable proxy for DQ8, allowing simplification of genotyping strategies, whereas DQA1*05 typing remains essential to discriminate DQ2.5 from other lower risk DQB1*02 carrying heterodimers. We also describe a saliva direct PCR approach showing a performance comparable to purified DNA based assays. These findings highlight the importance of population specific genetic data for optimizing CD screening strategies and foster the development of simplified, cost effective genotyping approaches for large scale applications.

Autoimmune diseases, such as systemic lupus erythematosus (SLE), are underscored by dysregulated B cell function including the production of autoantibodies, skewed population ratios, and aberrant signaling. Given that the family of nuclear factor kappa B (NF-{kappa}B) transcription factors govern responses to stimuli, survival, differentiation, and so forth understanding the intricate regulatory network of NF-{kappa}B in B cell biology is paramount for unraveling treatments for B cell-linked autoimmune diseases. Here, we focus on a negative regulator of NF-{kappa}B signaling, A20 (TNFAIP3), that deactivates NF-{kappa}B transcription factor translocation through the ubiquitination and deubiquitination of target proteins. Haploinsufficiency in A20 results in an autoimmune phenotype and mutations to A20 have been associated with SLE, suggesting implications to B cell function. To investigate the role of A20 in NF-{kappa}B in human B cells, we generated a TNFAIP3 knockout (KO) Raji cell line. Cells were stimulated with either anti-IgM or Resiquimod (R848) to activate distinct NF-{kappa}B signaling pathways. Using qRT-PCR, western blotting, and flow cytometry, we assessed differences in gene expression, protein production, and NF-{kappa}B activation. We observed key limitations in using Epstein-Barr virus transformed B cell lines to model inducible NF-{kappa}B signaling.

Background Diagnostic interpretation of kidney allograft biopsies using the Banff classification remains variable, but the determinants of this variability are not fully defined. We performed a global, fully digital multi-reader study to identify the principal drivers of disagreement in Banff-based assessment. Methods Thirty six kidney transplant biopsies were independently scored by 67 renal pathologists on a standardized digital platform. Readers assessed Banff lesions on hematoxylin and eosin, periodic acid Schiff, and Jones' silver stains; final diagnostic categories were assigned using prespecified Banff-based decision rules. Interobserver agreement was quantified with Gwet's agreement coefficient (AC) statistics. Determinants of diagnostic agreement were evaluated) using pairwise mixed-effects logistic regression, and reader similarity was examined by principal component analysis (PCA) with post hoc molecular annotation. Results Agreement for final diagnostic categories was moderate (Gwet's AC1, 0.55; 95% CI, 0.47 - 0.63). Lesion-level agreement varied substantially, with lowest agreement for selected threshold-dependent inflammatory or semi-quantitative lesions, including interstitial inflammation in areas of IFTA, peritubular capillaritis and arteriolar hyalinosis. Diagnostic concordance differed markedly across biopsies, indicating strong case-level heterogeneity. In pairwise models, differences in active inflammatory and vascular lesion scoring were the strongest correlates of diagnostic disagreement; reader experience and geography contributed minimally. Principal component analysis showed reader variation was organized along two dominant axes: a rejection-calling threshold axis linked mainly to tubulointerstitial inflammatory injury, and a T cell-mediated (TCMR/TI) and antibody-mediated/microvascular (AMR/MVI) inflammation-oriented phenotypic classification axis. Conclusion Interobserver variation in Banff-based kidney transplant biopsy assessment is structured rather than random and driven mainly by how readers threshold and integrate key inflammatory lesion compartments rather than experience or geographic location.

BACKGROUND In line with International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommendations for high-sensitivity cardiac troponin assays, analytical validation and reference limit assessments are required to confirm that an assay meets performance criteria. This study evaluated the analytical performance and established the 99th percentile upper reference limit (URL) for the SPINCHIP High-Sensitivity Cardiac Troponin I (SPINCHIP hs-cTnI) point-of-care assay. METHODS Analytical performance characteristics, including the limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ), were assessed. Additionally, 1,053 plasma samples and 1,055 whole-blood samples were used to determine the URL. Imprecision around the 99th percentile URL was evaluated as part of the analytical validation. High-sensitivity criteria were assessed by confirming measurable cTnI in [&ge;]50% of healthy individuals (n=432 plasma; n=431 whole blood) and achieving imprecision <10% at the 99th percentile (plasma, n=960; whole blood, n=480). RESULTS SPINCHIP hs-cTnI demonstrated a LoB of 0.3 ng/L; LoDs of 0.8 ng/L (plasma) and 0.9 ng/L (whole blood); and LoQs of 1.1 ng/L (plasma) and 1.4 ng/L (whole blood). The analytical measuring range was 1.1-9,000 ng/L. Imprecision at the common 99th percentile URL (14 ng/L) was 5.8%; for men (URL=16 ng/L) 5.6% and for women (URL=10 ng/L) 6.3%. Greater than 85.2% (94.0% and 76.1% in men and women, respectively) of healthy individuals showed measurable cTnI above the LoD. CONCLUSIONS The SPINCHIP hs-cTnI assay meets the IFCC high-sensitivity requirements, demonstrating <10% imprecision at the 99th percentile, reliable low-concentration precision and cTnI detection in more than half of healthy individuals.

