Journal of immunological methods最新文献

{"title":"Type-specific antibody detection of herpes simplex virus types 1&2 (HSV-1&2) in fingerstick blood at point-of-care sites by a rapid and sensitive lateral flow immunochromatographic assay","authors":"Nicholas Vafai,&nbsp;Sihan Chen,&nbsp;Winnie Tong,&nbsp;Clark Wang,&nbsp;Bret Sheffield,&nbsp;Natasha Griffin,&nbsp;Aubrey Kusi-Appiah,&nbsp;Emily Friedland,&nbsp;Veronika Atkinson,&nbsp;Patrick Vaughan,&nbsp;Melissa Farrell,&nbsp;Abbas Vafai","doi":"10.1016/j.jim.2026.114083","DOIUrl":"10.1016/j.jim.2026.114083","url":null,"abstract":"<div><div>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&amp;2 IgG antibody in fingerstick blood. Analytical and clinical analyses were performed to compare the performance characteristics of the HSV-1&amp;2 IgG LFA (VIZIHSV™) with Western Blot.</div><div>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).</div><div>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&amp;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.</div><div>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&amp;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.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114083"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148338811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of a versatile histidine-rich glycoprotein (HRG) measurement methodology bearing a highly compatible design for patients with sepsis in daily clinical practice","authors":"Takaoki Uchiumi,&nbsp;Ken-ichi Shimosako,&nbsp;Masahiro Nishibori,&nbsp;Hiroshi Morimatsu,&nbsp;Masahiko Oiwa,&nbsp;Yoko Inoue,&nbsp;Hiroshi Nishi,&nbsp;Norio Ota","doi":"10.1016/j.jim.2026.114085","DOIUrl":"10.1016/j.jim.2026.114085","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114085"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148421688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diagnostic performance of Pictor's targeted proteomics assay for extractable nuclear antigens compared with commercial line immunoassay and addressable laser bead immunoassay platforms","authors":"Jung-hyun Rho,&nbsp;Andrea Kinga,&nbsp;Bhoopika Shetty,&nbsp;Yoichi Furuya","doi":"10.1016/j.jim.2026.114087","DOIUrl":"10.1016/j.jim.2026.114087","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114087"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148455399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A semi-automated ELISA workflow for SARS-CoV-2 Nucleocapsid IgG detection significantly improves throughput and reduces hands-on time","authors":"Ruturaj R. Masvekar,&nbsp;Gabriela Mansano Do Nascimento,&nbsp;Aliyah Hannah A. Remoroza,&nbsp;Shweta Jain,&nbsp;Troy J. Kemp,&nbsp;Bruce K. Brown,&nbsp;Ligia A. Pinto","doi":"10.1016/j.jim.2026.114075","DOIUrl":"10.1016/j.jim.2026.114075","url":null,"abstract":"<div><h3>Background</h3><div>Enzyme-linked immunosorbent assay (ELISA) is widely used in clinical and research laboratories for applications such as disease surveillance, immunogenicity assessment, and vaccine evaluation. Traditional manual ELISA workflows, however, are labor-intensive, time-consuming, and susceptible to operator-dependent variability, which can limit throughput and reproducibility. Incorporating semi-automation into established protocols offers a potential strategy to improve efficiency while maintaining analytical performance.</div></div><div><h3>Methods</h3><div>A validated manual ELISA protocol for SARS-CoV-2 Nucleocapsid IgG detection was adapted into a semi-automated workflow using an automated multichannel pipette, microplate washer-dispenser, and plate stacker to streamline key steps. Performance was compared across 10 manual and 30 semi-automated plates. Sixteen pre-characterized serum samples were analyzed to evaluate precision, accuracy, and overall assay validity, and internal controls were monitored to ensure compliance with predefined acceptance criteria.