Journal of immunological methods最新文献

{"title":"NADPH oxidase subunit p22phox: A marker of oxidase-dependent oxidative stress and target for stress suppression in nonphagocytic cells","authors":"Kei Miyano ,&nbsp;Shuichiro Okamoto ,&nbsp;Fumiya Ojima ,&nbsp;Yasuhiro Takenouchi ,&nbsp;Risa Yamamoto ,&nbsp;Kimika Matsui ,&nbsp;Misaki Azuhata ,&nbsp;Mariko Inoue ,&nbsp;Mizuho Kajikawa ,&nbsp;Akira Yamauchi ,&nbsp;Futoshi Kuribayashi ,&nbsp;Shin-Ichiro Nishimatsu","doi":"10.1016/j.jim.2025.113850","DOIUrl":"10.1016/j.jim.2025.113850","url":null,"abstract":"<div><div>Reactive oxygen species (ROS)-producing NADPH oxidase (Nox) family proteins are involved in host defense. The overproduction of ROS leads to oxidative stress, which is associated with a myriad of diseases. The Nox subunit p22^{<em>phox</em>} is essential for Nox1–4 activity, and p22^{<em>phox</em>} and Nox2 proteins are mutually stabilized in phagocytic cells. This study investigated the suitability of p22^{<em>phox</em>} protein as a marker of Nox activity. To avoid contamination by phagocytic p22^{<em>phox</em>}, we developed global <em>Cybb</em> (encoding Nox2)-knockout mice and analyzed p22^{<em>phox</em>} stability and the expression profiles of Nox proteins in lysates of various tissues. We found that consistent with Nox2 in phagocytic cells, p22^{<em>phox</em>} protein was detected when Nox1–4 were coexpressed in nonphagocytic cells. Furthermore, p22^{<em>phox</em>} protein degradation was suppressed by Nox1–4, suggesting that p22^{<em>phox</em>} is a suitable marker of Nox family-dependent oxidative stress. Thus, we examined p22^{<em>phox</em>} protein levels in tissue lysates prepared from <em>Cybb</em>-knockout mice to avoid the contamination of phagocytic p22^{<em>phox</em>}. <em>Cybb</em>-knockout mice show moderately reduced p22^{<em>phox</em>} protein levels in lung tissue, suggesting that Nox2 and other Nox family members stabilized p22^{<em>phox</em>} protein. Paradoxically, this result implied that p22^{<em>phox</em>} knockdown concurrently suppressed various Nox family-dependent oxidative stress mechanisms, and this was confirmed by the suppression of Nox family-dependent directed migration in p22^{<em>phox</em>}<em>-</em>knockdown A549 human lung epithelial cells. Therefore, p22^{<em>phox</em>} not only served as a marker of Nox-dependent oxidative stress but also as a target to suppress this stress in various tissues and cells.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113850"},"PeriodicalIF":1.6,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143620864","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":"Optimization of IgG1 immune complexes to stimulate cytokine production in innate cells","authors":"Arpita Deb,&nbsp;Kailyn Lott,&nbsp;Audrey Miceli,&nbsp;Barbara L.F. Kaplan","doi":"10.1016/j.jim.2025.113851","DOIUrl":"10.1016/j.jim.2025.113851","url":null,"abstract":"<div><div>Fcγ receptors are key immunoreceptors, that when bound by IgG immune complexes, trigger activation of downstream signaling pathways. However, there are limited in vitro assays that stimulate innate cells via Fcγ receptors that allow for evaluation of drugs or chemicals on antibody-triggered signaling. Our study investigated the activation of Fcγ receptors in innate cells using immune complexes. Our findings revealed that immobilized IgG antibodies did not elicit a significant immune response, so we designed two IgG1 immune complexes: trinitrophenyl-bovine serum albumin (TNP-BSA)/TNP-IgG1 and streptavidin-biotinylated IgG1 (Strept-Biotin IgG1). Strept-Biotin IgG1 immune complex was particularly effective, significantly enhancing IL-6, TNFα, and C3a levels, whereas TNP-BSA/TNP-IgG1 immune complex showed a modest IL-6 increase. Both TNP-BSA/TNP-IgG1 and Strept-Biotin IgG1 stimulated CD86 marker expression on F4/80+ macrophages. We also confirmed the binding of Strept-Biotin IgG1 to innate cells with fluorochrome-conjugated streptavidin. To further understand the Fcγ receptor-mediated activation of innate cells, we blocked the downstream phosphatidylinositol 3-kinase (PI3K) pathway. We found out that the PI3K inhibitor successfully suppressed IL-6 cytokine release and C3a production. However, specific Fcγ receptor-blocking antibodies failed to block IL-6 cytokine production and only modestly suppressed TNFα cytokine release, suggesting either that the antibodies were not effective blockers or that these immune complexes use other receptors. Regardless, the use of the Strept-Biotin IgG1 immune complex to stimulate cytokine production and other immune signaling was consistent with Fcγ receptor activation on innate cells which might be useful in assessing the effects of drugs or chemicals in innate cells.