Botanical dietary supplements (like wheat, barley, teff, oats, white lupin, pumpkin seed, and chickpeas) may contain trace amounts of toxicants in addition to important micronutrients. Developing and validating a reliable protocol for the simultaneous quantification of Cu, Fe, Zn, Mo, Se, Mn, Pb, Al, Ni, and Cr using a PerkinElmer (NexIONTM2000 model) quadrupole ICP MS (including a He collision and reaction cell when needed) with closed vessel microwave digestion using (HNO3 + H2O2) was the aim of this study.The method was subsequently utilized in a sample survey, and the outcomes were evaluated against WHO/JECFA standards. From five study regions, twenty-seven farm-collected botanical powder samples representing seven species were acquired. To create one composite per species, field subsamples were cleaned, air dried, ground, and blended (nine subsamples per botanical: three grabs from each of three farms). HNO3/H2O2 was used to digest aliquots (0.250-0.500gm) in closed microwave containers. Internal standards, multi-point external calibration, procedural blanks, verified reference materials, matrix spikes, and duplicates were all used in ICP MSs multi-element quantitation. Method LODs/LOQs, accuracy (CRM recoveries), and precision (RSD) were calculated.The technique produced low LODs that were suitable for dietary evaluation (typical LOD ranges: Cu, Fe, Zn, Mn, Ni, Cr (0.001-0.01) mg/kg; Mo, Se, Pb, Al (0.002-0.05) mg/kg. For the majority of analytes, within-run RSDs were less than 5%, while CRM recoveries ranged from 88.9 to 110%. The concentrations of essential elements varied greatly (average mg/kg: Fe (280.7{+/-}25.6); Zn (6.0{+/-}0.541); Cu (2.8{+/-}0.269); Mn (398.3{+/-}23.8); {micro}gm/kg: Se (0.061{+/-}0.006); Mo (1.0 {+/-}0.022). Although some composites approached or exceeded conservative intake thresholds for Pb and Al under high consumption scenarios, toxic elements were generally low (mean mg/kg: Pb (0.062{+/-}0.007); Al(185.2{+/-}18.5); Ni(1.6{+/-}0.163); Cr(1.8{+/-}0.171).For the simultaneous nutritional and contaminant profiling of supplements derived from cereals and those not, the validated ICP- MS workflow with microwave HNO3 and H2O2 digestion is suitable. Accurate labeling and consumer safety can be supported by routine screening and supply chain controls.

Tuberculosis screening is not mandatory for prospective tissue donors. In 2021 and 2023, two different bone allograft products caused nationwide tuberculosis outbreaks. We assessed the morbidity and mortality of the second outbreak and reviewed donor and tissue screening to identify deficiencies. Thirty-six people residing in nine states received the product during spinal and dental procedures. Twenty-seven recipients had tuberculosis infection, 11 had microbiologic or imaging evidence of tuberculosis disease, and two died from tuberculosis within 12 months of outbreak detection. Another recipient died from tuberculosis nearly 3 years after product implantation. The bone donor died of pneumonia and septic shock. Polymerase chain reaction testing of the product before and after distribution did not detect Mycobacterium tuberculosis. Mycobacterial culture was not performed until after outbreak detection, when M. tuberculosis was isolated from 2 of 6 unused product units. This outbreak demonstrates persistent gaps in tissue transplant safety. Appropriate selection of donors and mycobacterial culture of donated tissues could reduce but not eliminate the risk of M. tuberculosis transmission. Therefore, it is important that clinicians monitor tissue recipients and promptly report adverse events to tissue establishments and health authorities.