</div></div><div><h3>Results</h3><div>The semi-automated workflow doubled assay throughput from 6 to 12 plates per analyst per day and reduced hands-on time by 75% (from 5.5 h to 1 h). Internal controls for all 40 plates met established acceptance ranges. Precision, measured as relative standard deviation, remained below 25% for all samples, and accuracy, expressed as percent error, remained below 20%. No loss of analytical performance was observed when transitioning from manual to semi-automated processing.</div></div><div><h3>Conclusion</h3><div>Semi-automation markedly increases ELISA throughput and reduces hands-on time while maintaining precision, accuracy, and overall assay validity. These results underscore its feasibility as an efficient workflow enhancement with potential for broader application across diverse ELISA platforms pending further validation.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114075"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148205252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Allotypes matter: Evaluation of twelve commercially available antihuman IgG2 antibodies for subclass specificity","authors":"Theo Rispens,&nbsp;Ninotska Derksen,&nbsp;Gerard van Mierlo,&nbsp;Sofie Keijzer,&nbsp;Steven de Taeye,&nbsp;Gestur Vidarsson","doi":"10.1016/j.jim.2026.114086","DOIUrl":"10.1016/j.jim.2026.114086","url":null,"abstract":"<div><div>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 (&gt;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.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114086"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148421632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CEI: A Clonal Expansion Identifier for T-cell receptor clones following SARS-CoV-2 vaccination","authors":"Yunbei Pan,&nbsp;Christian Hofmann,&nbsp;Barbara Banbury,&nbsp;Harsh Patel,&nbsp;Stephanie A. Bien,&nbsp;Tom Chou,&nbsp;Otto O. Yang","doi":"10.1016/j.jim.2026.114088","DOIUrl":"10.1016/j.jim.2026.114088","url":null,"abstract":"<div><div>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 <span><math><mrow><mo>∼</mo><mn>1</mn><msup><mrow><mn>0</mn></mrow><mrow><mn>5</mn></mrow></msup><mtext>–</mtext><mn>1</mn><msup><mrow><mn>0</mn></mrow><mrow><mn>6</mn></mrow></msup></mrow></math></span> 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 <em>in vivo</em>, illustrating the feasibility of quantifying the clone-specific dynamics of T-cell abundance changes following immunological perturbations.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"549 ","pages":"Article 114088"},"PeriodicalIF":1.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148455389","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exosomal survivin as a liquid biopsy biomarker in cancer","authors":"Wynne Wijaya ,&nbsp;Su Myat Phyu ,&nbsp;Wenshu Lu ,&nbsp;Shisong Jiang","doi":"10.1016/j.jim.2026.114064","DOIUrl":"10.1016/j.jim.2026.114064","url":null,"abstract":"<div><h3>Background</h3><div>Survivin, an inhibitor of apoptosis protein, is a tumour-associated protein linked to poor outcomes in cancer. Increasing evidence suggests that survivin is released by cancer cells within exosomes, meaning conventional serum measurements may underestimate its true circulating levels. Practical approaches to quantify exosomal survivin are therefore required to support its clinical utility as a liquid biopsy biomarker.</div></div><div><h3>Methods</h3><div>Exosome-enriched small extracellular vesicle (sEV) preparations were concentrated from conditioned media of HCT116 and MDA-MB-231 cancer cell models and from serum samples of 15 cancer patients and 5 healthy controls using polyethylene glycol (PEG) precipitation; Hs68 fibroblasts served as a low-survivin non-malignant control during lysis optimisation. Samples were analysed with and without membrane lysis prior to survivin measurement by ELISA</div></div><div><h3>Results</h3><div>1% NP-40 was the most effective tested detergent condition for membrane lysis, significantly improving survivin detection in cancer conditioned media (MDA-MB-231: 5.77 ± 0.61 vs 4.45 ± 0.41 ng/mL, <em>p</em> = 0.0348; HCT116: 14.55 ± 2.21 vs 8.30 ± 0.31 ng/mL, <em>p</em> = 0.0083) and patient serum (plain: 102.4 ± 7.63 vs 90.03 ± 9.35 pg/mL, <em>p</em> = 0.0309; PEG-precipitated: 132.7 ± 42.13 vs 102.7 ± 25.35 pg/mL, <em>p</em> = 0.0068). PEG precipitation enhanced exosomal marker recovery, as demonstrated by increased syntenin-1 expression.