Journal of immunological methods最新文献

{"title":"Development of a novel histidine-rich glycoprotein measurement system as a biomarker for sepsis","authors":"Takaoki Uchiumi ,&nbsp;Masahiro Nishibori ,&nbsp;Hiroshi Morimatsu ,&nbsp;Yoko Inoue ,&nbsp;Hiroshi Nishi ,&nbsp;Norio Ota","doi":"10.1016/j.jim.2025.113868","DOIUrl":"10.1016/j.jim.2025.113868","url":null,"abstract":"<div><div>The plasma histidine-rich glycoprotein concentration is a marker of sepsis severity. In this study, we generated selective and specific monoclonal antibodies against histidine-rich glycoprotein for use in a prototype enzyme-linked immunosorbent assay-based in vitro diagnostic system. First, we investigated the properties of monoclonal antibodies produced by 21 hybridomas that we developed using immunized mice, and we identified monoclonal antibodies 69-1A and 75-2D to be the most suitable combination for use in the sandwich enzyme-linked immunosorbent assay. Wild-type histidine-rich glycoprotein (Form-1, 75 kDa) with a proline residue at amino acid position 204 is the most common isoform of the protein in humans, followed by its variant (Form-2, 77 kDa), which has a serine residue at position 204.</div><div>The epitope mapping was examined for the HRG amino acid sequence with 69-1A and 75-2D mAbs to achieve the identification of respective specific binding domains, though the other kinds of mAbs showed considerably complex domains. The identified epitopes recognized by 69-1A and 75-2D monoclonal antibodies did not span position 204. Furthermore, immunoprecipitation-immunoblotting analysis showed that the 69-1A and 75-2D monoclonal antibodies could bind to both Form-1 and Form-2 in human plasma samples. Thus, these two new antibodies can be used to clearly detect both forms of histidine-rich glycoproteins in human plasma samples. In our analysis of clinical samples by enzyme-linked immunosorbent assays using various combinations of our newly synthesized antibodies, we found that the histidine-rich glycoprotein concentration was significantly lower in plasma samples from septic patients than in those from healthy volunteers (<em>p</em> &lt; 0.01). Thus, our novel analysis system using the new antibodies is expected to be a useful tool for sepsis research, and it may be adapted as an in vitro diagnostic tool for many other kinds of diseases in the future.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113868"},"PeriodicalIF":1.6,"publicationDate":"2025-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143936133","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":"Antibodies against chikungunya virus structural proteins reveal the presence of CHIKV virions in migrasomes","authors":"Shengnan Wang ,&nbsp;Xichi Tang ,&nbsp;Junxiang Zhao ,&nbsp;Haiyan Huang ,&nbsp;Leiliang Zhang","doi":"10.1016/j.jim.2025.113867","DOIUrl":"10.1016/j.jim.2025.113867","url":null,"abstract":"<div><div>Chikungunya fever (CHIKF) is a viral illness transmitted by mosquitoes, caused by the chikungunya virus (CHIKV), which has led to millions of infections globally. Antibodies against CHIKV serve as essential tools for research on this virus. In this study, we designed peptides corresponding to the E1, E2, E3, and capsid proteins of CHIKV to immunize New Zealand white rabbits, leading to the development of polyclonal antibodies. We characterized these antibodies through Western blot and immunofluorescence assays. Importantly, our findings using these antibodies revealed the localization of CHIKV structural proteins within migrasomes, thereby uncovering the presence of CHIKV virions in migrasomes, as confirmed by transmission electron microscopy (TEM). The antibodies generated in this study will provide a valuable resource for future investigations into CHIKV.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"540 ","pages":"Article 113867"},"PeriodicalIF":1.6,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143891328","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":"Divalent cations and SERPIN gene expression modulate horseshoe crab coagulation under LPS induction","authors":"Xinn Xian Kwong ,&nbsp;Fisal Ahmad ,&nbsp;Cheng Tzi Him ,&nbsp;Noor Azlina Kamaruding ,&nbsp;Ahmad Khusairi Azemi ,&nbsp;Noraznawati Ismail","doi":"10.1016/j.jim.2025.113866","DOIUrl":"10.1016/j.jim.2025.113866","url":null,"abstract":"<div><div>Horseshoe crabs (<em>Tachypleus gigas</em>) have a unique immune system used in enzyme research, particularly in coagulation studies. The coagulation process in these crabs involves serine protease enzymes. However, how these enzymes and their inhibitors regulate clotting under lipopolysaccharide (LPS) induction is unclear. This study aimed to examine the effects of divalent cations and pH on the gelation and proteolytic activity of trypsin-like serine protease in wild and captive horseshoe crab blood. It also compared the protein profiles of Tachypleus amoebocyte lysate (TAL) and measured SERPIN gene expression during LPS induction. The methods used included the gel clotting method to assess gelation, SDS-PAGE for protein profiling, LC-MS/MS for protein identification, and RT-PCR for gene expression analysis. Results showed that wild horseshoe crab lysate had higher clotting levels compared to captive crabs. Among 35 divalent cation combinations, 0.15 M MgCl2 + 0.15 M CaCl2 resulted in higher clotting in wild TAL. Fresh TAL had higher proteolytic activity, and four major proteins were detected. SERPIN gene expression increased fivefold in July compared to June. Overall, captivity reduced TAL quality and clotting efficiency.