Journal of immunological methods最新文献

{"title":"Automation and performance validation of the pooled donor basophil activation test for chronic urticaria using the Hamilton STAR system","authors":"Jessica Chavez,&nbsp;Evan W. McConnell,&nbsp;Rogger Alcalde,&nbsp;Ajay Grover,&nbsp;Deborah Boles,&nbsp;Christopher M. Shuford,&nbsp;Russell P. Grant,&nbsp;Andre Valcour,&nbsp;Saintedym Wills","doi":"10.1016/j.jim.2025.114009","DOIUrl":"10.1016/j.jim.2025.114009","url":null,"abstract":"<div><div>This study presents the automation of the pooled donor basophil activation test (PD-BAT) for chronic urticaria, transitioning from a previously published manual assay in tubes to an automated 96-well plate format using the Hamilton Microlab STAR system. The primary aim is to enhance throughput and robustness in a commercial lab environment while maintaining assay accuracy and reproducibility. Donor screening included 36 individuals, of which 16 were selected for pooled assays. A total of 90 sera were analyzed in parallel by manual and automated methods. The automated PD-BAT demonstrated 93.3% qualitative agreement (<em>r</em> &gt; 0.97) with the manual assay. Throughput improved through automation, a 66% reduction in hands-on time was observed between the manual (9 h/batch) and automated (3 h/batch) methods. Discordant results (6.7%) occurred only near the technical cutoff, highlighting both the robustness and limitations of the method. This automation represents a valuable advancement over the manual assay, optimizing laboratory workflows.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114009"},"PeriodicalIF":1.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145696084","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":"Subclass optimised antibodies as an effective tool to suppress protein A induced false positives in immunoassays detecting staphylococcal enterotoxin B","authors":"Paulin Dettmann,&nbsp;Hans Werner Mages,&nbsp;Martin Skiba,&nbsp;Daniel Stern,&nbsp;Martin B. Dorner,&nbsp;Brigitte G. Dorner","doi":"10.1016/j.jim.2025.114023","DOIUrl":"10.1016/j.jim.2025.114023","url":null,"abstract":"<div><div>Staphylococcal enterotoxins (SEs) belong to a family of highly potent superantigens produced by <em>Staphylococcus aureus</em> and play a significant role in food poisoning and toxic shock syndrome. Among the SE family type B (SEB) is unique as it is involved in naturally occurring diseases and also has a history of military use as an incapacitating agent. Thus, the accurate detection of SEs and particularly SEB is critical. However, immuno-based detection methods encounter intrinsic difficulties due to <em>S. aureus'</em> co-production of staphylococcal protein A (SpA), which has a strong affinity for immunoglobulins. This interaction can result in false-positive results in antibody-based assays, thereby complicating the interpretation of results with regard to presence and quantity of SEB. Commercially available detection methods seek to address this issue through SpA depletion by pre-incubating samples with animal serum. While these approaches mitigate the impact of SpA interference, they frequently result in diminished assay sensitivity. In this study, a previously established highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for SEB detection was optimised to maintain its low detection limit in the one-digit pg/mL-range while simultaneously abolishing its reactivity to SpA in <em>S. aureus</em> liquid-culture supernatants. The modifications were focused on direct alterations to existing detection antibodies by a) adapting the immunoglobulin subclass and generation of antibody fragments through recombinant technology, and b) the careful selection of control capture antibodies. This study can be taken as a blueprint for optimised ELISA strategies to overcome SpA related false results while maintaining high sensitivity for SE detection and quantification.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114023"},"PeriodicalIF":1.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145800089","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 cervical cancer screening: A novel in-house ELISA assay for the simultaneous detection of p16 and Ki-67","authors":"Sallam Al-Madhagi ,&nbsp;Patricia Khashayar ,&nbsp;Lieselotte Volckaert ,&nbsp;Jan Vanfleteren ,&nbsp;Mieke Adriaens","doi":"10.1016/j.jim.2025.113998","DOIUrl":"10.1016/j.jim.2025.113998","url":null,"abstract":"<div><div>Immunostaining and cytology, commonly used to detect cervical cancer, are time-consuming and pathologist dependent. In this work, for the first time, we have developed an ELISA assay for the extraction and co-measurement of p16 and Ki-67 in both vaginal and cervical samples for cervical cancer screening. Novel custom-made antibodies were developed, and a competitive ELISA assay was developed and optimized based on the concentration and incubation time of different steps to improve the sensitivity and specificity of the final assay in assessing cervical samples. The as-developed ELISA assay shows high sensitivity and selectivity in different collection media. The LoD of the test is 0.00004 and 0.0058 μg/mL for p16 and Ki-67, respectively. The as-developed assay can be used in future studies to determine protein thresholds for different lesion grades of cervical cancer for samples collected in different settings. These assays can revolutionize the cervical cancer screening management process in the near future.