</div></div><div><h3>Conclusions</h3><div>NP-40 combined with PEG precipitation significantly improves survivin detection in cancer samples. This practical approach can support quantification of exosome-associated survivin and may be adapted to other exosome-associated proteins in translational cancer research, pending validation in larger cohorts with orthogonal EV characterisation.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114064"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147982124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An improved peptide-ELISA protocol for serological identification of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii","authors":"Antonio C.G. Foddai ,&nbsp;Rosa M.M. Gynthersen ,&nbsp;Mette F. Hansen ,&nbsp;Karen A. Krogfelt ,&nbsp;Peter Wilhelmsson ,&nbsp;Per-Eric Lindgren ,&nbsp;Jeremy M. Sternberg ,&nbsp;Anne-Mette Lebech ,&nbsp;Alan S. Bowman","doi":"10.1016/j.jim.2026.114061","DOIUrl":"10.1016/j.jim.2026.114061","url":null,"abstract":"<div><div>The performance of 21 synthetic peptides for serological identification of <em>Borrelia burgdoferi</em> sensu stricto, <em>B. garinii</em> and <em>B. afzelii</em> was assessed in this study using two ELISA protocols based on (1) conventional passive binding onto 96-well Greiner Microloon<em>®</em>600 High-binding microplates and (2) covalent binding onto 96-well surfaced-activated Nunc™ Immobilizer Amino Plates. Sensitivity, specificity and accuracy was initially assessed testing 13 follow-up positive sera from seroconverted patients bitten by ticks that had tested positive for <em>Borrelia burgdorferi</em> sensu stricto (<em>N</em> = 2), <em>B. garinii</em> (N = 2), <em>B. afzelii</em> (<em>N</em> = 9). The optimized protocol was then applied to two cohorts of samples including plasma from Danish patients with Lyme borreliosis (LB) (<em>N</em> = 32) and positive samples from Danish healthy blood donors (HBD) (<em>N</em> = 44).</div><div>Use of covalent binding resulted in higher OD values and significantly increased the accuracy of the test. Results of the seroprevalence study applied to LB and HBD samples showed different distribution of the three <em>Bb</em>sl. <em>Borrelia burgdorferi</em> sensu stricto (<em>Bb</em>ss), <em>B. garinii</em> and <em>B. afzelii</em> were identified in 3 out 24 (12.5%), 13 out of 24 (54.2%) and 7 out of 24 (29.2%) LB samples, respectively. <em>B. garinii</em> was confirmed to be to the most prevalent species also in the second cohort of HBD samples as it was identified in 20 out 32 samples (62.5%) followed by <em>Borrelia burgdorferi</em> sensu stricto and <em>B. afzelii</em> and which were identified in 5 out of 32 (15.6%) and 4 out of 32 (12.5%) samples, respectively.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114061"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147959813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comparison of monocyte-derived macrophages with and without prior CD14+ magnetic sorting","authors":"Julia Thomé ,&nbsp;Johannes Zeller ,&nbsp;Thierry Christmann ,&nbsp;Balazs Bogner ,&nbsp;Maike Lind ,&nbsp;Marie Dreck ,&nbsp;Teresa Z. Brose ,&nbsp;Laura Schneider ,&nbsp;Franziska Pankratz ,&nbsp;Sheena Kreuzaler ,&nbsp;Steffen U. Eisenhardt","doi":"10.1016/j.jim.2026.114058","DOIUrl":"10.1016/j.jim.2026.114058","url":null,"abstract":"<div><h3>Introduction</h3><div>For in vitro studies involving macrophages, an efficient protocol for isolation and cell culture of monocyte-derived macrophages (MDM) is essential. However, it remains unclear how far isolation steps improve purity or provoke cell changes. Here we compare the characteristics and functionality of macrophages isolated from leukocyte reduction (LRS) chambers by either CD14+ magnetic sorting (sMac) or by seeding PBMC with separation by plastic adhesion and subsequent washing steps (uMac) to evaluate the necessity of magnetic sorting.