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"540 ","pages":"Article 113866"},"PeriodicalIF":1.6,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143870724","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 laboratory validation of an electrochemiluminescence ELISA technique for measuring infliximab concentrations and anti-drug antibodies","authors":"Tracie Profaizer ,&nbsp;Marc G. Elgort ,&nbsp;Julio C. Delgado","doi":"10.1016/j.jim.2025.113865","DOIUrl":"10.1016/j.jim.2025.113865","url":null,"abstract":"<div><div>Infliximab is a monoclonal biologic medication used to treat inflammatory and autoimmune disorders. Effective patient management involves quantifying the drug's therapeutic levels and timely detection of anti-drug antibodies (ADA). In this study, we show results of the development and clinical laboratory validation of an electrochemiluminescence immunoassay (ECLIA). The ADA assay employs a bridging ELISA format with biotinylated infliximab as the capture molecule and ruthenium-labeled infliximab as the reporter. For the drug assay, a sandwich format was developed using a biotinylated tumor necrosis factor as the capture molecule and an anti-idiotypic antibody fragment specific to infliximab as the reporter. During assay development, the optimal concentrations for the capture and reporter reagents in both ADA and drug assays were determined. The validation assessed accuracy through two methods: a reporter gene assay (RGA) and sending residual clinical samples to a peer lab utilizing ECLIA methodology. The ADA assay showed a combined 96.1 % qualitative agreement with both RGA and peer ECLIA methods. For the drug assay, comparison with the peer ECLIA method revealed a positive correlation (R² = 0.79) with a Deming Regression slope of 1.02 (95 % CI, 0.86–1.18). Despite also having a positive correlation (R² = 0.82) with the RGA, the drug assay slope was only 0.71 (95 % CI, 0.60–0.83), indicating functional differences between methodologies. Both assays displayed inter- and intra-assay precision below 25 % coefficient of variance. Linearity studies and analytical sensitivity tests indicated the analytical measurement range was 20–1500 ng/mL for the ADA assay and 0.5–40 μg/mL for the drug assay. In conclusion, validation of the infliximab ADA and drug assays demonstrated high sensitivity, precision, and accuracy, meeting criteria for the detection and quantification of infliximab and related antibodies in clinical patient testing.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"540 ","pages":"Article 113865"},"PeriodicalIF":1.6,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859065","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":"Inhibition of RANKL is critical for accurate assessment of anti-drug antibody incidence to denosumab in clinical studies","authors":"Mieke Jill Miller ,&nbsp;Mark A. Kroenke ,&nbsp;Troy Barger ,&nbsp;Marta Starcevic Manning ,&nbsp;Winnie Sohn ,&nbsp;Kevin Graham ,&nbsp;Daniel T. Mytych ,&nbsp;Shalini Gupta","doi":"10.1016/j.jim.2025.113864","DOIUrl":"10.1016/j.jim.2025.113864","url":null,"abstract":"<div><div>Denosumab is an approved monoclonal antibody therapeutic for the treatment of bone loss in the postmenopausal osteoporosis and oncology settings and acts by binding and neutralizing the activity of receptor activator of nuclear factor-kappa-B ligand (RANKL). Anti-drug antibodies (ADAs) to denosumab are measured <em>via</em> a standard electrochemiluminescence- (ECL) based bridging assay. In this format, the presence of soluble RANKL (sRANKL) in clinical samples can lead to false positive results. In a denosumab bioequivalence study, approximately 50 % of the serum samples showed reactivity to denosumab in the absence of a specific reagent to sequester the sRANKL. However, upon addition of osteoprotegerin (OPG) as a reagent to neutralize the sRANKL, the overall ADA incidence was lowered to &lt;1 %. To address this RANKL reactivity over the long-term use of this assay, the performance of 3 RANKL inhibitors was evaluated using healthy donor sera samples spiked with different concentrations of positive control ADA, sRANKL, or both, in an ECL based immunoassay utilizing the Meso Scale Discovery (MSD) platform. Based on these data, the denosumab antibody assay was modified to include a neutralizing anti-RANKL monoclonal antibody to eliminate the assay reactivity or false positivity due to sRANKL. Use of an anti-RANKL antibody did not impact the ADA-specific signal but inhibited the false positive assay signal due to sRANKL, resulting in an accurate detection of ADA incidence. Therefore, inhibition of interference posed by sRANKL in study samples is critical for the accurate assessment of ADA incidence towards denosumab and any biosimilars for this product that are undergoing clinical development.