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113851"},"PeriodicalIF":1.6,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143624607","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":"ELISA protein detector (EPD): A Python-based ELISA tool for accurate low-level protein quantification","authors":"You Lu ,&nbsp;Li Qi ,&nbsp;QinZheng Xu ,&nbsp;ZhuoHuan Li ,&nbsp;Hao Duan ,&nbsp;Fei He ,&nbsp;Na Zhao ,&nbsp;James M. Hyman","doi":"10.1016/j.jim.2025.113847","DOIUrl":"10.1016/j.jim.2025.113847","url":null,"abstract":"<div><div>The enzyme-linked immunosorbent assay (ELISA) is a cornerstone technique for quantifying protein secretion in biological research. However, the built-in software provided by ELISA plate readers often struggles to accurately detect low-concentration proteins, particularly in the sub-nanogram/mL range, due to limitations in calibration curve fitting. We developed the ELISA Protein Detector (EPD) to overcome these challenges. This open-source Python-based software employs advanced optimization algorithms to enhance curve fitting precision, particularly at low detection thresholds.” EPD features an intuitive user interface, requires minimal technical expertise, and supports robust cross-validation to enhance the reliability of ELISA data analysis. Tested on Windows systems, this tool provides a cost-effective and versatile solution for researchers, enabling accurate quantification of low-level protein concentrations and addressing the shortcomings of standard ELISA software in diverse biological and clinical applications.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113847"},"PeriodicalIF":1.6,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596882","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":"Optimization of blood sampling time for isolation of antigen binding single-domain antibodies from immunized llamas","authors":"Michiel M. Harmsen,&nbsp;Frank Harders,&nbsp;Sandra van de Water,&nbsp;Conny S. van Solt,&nbsp;Ad P. Koets,&nbsp;Fimme J. van der Wal,&nbsp;Nishi Gupta","doi":"10.1016/j.jim.2025.113844","DOIUrl":"10.1016/j.jim.2025.113844","url":null,"abstract":"<div><div>Single-domain antibody fragments (VHHs) binding specific antigens are often isolated by llama immunization and phage display selection. We determined the optimal blood sampling time for generating phage display libraries with a high frequency of antigen-binding VHHs. Two llamas were immunized with 7 antigens. Blood samples collected with 2–3 day interval up to 11 days post immunization were used for deep sequencing and phage display selection of VHHs. The frequency of VHH clones was determined by the identification of unique VHH sequences in deep sequenced libraries. On average, the highest clone frequency was found in libraries generated 5 days post immunization. Polyclonal phage ELISA also suggested day 5 as the optimal blood sampling time.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113844"},"PeriodicalIF":1.6,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143563409","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":"Anti-chicken and anti-pigeon immunoglobulin G testing in patients with bird-related fibrotic hypersensitivity pneumonitis","authors":"Ryo Okuda ,&nbsp;Tamiko Takemura ,&nbsp;Toshihiro Misumi ,&nbsp;Eri Hagiwara ,&nbsp;Takashi Ogura","doi":"10.1016/j.jim.2025.113846","DOIUrl":"10.1016/j.jim.2025.113846","url":null,"abstract":"<div><h3>Background</h3><div>One of the inciting antigens for fibrotic hypersensitivity pneumonitis (HP) is avian. It is controversial whether chickens are the inciting antigen of bird-related fibrotic HP; the anti-chicken immunoglobulin (Ig) G testing for bird-related fibrotic HP patients was investigated.