</div></div><div><h3>Methods</h3><div>PBMC were isolated from LRS chambers using density gradient centrifugation. sMac were enriched from PBMC by magnetic separation removing lymphocytes, uMac were seeded and separated from lymphocytes via plastic adhesion and washing steps. Both groups were plated under matched conditions to allow monocyte-to-macrophage differentiation after adhesion. Morphology, purity, cell viability, surface marker expression, and cytokine production upon stimulation were assessed using flow cytometry, qPCR, brightfield and confocal microscopy.</div></div><div><h3>Results</h3><div>MDM were successfully cultured from both uMac and sMac in over 75%. Cell viability was similar between uMac (54%) and sMac (48%). CD14 expression was significantly higher in sMac (89.48%) compared to uMac (65.91%), the mean fluorescence intensity of CD14 did not differ significantly. No significant differences were found in CD80 and CD163 expression, nor in the production of pro-inflammatory cytokines upon stimulation.</div></div><div><h3>Conclusions</h3><div>LRS chambers serve as a valid source for culturing of MDM with high reliability and yield. <em>Since both methods produced macrophages of comparable phenotype and function, our findings indicate that magnetic CD14+ sorting is not mandatory for most</em> in vitro <em>applications.</em></div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114058"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147816197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development and clinical application of a highly sensitive serological assay for detecting thrombospondin type-1 domain-containing 7A antibodies in idiopathic membranous nephropathy","authors":"Tianyu Zheng ,&nbsp;Xuanli Tang ,&nbsp;Shangbin Kao ,&nbsp;Nannan Tian ,&nbsp;Yufeng Yan ,&nbsp;Dian Li ,&nbsp;Canlong Yan ,&nbsp;Yuan Qin ,&nbsp;Xiumei Zhou ,&nbsp;Yigang Wang ,&nbsp;Guoqin Wang ,&nbsp;Biao Huang","doi":"10.1016/j.jim.2026.114065","DOIUrl":"10.1016/j.jim.2026.114065","url":null,"abstract":"<div><h3>Objective</h3><div>To establish a highly sensitive serological assay for detecting serum thrombospondin type-1 domain-containing 7 A antibodies (THSD7A-Ab) in patients with idiopathic membranous nephropathy (IMN), thereby addressing the current gap in available diagnostic biomarkers.</div></div><div><h3>Methods</h3><div>A time-resolved fluorescence immunoassay (TRFIA) was established by immobilizing THSD7A protein on the solid phase, which captured THSD7A-Ab present in serum samples. Detection was performed using a europium (Eu³⁺)-labeled secondary antibody. Serum THSD7A-Ab levels were measured in both THSD7A-positive IMN patients and M-type phospholipase A2 receptor (PLA2R)-negative IMN patients, and the corresponding seropositivity rates were determined.</div></div><div><h3>Results</h3><div>The TRFIA-THSD7A-Ab assay demonstrated a sensitivity of 0.13 RU/mL, with a linear range from 0.13 to 600 RU/mL. The intra-assay precision ranged from 3.35% to 4.63%, while the inter-assay precision ranged from 5.21% to 9.07%. The recovery rate was 93.78% to 107.20%. Serum THSD7A-Ab levels in THSD7A-associated IMN patients (176.54 ± 224.77 RU/mL) were significantly higher PLA2R-associated IMN patients (11.38 ± 4.76 RU/mL, <em>P</em> &lt; 0.0001) and healthy controls (7.86 ± 2.92 RU/Ml, P &lt; 0.0001). The cut-off value for distinguishing THSD7A-associated IMN was determined to be 13.70 RU/mL. Among PLA2R-negative IMN patients, the positive rate of THSD7A-Ab was 1.6%.</div></div><div><h3>Conclusion</h3><div>A highly sensitive TRFIA method for detecting serum THSD7A-Ab was successfully established for the first time, demonstrating excellent discriminative capability for identifying THSD7 A-positive patients.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114065"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147982001","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}