Metastasis remains the major cause of cancer-related mortality, and circulating tumor cells (CTCs) are both candidate liquid-biopsy biomarkers and plausible intermediates of metastatic dissemination. Because CTCs are extremely rare in peripheral blood, platform comparisons have often focused solely on recovery. That focus is insufficient for applications that depend on the quality of the recovered material, including single-cell profiling, short-term culture, and functional testing. Here, we compared four CTC isolation approaches: TellDx CTC System, Genesis System, RosetteSep, and flow cytometry, using spike-in experiments in human blood. Capture efficiency was evaluated across all four platforms; purity was assessed for TellDx, Genesis, and RosetteSep; and post-isolation GFP signal persistence in culture was assessed for TellDx and Genesis as an exploratory proxy for short-term post-isolation preservation. Under the conditions tested, TellDx showed the highest recovery (88.1% {+/-} 3.7%), followed by Genesis (40.6% {+/-} 12.1%), RosetteSep (36.5% {+/-} 9.0%), and flow cytometry (7.6% {+/-} 4.5%). TellDx also showed the highest purity score (3.76), whereas Genesis (2.25) and RosetteSep (2.09) did not differ substantially. In the short-term culture assay, TellDx-derived samples retained a higher normalized GFP signal than Genesis-derived samples at 48 h and 72 h. To synthesize these readouts, we propose the Recovery Performance Index (RPI), a composite score integrating recovery, purity, and post-isolation signal persistence. Within this experimental framework, TellDx achieved the highest RPI. These data support two conclusions. First, platform benchmarking for CTC workflows benefits from multidimensional evaluation rather than recovery alone. Second, under this spike-in model and within the specific workflows used here, TellDx performed best among the platforms tested. The principal contribution of this study is therefore the establishment of a practical benchmarking framework that can be expanded in future work using clinical samples, multiple CTC phenotypes, and orthogonal viability assays.

Cell-free DNA (cfDNA), released from apoptotic and necrotic cells into body fluids, represents a non-invasive source of genetic information for disease prediction, diagnosis, and monitoring. However, its low physiological abundance makes cfDNA highly susceptible to pre-analytical influences. In particular, genomic DNA (gDNA) released from lysed white blood cells (WBCs) can contaminate plasma and compromise downstream cfDNA analyses. This study evaluated the impact of different blood collection tubes and isolation methods on cfDNA stability and yield. Blood samples from 13 healthy donors were collected using cfDNA-stabilizing tubes (Cell-Free DNA BCT, Streck; S-Monovette cfDNA Exact, Sarstedt) and stored at room temperature for 1, 5, or 10 days before plasma isolation. CfDNA was extracted using either a magnetic bead-based method or a silica column-based approach. DNA quantity and quality were assessed by fluorometric quantification, automated fragment analysis, and gene-specific quantitative PCR. Streck-based workflows maintained stable cfDNA yields and characteristic mononucleosomal fragmentation profiles across all storage times. In contrast, Sarstedt tubes showed reduced cfDNA concentrations after 5 days and a pronounced increase at 10 Days, accompanied by high-molecular weight DNA patterns consistent with WBC lysis. These trends were largely independent of the extraction method. Overall, the results demonstrate that blood collection tube chemistry critically influences cfDNA integrity during delayed processing. Streck tubes, particularly when combined with QIAamp, provided the most robust and reproducible workflow for routine molecular diagnostics, whereas Sarstedt tubes produced physiologically implausible results after extended storage.

Multiplex bead-based immunoassays (MIAs) are promising tools for simultaneously detecting humoral immunity to multiple targets, potentially playing a crucial role in serosurveillance and vaccine response assessments. However, evaluation of assay performance is paramount prior to widespread use. This study presents a performance evaluation of a pan-filovirus MIA through characterization of the analytical range for the EBOV glycoprotein (GP) target and assessments of assay precision and antigen discrimination. The precision of the MIA was evaluated by comparing the detection of anti-filovirus antibodies at two independent laboratory sites: the University of Hawaii, Honolulu (UH), and the Institut National de Recherche Biomedicale (INRB) in Kinshasa, Democratic Republic of the Congo (DRC). Forty-six samples from Yambuku, DRC, including Ebola virus Disease (EVD) survivors and close contacts, were tested at both sites. Additionally, 858 samples were tested in DRC before and after vaccination with a prophylactic EVD vaccine, ERVEBO. Results demonstrated low variability between laboratories, with intra-assay and inter-laboratory coefficients of variation below predefined thresholds for all filovirus targets included in the multiplex panel. Analyte correlations between sites were high (r2=0.86-0.92). Longitudinal analysis detected increased EBOV GP reactivity following vaccination, while reactivity to non-vaccine filovirus antigens remained stable, consistent with minimal cross-reactivity in a vaccinated cohort. These findings suggest that this pan-filovirus MIA produces reproducible results across distinct laboratory settings and may serve as a useful tool for comparative serologic investigations, serosurveillance, and evaluation of EBOV vaccine-associated antibody responses. IMPORTANCE STATEMENTThis study investigates the functionality and intra-laboratory consistency of a novel multiplex bead-based immunoassay with pan-filovirus targets. As part of the evaluation process, the feasibility of using the assay in resource-limited settings was demonstrated in the Democratic Republic of the Congo. This assay holds significant promise as a tool for detecting filovirus-specific antibody responses. By leveraging its multiplex capabilities, it may be used for widespread serosurveillance of high-consequence pathogens, including the pan-filovirus antigens such as Ebolavirus and Marburg virus already incorporated in the assay, as well as other targets of interest.