</div></div><div><h3>Methods</h3><div>Anti-pigeon IgG and anti-chicken IgG antibodies by mainly enzyme-linked immunosorbent assay (ELISA) were measured in patients with bird-related fibrotic HP. We included bird-related fibrotic HP patients who had undergone histopathological examination and who had a positive inhalation challenge test.</div></div><div><h3>Results</h3><div>We included 44 patients with bird-related fibrotic HP and 48 with fibrotic interstitial lung diseases other than fibrotic HP. The mean titers of anti-pigeon IgG antibody by ELISA were 0.659 ± 0.381 and 0.494 ± 0.187 in the bird-related fibrotic HP and control groups, respectively (<em>p</em> = 0.012). Those of anti-chicken IgG antibody were 0.345 ± 0.189 and 0.376 ± 0.167, respectively (<em>p</em> = 0.457). No significant correlation was found between anti-pigeon IgG testing by ELISA and anti-chicken IgG testing (<em>r</em> = 0.45, <em>p</em> = 0.45). In patients with bird-related fibrotic HP, annual FVC changes were significantly different (<em>p</em> = 0.001), with the higher titer group improving by 0.4 % and the lower titer group decreasing by 4 %. Similarly, no significant differences in annual FVC changes were observed between the higher and lower anti-chicken IgG antibody titer groups among bird-related fibrotic HP patients.</div></div><div><h3>Conclusion</h3><div>Serum IgG testing for chicken serum was less effective in diagnosing or predicting the disease progression of bird-related fibrotic HP than that for pigeon serum.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113846"},"PeriodicalIF":1.6,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143573244","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":"Highly sensitive detection of carcinoembryonic antigen via an electrochemical platform fabricated by CCLP- gold nanocomposite","authors":"N. Krithiga ,&nbsp;V.S. Vasantha ,&nbsp;A. Jayachitra","doi":"10.1016/j.jim.2025.113848","DOIUrl":"10.1016/j.jim.2025.113848","url":null,"abstract":"<div><div>An electrochemical CCLP-AuNPs (Calcium Cross Linked Pectin with Gold nanoparticles)-based biosensor was developed for sensitive detection and discrimination of carcinoembryonic antigen (CEA) based on dual signal amplification of gold nanoparticles tagged enzymatic catalysis. The electrochemical biosensor was fabricated by assembly of CCLP-AuNp modified GCE and subsequently anti CEA was immobilized on the modified GCE followed by the addition of CEA protein and then forming a sandwich-type conjugate was formed when the biosensor was successively reacted with Au-Anti CEA-HRP(Gold nanoparticles tagged with the antibody Carcinoembryonic antigen labelled with Horse radish peroxidase). In the presence of hydroquinone and hydrogen peroxide, in the PB(phosphate buffer) solution resulted in the fabrication of biosensor. The concentration of CEA in the range of 0.1 ng/mL to 10 ng/mL with low detection limit of 0.08 ng/mL thus the fabricated biosensor proves to be feasible tool for clinical diagnosis in complex biological systems and shows good for the early diagnosis of cancer.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113848"},"PeriodicalIF":1.6,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143563410","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":"Chimeric protein A/G conjugate based lateral flow assay for the rapid detection of brucellosis in multiple animal species","authors":"Rajeswari Shome ,&nbsp;Sudipta Patra ,&nbsp;Muneera Mohamed Sahib ,&nbsp;G. Shanmugam ,&nbsp;Somy Skariah ,&nbsp;Samer Shamshad ,&nbsp;Nagendra Nath Barman ,&nbsp;Durlav Prasad Bora ,&nbsp;Arijit Shome ,&nbsp;Triveni Kalleshamurthy ,&nbsp;Nagalingam Mohandoss ,&nbsp;Bibek Ranjan Shome","doi":"10.1016/j.jim.2025.113845","DOIUrl":"10.1016/j.jim.2025.113845","url":null,"abstract":"<div><div>The study highlights diagnostic efficacy of chimeric protein A/G based lateral flow assay (LFA) for on-spot diagnosis of brucellosis in various livestock species. The test device was developed using chimeric protein A/G conjugated with 40 nm colloidal gold nanoparticles as detection reagent