Journal of immunological methods最新文献

{"title":"Identification of polyreactive antibodies by high throughput enzyme-linked immunosorbent assay and surface Plasmon resonance.","authors":"Luis Antonio Rodriguez Carnero, Daniel Bedinger, Simon Cocklin, Jianquan Li, M Frank Erasmus, Sara D'Angelo, Camila Leal-Lopes, Andre Azevedo Reis Teixeira, Fortunato Ferrara, Andrew Raymon Morton Bradbury","doi":"10.1016/j.jim.2025.113855","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113855","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113855"},"PeriodicalIF":1.6,"publicationDate":"2025-03-27","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":"","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}

{"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, C A Shaw, M Chandra, C S Forconi, A M Moormann, V Konduri, V N Tubman, W K Decker","doi":"10.1016/j.jim.2025.113852","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113852","url":null,"abstract":"","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113852"},"PeriodicalIF":1.6,"publicationDate":"2025-03-21","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MARMOT: A multifaceted R pipeline for analysing spectral flow cytometry data from subcutaneously growing murine gastric organoids.","authors":"Lydia Kirsche, Jiazhuo He, Anne Müller, Peter Leary","doi":"10.1016/j.jim.2025.113854","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113854","url":null,"abstract":"<p><p>The analysis of murine immune cell types is a critical component of immunological research, necessitating precise and reproducible methodologies. Here, we present a comprehensive protocol and pipeline designed to streamline the process from murine gastric organoid transplant sample preparation to figure generation. This pipeline includes a detailed staining panel tailored for murine immune cells, ensuring accurate and comprehensive identification of various cell types. Additionally, it integrates an R-based analysis script (MARMOT Pipeline), encompassing data processing and visualisation. A key feature of this pipeline is its ability to produce publication-quality figures with minimal direct R coding, thus making advanced data analysis accessible to researchers with limited programming experience. Additionally, figures can be customised using a provided Shiny application. This approach both enhances the efficiency of data analysis and enables the reproducibility required for high-quality scientific research.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113854"},"PeriodicalIF":1.6,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692416","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":"The generation of a novel HER2-targeting nano PE25 immunotoxin with superior anti-tumor activity and high productivity","authors":"Jianguo Xiao ,&nbsp;Xianfei Chen ,&nbsp;Yibin Wan ,&nbsp;Zhenhua Guo ,&nbsp;Xiu Ren ,&nbsp;Haomin Huang","doi":"10.1016/j.jim.2025.113849","DOIUrl":"10.1016/j.jim.2025.113849","url":null,"abstract":"<div><div>Immunotoxins have the potential to be developed into anti-tumor drugs due to their targeting and strong tumor-killing activity. However, at present, issues such as dose limitations due to off-target toxicity and immunogenicity hinder the clinical use of immunotoxins. A novel HER2-targeted immunotoxin F02 was devised in this study. F02 links an anti-HER2 single-domain camelid antibody to a domain III mutant of PE toxin via a cleavable linker. The PE domain III mutant has seven point-mutations, which potentially remove B-cell and T-cell binding epitopes, thus reducing immunogenicity risks. F02 maintains anti-tumor activity in vivo and in vitro. In animals, F02 effectively inhibited the growth of NCI-N87 tumors at 1.0 mg/kg, and showed a dose-effect relationship, as the effect of completely removing tumors could be achieved at doses above 2.5 mg/kg. F02 also has low toxicity. In cynomolgus monkey models, good tolerability was observed even at 5 mg/kg, a much higher dose than the effective dose. In addition, the molecule has good druggability and can achieve soluble expression in <em>E. coli</em> with a high expression level. Thus, this new molecule has the potential to become a new option for treatment of HER2-positive tumors.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113849"},"PeriodicalIF":1.6,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143628571","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":"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}