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"540 ","pages":"Article 113864"},"PeriodicalIF":1.6,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143839487","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":"Advancing multiplex diagnostics: A novel framework for simultaneous detection of diverse pathogens using respiratory RNA viruses as a model system","authors":"M. Caputo ,&nbsp;S. Ginart ,&nbsp;L. Garrigos ,&nbsp;D. Corach ,&nbsp;F. Remes Lenicov ,&nbsp;A. Sala","doi":"10.1016/j.jim.2025.113857","DOIUrl":"10.1016/j.jim.2025.113857","url":null,"abstract":"<div><div>Numerous infectious pathologies share clinical presentation but require different management. The search for multiple pathogens in a single test could improve the resolution of these cases. For example respiratory infections, caused either by viral, bacterial or fungal agents. Rapid differential detection of common respiratory RNA viruses could be benefit by such a strategy.</div><div>We present a diagnostic method based on real-time quantitative PCR (qPCR) coupled with high-resolution melt (HRM) analysis. While applicable to diverse pathogens, this study focuses on detecting three respiratory RNA viruses: influenza A (H1N1), respiratory syncytial virus (RSV), and SARS-CoV-2.</div><div>To develop a rapid, specific, and cost-effective system, we designed a multiplex assay using SYTO 9 fluorescent dye and pathogen-specific primers. Post-amplification HRM analysis generated melt curves, with first-derivative plots enabling discrimination of the three viruses.</div><div>Samples tested in quintuplicate (10¹–10³ copies/μL) yielded distinct melt peaks. Mean melting temperatures (average Tm ± SD) were 83.64 ± 0.08 °C (SARS-CoV-2), 80.45 ± 0.07 °C (influenza H1N1), and 75.99 ± 0.07 °C (RSV). The system robustly differentiated all three viruses (100 % accuracy). Preliminary validation with clinical samples showed strong agreement with a commercial kit: κ = 0.87 (95 % CI: 0.61–1.00) for SARS-CoV-2 and κ = 1.00 (95 % CI: 0.74–1.00) for RSV.</div><div>Therefore, the designed method represents a rapid and cost-effective alternative for differential diagnosis in a single reaction tube.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"540 ","pages":"Article 113857"},"PeriodicalIF":1.6,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795603","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 novel combination mode for inhalant allergen screening based on 8-well microplates by LiCA","authors":"Yixian Li ,&nbsp;Xiaoming Cui ,&nbsp;Xiaohui Yang ,&nbsp;Qinqin Liu ,&nbsp;Yuanmin Sun ,&nbsp;Xue Li ,&nbsp;Huiqiang Li ,&nbsp;Yang Yu","doi":"10.1016/j.jim.2025.113856","DOIUrl":"10.1016/j.jim.2025.113856","url":null,"abstract":"<div><h3>Objective</h3><div>Currently, allergen detection is primarily classified into two types: single detection and multiple combined detection. The challenges include large volume of serum required, high testing costs, and fixed combinations of test items. A cost-effective method that uses a small amount of serum and allows for the flexible combination of allergen screening is necessary. Light-initiated Chemiluminescence Assay (LiCA) requires no washing, only need little serum, and supports flexible combination detection of allergens.</div></div><div><h3>Methods</h3><div>Based on the advantages of LiCA technology, which does not require washing, the ‘4 + 3X + 1’ allergen detection mode was established in this study. The antigens are coated on chemibeads, which are selectively added into the detection wells to achieve flexible combination, supporting the simultaneous detection of eight allergens.</div></div><div><h3>Results</h3><div>This study introduces a new allergen screening model termed “4 + 3X + 1”, where “1” represents tIgE, “4” comprises four commonly encountered indoor inhalable allergens, and “3X” encompasses a variety of frequent inhalable allergens. Allergens from “3X” could be selected based on regional characteristics and patient-specific conditions. We tested the intermediate precision(3.59 %–9.71 %) and repeatability(2.81 %–9.31 %) of the four allergens in “4-fixed”, and LoB(0.035–0.066 kU_A/L), LoD(0.092–0.156 kU_A/L), and LoQ(0.135–0.199 kU_A/L) all showed good results. We compared the consistency of LiCA and ImmunoCAP/Dot-ELISA, each of which was greater than 0.8828. In addition, two allergens were selected from “4-fixed” and “3X-optional “respectively to detect the sensitivity(80 %–97.72 %) and specificity(93.75 %–100 %).