having a strong binding affinity specifically IgG isotypes in multiple livestock species. Known positive and negative control sera samples from three livestock species and 36 Gram-negative bacterial cross-reactive hyper immune sera were used for determining the analytical sensitivity (ASn) and specificity (ASp), respectively. The diagnostic performance of LFA was evaluated in comparison with RBPT (Rose Bengal Plate Test) and indirect enzyme linked immunosorbent assay (iELISA) using 652 small ruminants, 532 bovine and 241 pig samples. The analytical sensitivity of LFA was observed up to 1:1280, 1:2560, and 1:10240 dilutions in bovine, small ruminants and pigs samples, respectively. Similarly, non-reactive to 35 Gram-negative bacterial cross-reactive hyper immune sera showed high ASp for LFA test except mild reactivity with <em>Y. enterocolitca</em> O9. Considering RBPT as gold standard, the diagnostic sensitivity (DSn) of protein A/G based LFA in infected farm samples was 93.9 (86.3–97.9) in small ruminants, 84.6 (54.5–98.1) in cattle and 96.5 (82.2–99.9) in swine. Considering iELISA as gold standard, the DSn of LFA was 96.3 (89.4–99.2) in small ruminants, 90 (55.5–99.7) in cattle and 96.7 (82.8–99.9) in swine. The higher performance efficacy of the LFA tests developed in this study outlines the practicality of using these rapid, easy to perform, highly sensitive and specific devices for on-spot screening of brucellosis in livestock species, which in-turn facilitate prevention, control and management of disease transmission in farms.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113845"},"PeriodicalIF":1.6,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143573245","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":"CHO-S expression of a novel human recombinant IgG1 of anti-ALD antibody isolated by phage mutation display","authors":"Guilin Li ,&nbsp;Jiazhen Liu ,&nbsp;Zhenzhen Guan ,&nbsp;Sujuan Wang ,&nbsp;Songrui Li ,&nbsp;Zhiqiang Wang ,&nbsp;Yanna Dong ,&nbsp;Yamin Cui ,&nbsp;Yaya Li ,&nbsp;Weitao Zhang ,&nbsp;Xiaoping Tian ,&nbsp;Qiaohui Zhao","doi":"10.1016/j.jim.2025.113843","DOIUrl":"10.1016/j.jim.2025.113843","url":null,"abstract":"<div><div>Aldosterone (ALD) is elevated in the serum and plasma of patients with various metabolism-related diseases, and anti-ALD antibodies are important materials in early diagnostic kits. However, current anti-ALD antibodies for clinical diagnosis are not specific, resulting in high false positives. We have developed an anti-ALD-601# rabbit monoclonal antibody that is commercially available and licensed for diagnosis. To obtain monoclonal antibodies with better stability and affinity, a phage mutation library was constructed by site-specific mutagenesis of the CDR3 region in the V_L and V_H domain of the anti-ALD-601# antibody. Anti-ALD3302#, a single-chain variable fragment (scFv), with enhanced affinity, was screened by enzyme-linked immunoassay (ELISA). To improve the less stable and shorter half-lives of scFvs, anti-ALD-scFv-3302# was inserted into a eukaryotic expression vector (pCHO1.0-Fc) and then transfected into CHO-S cells to express anti-ALD-scFv-Fc. Then, cells were cultured by reducing culture temperature (33.5 °C) and application of CuSO₄ (50–200 μM) treatment. The expression-purified antibody was detected with competitive-ELISA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and capillary electrophoresis. Non-reducing SDS-PAGE electrophoresis showed a single band and the percentage of the highest peaks in capillary electrophoresis was more than 90 %. The coefficient of variation in the competitive-ELISA assay was reduced to 2 % from 5 % before optimization. The correlation between the assay using this monoclonal antibody and the HPLC-FLD determination was also greater than 0.99. In conclusion, the designed anti-ALD-specific antibody (3302#) has been successfully developed for use in metabolism-related diseases to detect the concentration of ALD in early diagnostic.