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 113998"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145420287","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":"Cell-based potency assay for anti-CD3-anti-CD19 diabody","authors":"Tania L. Weiss ,&nbsp;Justine Paniagua ,&nbsp;Tonny Johnson ,&nbsp;Timothy P. Cripe ,&nbsp;Peter Ralph","doi":"10.1016/j.jim.2025.114004","DOIUrl":"10.1016/j.jim.2025.114004","url":null,"abstract":"<div><div>A cytotoxicity assay was developed to measure the potency of GP101, a single chain diabody. GP101 is comprised of two linked Fv domains, with one that binds CD3 (expressed on T cells), and the other that binds CD19 (expressed on B cells). GP101 directs CD3 positive T lymphocytes to CD19 positive B lymphocytes. This immunotherapy redirects CD3+ T cells to CD19-expressing B-cell malignancies, enabling T-cell-mediated tumor cell lysis. We developed a GMP cell-based potency assay to satisfy the FDA requirements. The potency assay uses a cytotoxic T cell line, and a B cell lymphoma cell line as the target. The target lymphoma B cell line is prelabeled with a fluorescent dye, and upon T cell mediated killing, the fluorescence dye is released and detected using a fluorometer. The emitted fluorescence is proportional to the dose of GP101. Greater than 90 % of the target B cells were killed within two hours exposure in vitro with the lowest amount of detectable killing at 60 pg/mL GP101. The assay is suitable for measuring purified GP101, or GP101 expressed by cells transduced by GP101 plasmid or AAV preparations, and bioactivity in animal or human blood. This novel assay met GMP/GLP compliance, allowing a quantifiable and reproducible measure of efficacy, ensuring batch-to-batch consistency, and met safety and effectiveness regulatory requirements. This potency assay may be applicable for testing other CD3-CD19 T cell engagers and suitable for developing other diabody mediated potency assays with appropriate antigens.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114004"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145512989","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 sensitive sandwich ELISA for the envelope domain III protein of dengue virus type 2","authors":"Ziting Ma ,&nbsp;Jingyi Su ,&nbsp;Zelin Han ,&nbsp;Ximing Ren ,&nbsp;Jinnian Guo ,&nbsp;Lu Jiang ,&nbsp;Suqing Zhao","doi":"10.1016/j.jim.2025.114000","DOIUrl":"10.1016/j.jim.2025.114000","url":null,"abstract":"<div><div>Dengue, caused by the dengue virus (DENV), is a rapidly spreading infectious disease that poses a significant global health challenge. Rapid and accurate diagnosis is critical for effective disease management and epidemic control, as dengue is a kind of vector-borne disease and lacks definitive medical treatments. Current antibodies and antigens for DENV immunoassay, including immunoglobulin G (IgG), immunoglobulin M (IgM), and non-structural protein 1 (NS1), have drawbacks like cross-reaction and false-negative results in the late stages of primary infection or secondary infection. Envelope domain III (EDIII) protein, which is immunogenic and is a recognition region and binding site for DENV receptors, can also be the potential detection target of DENV infection. However, few studies of EDIII protein as a detection target have been reported. Therefore, this study developed a sensitive and specific ELISA for DENV-2_EDIII protein detection. Specific polyclonal antibodies (pAbs) against the DENV-2_EDIII protein were generated by immunizing the New Zealand White rabbits with the recombinant DENV-2_EDIII protein. Using these pAbs, a highly sensitive and specific ELISA method was developed. The assay demonstrated a wide detection range (7.8 ng/mL to 500 ng/mL) and a low limit of detection (LOD) of 0.156 ng/mL and showed no cross-reactivity with other orthoflaviviruses. These findings may provide a new methodological framework and research direction for the diagnosis of dengue after further clinical verifications.