</div></div><div><h3>Conclusions</h3><div>The proposed combined test model offers a new, economical, and rapid option for allergen screening that requires low blood volumes and can be customized for individual patients.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"540 ","pages":"Article 113856"},"PeriodicalIF":1.6,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795601","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":"Corrigendum to “Comprehensive normalization and binary classification methods for enhanced sensitivity and reproducibility in Luminex assay quantitation” [Journal of Immunological Methods 538 (2025) 113826]","authors":"B.A. Burns ,&nbsp;C.A. Shaw ,&nbsp;M. Chandra ,&nbsp;C.S. Forconi ,&nbsp;A.M. Moormann ,&nbsp;V. Konduri ,&nbsp;V.N. Tubman ,&nbsp;W.K. Decker","doi":"10.1016/j.jim.2025.113852","DOIUrl":"10.1016/j.jim.2025.113852","url":null,"abstract":"","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113852"},"PeriodicalIF":1.6,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692411","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":"Identification of polyreactive antibodies by high throughput enzyme-linked immunosorbent assay and surface Plasmon resonance","authors":"Luis Antonio Rodriguez Carnero ,&nbsp;Daniel Bedinger ,&nbsp;Simon Cocklin ,&nbsp;Jianquan Li ,&nbsp;M. Frank Erasmus ,&nbsp;Sara D'Angelo ,&nbsp;Camila Leal-Lopes ,&nbsp;Andre Azevedo Reis Teixeira ,&nbsp;Fortunato Ferrara ,&nbsp;Andrew Raymon Morton Bradbury","doi":"10.1016/j.jim.2025.113855","DOIUrl":"10.1016/j.jim.2025.113855","url":null,"abstract":"<div><div>The assessment of polyreactivity is usually carried out by enzyme linked immunosorbent assay (ELISA) using biochemically diverse target antigens with different biochemical properties, including charge and hydrophobicity, and comprising proteins, carbohydrates, nucleic acids and lipids, some of which are heterogenous in nature. Here we explored polyreactivity ELISAs based on probes of defined molecular weight, which we were also able to directly transition to a polyreactivity assay using surface plasmon resonance (SPR). Using a panel of previously characterized clinical antibodies we obtain results compatible with previous polyreactivity studies, but with potential for high throughput analysis following kinetic measurements in the early discovery process. We find ELISA is more sensitive for the detection of polyreactivity in antibodies, and with potential lower throughput, compared to SPR, but may lack the linear sensitivity of SPR.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113855"},"PeriodicalIF":1.6,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143743167","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":"Flow cytometry reveals constant lymphocyte proportions after long-term cryopreservation of whole blood in TransFix® cell stabilization reagent","authors":"Maria Faresjö ,&nbsp;Junko Johansson ,&nbsp;Ulrika Islander ,&nbsp;Andrea Tompa","doi":"10.1016/j.jim.2025.113853","DOIUrl":"10.1016/j.jim.2025.113853","url":null,"abstract":"<div><div>Flow cytometry is an important technique for characterization of immune cells, with accurate lymphocyte profiling being essential for clinical diagnostics and research applications. While immediate processing of blood samples is ideal, long-term storage solutions are needed for large-scale studies or settings without immediate access to laboratory facilities. TransFix® is a chemical stabilization solution that allows delayed analysis by preserving cell morphology and surface markers. However, the impact of long-term cryopreservation in TransFix® on lymphocyte integrity remains underexplored. In this study, we evaluated the efficacy of cryopreservation in TransFix® for maintaining the proportions of key lymphocyte subsets, including CD3⁺ T cells, CD3⁺CD4⁺ T helper cells, CD3⁺CD8⁺ cytotoxic T cells, CD19⁺ B cells, and CD16⁺/CD56⁺ natural killer cells. Blood samples were cryopreserved in TransFix® for varying time periods, up to 48 months, and compared to fresh samples using flow cytometry. The results show that the proportions of lymphocyte subsets remain stable during cryopreservation for up to 48 months, with no significant differences observed between fresh and cryopreserved samples. This suggests that TransFix® can successfully preserve lymphocyte integrity for long-term storage, providing a reliable option for delayed analysis. These results highlight the usefulness of TransFix® for studies that require extended storage, making it easier to conduct immune monitoring in a wide range of settings.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113853"},"PeriodicalIF":1.6,"publicationDate":"2025-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143697791","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}