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113843"},"PeriodicalIF":1.6,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143523681","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":"Preparation and specific identification of monoclonal antibodies targeting the VP2 structural protein of minute virus of canines","authors":"Xiang Ren ,&nbsp;Zhiping Hei ,&nbsp;Kai Ji ,&nbsp;Jianhui Guo ,&nbsp;Yan Yan ,&nbsp;Yuning Sun","doi":"10.1016/j.jim.2025.113833","DOIUrl":"10.1016/j.jim.2025.113833","url":null,"abstract":"<div><div>The Minute Virus of Canines (MVC), classified under the genus Bocaparvovirus, causes severe respiratory and gastrointestinal symptoms in neonatal canines worldwide. The structural protein VP2 is essential for the attachment, infection, uncoating, and induction of the host immunological response to MVC. This study aimed to prepare a monoclonal antibody (mAb) against VP2 using the hybridoma technique. AlphaFold and CavityPlus bioinformatics analysis revealed that the N-terminal region of VP2 (amino acids 1–300) possesses structural characteristics that make it the most suitable target for effective antibody generation. The recombinant plasmid pET-32a(+)-VP2(N300) with fused Trx and His tags was successfully constructed. After immunizing mice, nine hybridoma cell lines were obtained, namely 1G5, 1G5–1, 1I24, 1I24–1, 2E6–1, 2 N9, 3C12–1, 4 M1, and 4 M1–1. The ascitic antibody titers of all cell lines were above 1:100,000. Western blot analysis of Walter Reed canine cells infected with MVC indicated the selection of three strains of monoclonal antibodies with strong specificity: 1G5, 3C12–1, and 4 M1. These three strains can be employed in immunofluorescence (IF) and immunoprecipitation (IP) tests for detecting VP2 protein. The monoclonal antibody mAb VP2 prepared in this study may serve as a valuable tool for detecting MVC and beneficial for investigating the mechanisms of MVC infection.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113833"},"PeriodicalIF":1.6,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143437190","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":"Comprehensive normalization and binary classification methods for enhanced sensitivity and reproducibility in Luminex assay quantitation","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.2025.113826","DOIUrl":"10.1016/j.jim.2025.113826","url":null,"abstract":"<div><div>The Luminex assay is a powerful tool for large-scale quantitation of antibody levels and cytokines, but its utility can be limited by issues of specificity, sensitivity, and reproducibility. The corrections for background fluorescence and machine drift are essential steps in the normalization process. However, traditional methods often oversimplify these steps, failing to account for the complexity of the data, leading to the introduction of error and decreasing the sensitivity and reproducibility of the analysis. Furthermore, conventional methods to determine cut-points in binary measures do not consider the true distribution of the data, leading to arbitrary cut-points that compromise the integrity of the analysis. Here, we present a novel approach to normalize Luminex data and split the normalized bimodal data. Our method uses orthogonal regression of the measured fluorescence of a negative control bead and a blank bead to correct for background fluorescence, enhancing accuracy by preventing overcorrection due to cross-reactivity. To account for machine drift, we use a generalized additive model (GAM) on the standard curves to calculate a plate correction, thus reducing error and improving reproducibility. To distinguish between positive and negative results in bimodal measures, we use a clustering analysis to accurately split the data based on distribution. Finally, we developed a web application to easily carry out the developed method. These methods collectively increase sensitivity, specificity, and reproducibility of Luminex assay data analysis by effectively addressing the limitations of current normalization techniques, correcting for background fluorescence and machine drift, and improving the specificity and accuracy in splitting bimodal data.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113826"},"PeriodicalIF":1.6,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143391098","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}