Journal of immunological methods最新文献

{"title":"Validation for soluble C5b-9 detection and comparative analysis of three quantification methods","authors":"Tracie Profaizer ,&nbsp;Abdulrahman Saadalla ,&nbsp;Vijayalakshmi Nandakumar","doi":"10.1016/j.jim.2025.113882","DOIUrl":"10.1016/j.jim.2025.113882","url":null,"abstract":"<div><h3>Background</h3><div>Soluble C5b-9 (sC5b-9) is a biomarker of complement activation, representing the soluble form of the Terminal Complement Complex (TCC), which is released into circulation rather than embedding in the cell membrane. Measurement of sC5b-9 is increasingly utilized for detecting complement activation, particularly in conditions like thrombotic microangiopathy and during complement inhibitor therapy. Accurate detection of sC5b-9 can be useful for guiding therapeutic decisions, especially in patients receiving anti-C5 inhibitors. This study aimed to validate the Quidel sC5b-9 ELISA kit and compare its performance with two other assays, Hycult and SVAR, focusing on assay linearity and therapeutic monitoring.</div></div><div><h3>Methods</h3><div>The analytical performance of the Quidel sC5b-9 ELISA kit was evaluated using 60 split samples over a wide concentration range, in collaboration with a peer laboratory. Precision, sensitivity, linearity, and reference limits were assessed, mainly using artificially activated samples with magnesium chloride and Zymosan, covering a broad range of sC5b-9 concentrations. Four patient samples with complement-mediated diseases were also tested. The correlation between the Quidel, Hycult, and SVAR assays was analyzed with 40 samples, while linear ranges were compared using 11 proportionally diluted samples. Additionally, the impact of anti-C5 inhibitors on sC5b-9 concentrations was evaluated by spiking anti-C5 biosimilar drugs into activated plasma.</div></div><div><h3>Results</h3><div>The Quidel sC5b-9 assay showed accuracy, with an R² of 0.90 and a qualitative concordance of 93.3 %. Very few discrepancies were noted in samples near the reference limit. Both intra- and inter-assay precision were below 20% CV for samples above the Limit of Quantification (LOQ) of 170 ng/mL. Linearity studies defined the reportable range for Quidel's assay as 220–1800 ng/mL. Comparisons among Quidel, SVAR, and Hycult assays revealed strong correlations, with the Hycult kit demonstrating a broader linear range, maintaining linearity even at concentrations 10 times higher than its highest calibrator. All assays showed a significant reduction in sC5b-9 concentrations after the addition of 50 μg/mL of Eculizumab biosimilar, with SVAR and Quidel reaching values close to their lowest calibrators. In contrast, the Hycult assay maintained a wide dynamic range even at 100 μg/mL of anti-C5 inhibitor addition.</div></div><div><h3>Conclusions</h3><div>This study confirms that the Quidel sC5b-9 ELISA kit is a reliable tool for detecting complement activation and inhibition. However, the Hycult assay offers a broader dynamic range which can be an important consideration when assessing assay suitability for for disease monitoring and therapeutic assessments.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113882"},"PeriodicalIF":1.6,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144138956","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":"Chlamydia trachomatis pgp3 ELISA","authors":"Filomeno Coelho da Silva ,&nbsp;Gathoni Kamuyu ,&nbsp;Kazutomo Yokoya ,&nbsp;Jessica Edney ,&nbsp;Kate Soldan ,&nbsp;Simon Beddows","doi":"10.1016/j.jim.2025.113879","DOIUrl":"10.1016/j.jim.2025.113879","url":null,"abstract":"<div><div><em>Chlamydia trachomatis</em> (Ct) serology is an important tool to support infection and disease surveillance of the population but there are currently no formal reference reagents and few commercial assays available. Our objective was to develop an internally standardized ELISA capable of estimating antibody levels against the virulence factor antigen, pgp3, that could be used for Ct serosurveillance. Purified trimeric pgp3 antigen was stable and the internal standard, quality controls, intra- and inter-assay repeatability metrics were robust. For example, retesting a panel of serum samples (<em>n</em> = 203) resulted in 12 % and 13 % seropositivity for the initial and repeat tests, respectively (Kappa 0.933, 95 %CI 0.857–1.000). A comparison of serum samples from children who would not be expected to have had exposure to Ct and samples from females with a history of chlamydia infection, though not necessarily antibody positive at the time of sampling, generated excellent specificity (99.4 %) and adequate sensitivity (67.9 %) for surveillance purposes. The pgp3 ELISA reports results in antibody levels (AU/mL) which are amenable to recalibration should an international reference reagent be approved in the future.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113879"},"PeriodicalIF":1.6,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144072438","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":"Comparison of mixed modeling regression methods for the assessment of longitudinal CyTOF® data","authors":"Tyson H. Holmes,&nbsp;Caroline Duault","doi":"10.1016/j.jim.2025.113880","DOIUrl":"10.1016/j.jim.2025.113880","url":null,"abstract":"<div><div>A simulation study was conducted to assess Type I error and statistical power of linear mixed models, generalized linear mixed models, and linear quantile mixed models for the analysis of longitudinal cytometry by time-of-flight data. Findings indicate that, while generalized linear mixed models have superior statistical power, they also suffer from inflated Type I error rates. Linear mixed models can have substantially lower statistical power than the other methods at larger effect sizes. While linear quantile mixed models have slightly lower statistical power at smaller effect sizes, they have intermediate statistical power at larger effect sizes and good Type I error control. Taken altogether, these results generally recommend the use of linear quantile mixed models for longitudinal datasets such as for cytometry by time-of-flight data, although linear mixed models may be slightly more useful at small effect sizes and sample sizes.