IntroductionAccurate in silico evaluation of primers and probes is essential for the rational design of molecular multi-parameter assays. We present Assay-BLAST v2 to automate and simplify this process for extensive assay designs. ResultsA newly integrated strand and proximity check enables precise validation of corresponding oligonucleotides, ensuring correct orientation and spacing for efficient amplification. Based on predicted oligonucleotide interactions, Assay-BLAST v2 estimates amplification outcomes, offering a computational benchmark for downstream wet-lab validation and performance correlation. Additionally, the updated software integrates an adaptive BLAST parameter optimization that dynamically scales with database size, thereby improving both analytical sensitivity and computational performance. These improvements are supported by a comparative evaluation against the previous version of AssayBLAST. ConclusionsCollectively, these enhancements streamline the assay development workflow, reduce costs associated with suboptimal primer and probe synthesis, and increase the robustness and reliability of molecular diagnostics and research applications.

Background: The N-terminal ectodomain (NTE) of the SARS-CoV-2 membrane (M) glycoprotein is a short, flexible region that remains exposed on the virion surface and exhibits immunogenic potential across multiple coronaviruses. Despite its small size and conformational plasticity, this region contains conserved linear epitopes that may serve as practical surrogates for full-length proteins in serological diagnostics. Objective: To develop and evaluate a synthetic peptide-based diagnostic assay targeting the NTE of the SARS-CoV-2 M protein. Methods: Epitope prediction, peptide synthesis, and antibody affinity assays were performed to design homomultivalent peptide analogs that exploit avidity effects through disulfide polymerization. The resulting peptide antigens were tested in an enzyme-linked immunosorbent assay (ELISA) using clinical samples from RT-PCR-confirmed COVID-19 patients and biobanked controls. Results: The selected peptide analogs (M1, M1i, M1s) corresponded to a conserved surface-exposed motif of the SARS-CoV-2 M protein. Polymeric M1 exhibited a twofold gain in apparent affinity (Kdapp = 4.33 nM) compared with the monomeric form (Kdapp = 8.00 nM). Clinical validation using 1,222 patient samples yielded a sensitivity of 95.26% and specificity of 52.27%, with an overall diagnostic accuracy of 88.70%. Conclusion: The M peptide analogs demonstrate that synthetic peptide antigens can serve as stable, high-sensitivity surrogates for whole-protein assays. This design principle may be applied to other emerging pathogens where rapid assay development and scalability are critical. Keywords: Peptides, Antibodies, COVID-19, Enzyme-Linked Immunosorbent Assay, Protein Binding

African swine fever virus (ASFV) poses a major transboundary threat to global swine production, underscoring the need for rapid and field-deployable diagnostic tools. Although quantitative polymerase chain reaction (qPCR)-based assays are the standard molecular assay for ASFV detection, their reliance on centralized laboratory infrastructure, multi-step sample preparation, and trained personnel limit their utility for timely decision-making at the point of need (PON). Here, we report a portable molecular diagnostic platform that enables colorimetric quantitative loop-mediated isothermal amplification (qLAMP) directly from diluted whole blood on microfluidic paper-based analytical devices ({micro}PADs). The assay targets the conserved ASFV viral protein 72 (VP72) and topoisomerase II (TOPII) genes and incorporates objective image-based colorimetric signal analysis to reduce user-dependent interpretation. Using plasmid DNA spiked into whole blood diluted to 5% (v/v) in 5% D-mannitol, the {micro}PAD-LAMP assay achieved a limit of detection (LOD) of 25 copies per reaction (67 copies/{micro}L of whole blood sample) for VP72 targets with no observed cross-reactivity against nine common swine pathogens, demonstrating 100% analytical sensitivity and specificity during in-house testing and 90% and 92% analytical sensitivity and specificity respectively in an external laboratory evaluation. The complete assay was performed within 60 minutes using a portable heating and imaging platform. Together, these results demonstrate a simple, DNA extraction-free molecular diagnostic approach that enables rapid and reliable ASFV detection from whole blood applicable to field-relevant conditions.