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114000"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145438300","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":"Variation in DNA enrichment among deuterium labeling studies is largely explained by different background corrections","authors":"Erdem Şanal ,&nbsp;José A.M. Borghans ,&nbsp;Rob J. de Boer","doi":"10.1016/j.jim.2025.113987","DOIUrl":"10.1016/j.jim.2025.113987","url":null,"abstract":"<div><div>Heavy water (<span><math><mrow><msub><mrow><mi>D</mi></mrow><mrow><mn>2</mn></mrow></msub><mi>O</mi></mrow></math></span>) labeling can be used to track the dynamics of circulating cells <em>in vivo</em>. <span><math><mrow><msub><mrow><mi>D</mi></mrow><mrow><mn>2</mn></mrow></msub><mi>O</mi></mrow></math></span> is administered via the drinking water, dividing cells become labeled by incorporating deuterium in their newly formed DNA, and the accrual of labeled deoxyribose molecules in the DNA is measured by gas-chromatography mass-spectrometry (GC–MS). Deuterium labeling studies need to correct for a background enrichment that is due to several naturally occurring isotopes, but the procedure for performing this correction differs between studies. We show that a background correction that is based upon subtracting the background enriched <em>fraction</em> (i.e., the atom percent excess, APE) underestimates the true enrichment, whereas one that is based upon subtracting the background enriched <em>ratio</em> (i.e., the tracer-to-tracee ratio, TTR) provides an enrichment that correctly describes the additional effect of the deuterium labeling. This difference is reflected in the so-called ‘amplification factor’, which quantifies the relative enrichment of deoxyribose in a fast reference population of cells to the enrichment in the body water. Using mechanistic binomials we reanalyze deuterium labeling data and show that previous studies based upon an APE-based background correction have underestimated this amplification factor by about 25%, which explains most of the variation among studies in their average amplification factor. We propose a novel model in which the amplification factor is defined by a binomial expression allowing for metabolic differences between volunteers in the contribution of body water to the hydrogens incorporated into deoxyribose during the synthesis of DNA.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 113987"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145438359","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":"Long-term cell storage of RAW 264.7 cells in a deep freezer dampens M2 marker expression","authors":"Tomonori Makiguchi ,&nbsp;Akio Nakane ,&nbsp;Sadatomo Tasaka","doi":"10.1016/j.jim.2025.114001","DOIUrl":"10.1016/j.jim.2025.114001","url":null,"abstract":"<div><div>RAW 264.7, a well-established cell line, is widely used in research; however, it is easily polarized upon subtle changes in environmental conditions or repeated passages. As the effects of cell storage conditions and duration on their phenotypic changes remain unknown, we investigated the effects of long-term storage at −80 °C on M2 marker expression in RAW 264.7 cells after stimulation with IL-4. Cells, stored in a − 80 °C deep freezer for a few years (defined as “long-stored cells”) and newly purchased cells (referred to as “new cells”), were compared through flow cytometry for CD68-positive naïve macrophages (M0) and the expression of three types of M2 markers, CD206-FITC, CD206-PE, and Arginase-1-Alexa Fluor 488, in the M2 state. CD68 positivity was similar between the M0 macrophage types. After stimulation with IL-4, the proportion of CD206-FITC-positive cells was 5.0 ± 0.7 % and 61.5 ± 3.1 % in long-stored cells and new cells, respectively (<em>p</em> &lt; 0.0001), while that of CD206-PE-positive cells was 9.2 ± 0.2 % and 69.8 ± 0.6 %. Similarly, the proportion of Arginase-1- Alexa Fluor 488-positive cells was 3.0 ± 0.1 and 9.0 ± 9.6 %, respectively (<em>p</em> = 0.0006). Overall, these results demonstrate that long-term storage of RAW 264.7 in a deep freezer affects their M2 marker expression. These results highlight the need to consider the effect of cell storage conditions and duration on experimental results.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114001"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471167","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":"Preliminary evaluation of a whole blood dual-platform flow cytometry protocol for CAR-T cell quantitation: Toward accreditation and clinical routine application","authors":"Rémi Pescarmona ,&nbsp;Guillaume Monneret ,&nbsp;Eleonore Micoud ,&nbsp;Christophe Malcus ,&nbsp;Emmanuel Bachy ,&nbsp;Pierre Sesques ,&nbsp;Lionel Karlin ,&nbsp;Fabienne Venet ,&nbsp;Morgane Gossez","doi":"10.1016/j.jim.2025.113986","DOIUrl":"10.1016/j.jim.2025.113986","url":null,"abstract":"<div><div>Chimeric Antigen Receptor T-cells, commonly known as CAR-T cells, have represented a major scientific breakthrough in cancer cell therapy revolutionizing the treatment of haematological disorders. Pharmacokinetic studies highlighted the importance of peripheral blood CAR-T cell monitoring to predict therapeutic outcomes and manage side effects. This study aimed to establish and evaluate on three different flow cytometers (DxFLEX, Navios and Navios EX) the performances of a dual-platform protocol for CAR-T quantitation method