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113880"},"PeriodicalIF":1.6,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144068668","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":"Target tolerance improvement of the immunogenicity assay developed for analysing samples from clinical trials of RGB-14, a proposed biosimilar to Prolia/Xgeva","authors":"Tímea Pap,&nbsp;Nikolett Király,&nbsp;Anna Fekete,&nbsp;János Pál Tóth,&nbsp;Attila Kónya","doi":"10.1016/j.jim.2025.113878","DOIUrl":"10.1016/j.jim.2025.113878","url":null,"abstract":"<div><div>An immunogenicity assay was developed to support the clinical program of RGB-14, a proposed biosimilar to Prolia/Xgeva. The ADA assay was successfully validated following current guidelines and white papers. The validation included an assessment of target (RANKL) interference, and the assay passed the test up to a 30 pg/mL RANKL level. This concentration was anticipated to be the maximum level present in clinical samples. Despite the successful validation, most of the analysed samples from the RGB-14 Phase I clinical trial yielded false-positive results for anti-denosumab antibodies. The investigation concluded that unexpectedly high levels of RANKL were present in the samples and caused interference in the assay. Consequently, the assay was re-developed by adding target-specific reagent, eliminating acid dissociation step and doubling the assay dilution to enhance target tolerance to 10,000 pg/mL RANKL, a requirement for clinical sample testing. The re-developed assay underwent successful validation, and the samples from the clinical trials of RGB-14 were subsequently analysed with the improved assay. The article outlines a strategy for enhancing the target (RANKL) tolerance of the ADA assay. It also underscores the importance of monitoring clinical sample analysis results, even if a validated assay is used for sample testing, as clinical samples may differ from the spiked samples prepared for validation. If unexpected results are obtained, an investigation should be initiated, and the assay should be improved if needed to ensure the reliability of the results.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113878"},"PeriodicalIF":1.6,"publicationDate":"2025-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144072238","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 of a high-sensitivity assay for direct quantitation of EGFr on extracellular vesicles in plasma","authors":"Dorte Aalund Olsen ,&nbsp;Torben Frøstrup Hansen ,&nbsp;Ivan Brandslund ,&nbsp;Jonna Skov Madsen","doi":"10.1016/j.jim.2025.113877","DOIUrl":"10.1016/j.jim.2025.113877","url":null,"abstract":"<div><div>Extracellular vesicles (EVs) play an important role in intercellular communication and hold promise for cancer diagnostics and therapeutic monitoring, particularly in Epidermal Growth Factor receptor (EGFr)-driven malignancies. This study aims to develop a method to quantitate EGFr on EVs directly in plasma samples without using an EV purification step first. Additionally assays for quantitating the total concentrations of EVs were established. The assays were developed using Single molecule array (Simoa) technology with antibodies targeting the EV surface markers EGFr, CD9, CD63 and CD81. Plasma samples from healthy individuals and cancer patients were used for assay development, validation and testing. In this pilot study we observed a lower concentration of EGFr on EVs in cancer patients as compared to healthy individuals, though the difference was not statistically significant. The total EV concentrations were significantly increased in plasma from cancer patients compared to healthy individuals (<em>p</em> &lt; 0.0001). Positive correlations were observed among the total EV assays (<em>r</em> = 0.6, p &lt; 0.0001). The developed EV assays present non-invasive methods for quantitating both total EVs and EGFr on EVs directly in plasma samples. Next step will be a comprehensive validation using large cohorts of cancer samples to explore the clinical relevance.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113877"},"PeriodicalIF":1.6,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143936135","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":"Deciphering the autoreactome: Massively parallelized methods for autoantibody detection in humans","authors":"Nicolai V. Hörstke,&nbsp;Thomas Vogl","doi":"10.1016/j.jim.2025.113876","DOIUrl":"10.1016/j.jim.2025.113876","url":null,"abstract":"<div><div>Autoantibodies have a substantial impact on human health ranging from autoimmune diseases to cancer diagnostics. Knowledge of the antigens recognized can allow for more accurate diagnostics, a better understanding of pathogeneses and thus improved prevention, as well as laying the foundation for the development of new therapies. A critical step to acquire this knowledge is to detect the exact self-antigens targeted by autoantibodies out of the pool of 20,000 human proteins against which reactivities could be observed.</div><div>Here, we review established and emerging methods for highly parallelized autoantigen detection such as human proteome microarrays, serological identification of antigens by screening of cDNA expression libraries (SEREX), serological proteome analysis (SERPA), phage display immunoprecipitation sequencing (PhIP-Seq), parallel analysis of translated ORFs (PLATO), and rapid extracellular antigen profiling (REAP). We highlight advantages and limitations of these methods, aiming to give a guideline to choose the appropriate method for a certain application.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"541 ","pages":"Article 113876"},"PeriodicalIF":1.6,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143936134","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 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}