using flow cytometry in a context of hospital routine use. Fresh whole blood was stained using a biotinylated CD19 or BCMA recombinant protein and a PE-coupled anti-biotin antibody. CAR-T cells were identified among CD45+ CD3+ cells after doublets elimination, using a control tube to define CAR-T positive expression threshold among T lymphocytes. Results were expressed as percentages of CAR T cells among CD3+ T lymphocytes. Optilyse C and Versalyse red blood cell lysis reagents (Beckman Coulter) gave comparable results in terms of CAR T cell percentage among T lymphocytes as well as using 0.5 μL CAR detection reagent compared to the 2 μL recommended by the manufacturer. Using this protocol, we did not observe any cross-reactivity between CD19 and BCMA CAR detection reagents or decreased signal even up to &gt;3000 circulating CAR-T cells/μL. The coefficient of variations for repeatability and intermediate precision were systematically below 10 % whatever the flow cytometer, the type of CAR reagent detection used and the proportion of circulating CAR-T cells. All three flow cytometers returned correlated and comparable results. Finally, concerning whole blood sample and stained cell storage duration, we observed a significant decreased of CAR-T percentages when staining was delayed for more than one day after sampling, and after three days of stained cells storage at 4 °C. In conclusion, we propose a dual-platform protocol for CAR-T cell enumeration which demonstrated consistent and reliable performances for BCMA and CD19 CAR-T cell quantification.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 113986"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145368141","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":"CpG-ODN from Streptococcus thermophilus as a tool for inducing extrinsic apoptosis via caspase-8 activation in mouse splenocytes","authors":"Md. Rayhan Chowdhury ,&nbsp;Takuma Okajima ,&nbsp;Aito Murakami ,&nbsp;Ariful Islam ,&nbsp;Takeshi Shimosato","doi":"10.1016/j.jim.2025.113999","DOIUrl":"10.1016/j.jim.2025.113999","url":null,"abstract":"<div><div>Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9), thereby triggering a diverse range of immunomodulatory responses. Although the immune-stimulating properties of CpG-ODNs have been extensively studied, their potential application as apoptosis-inducing tools remains underexplored, particularly for probiotic-derived sequences. In this study, we established a method for inducing extrinsic apoptosis in mouse splenocytes using MsST, a CpG-ODN identified from the <em>lacZ</em> gene of <em>Streptococcus thermophilus</em> ATCC19258. In genomic DNA fragmentation assays, MsST induced moderate but significant internucleosomal DNA fragmentation. Flow cytometric analysis using annexin V/7-aminoactinomycin D staining confirmed a time-dependent increase in the apoptotic cell population following MsST treatment. Furthermore, Western blot analysis revealed elevated levels of cleaved caspase-8, thus verifying activation of the extrinsic apoptosis pathway. Compared to a representative CpG-ODN and the apoptosis inducer actinomycin D, MsST exhibited distinct apoptosis-inducing characteristics. These findings highlight MsST as a potential tool for controlled apoptosis induction, providing a methodological basis for further exploration of CpG-ODNs in immunological and therapeutic applications, including cancer immunotherapy and the treatment of autoimmune diseases and inflammatory disorders.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 113999"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426812","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":"Detection of human IgM and IgG antibodies reactive to Dengue virus using magnetic bead immunoassay","authors":"Marcelo S. Conzentino ,&nbsp;Thais A. Silva ,&nbsp;Ana Carolina A. Gonçalves ,&nbsp;Fabiane G.M. Rego ,&nbsp;Vera J.L. Chicora ,&nbsp;Stephanie M.S. Lo ,&nbsp;Fernanda L. Martiny ,&nbsp;Fabio O. Pedrosa ,&nbsp;Luciano F. Huergo","doi":"10.1016/j.jim.2025.114002","DOIUrl":"10.1016/j.jim.2025.114002","url":null,"abstract":"<div><div>Here we describe a magnetic bead-based indirect ELISA that enables the rapid and scalable detection of human IgM and IgG antibodies reactive to dengue virus (DENV). The assay can be performed in 12 min in the chromogenic format to IgM or IgG detection. The system can also handle simultaneous detection of IgM and IgG within less than 8 min when combined with fluorescent-labelled secondary conjugated IgM and IgG detection antibodies. The sensitivity and specificity of the assay are in the same range as described for commercially available ELISA kits. This simple and ultrafast assay for dengue seroconversion detection is an efficient alternative for the rapid track dengue cases.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114002"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145482327","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}