Journal of immunological methods最新文献

{"title":"A comparison of monocyte-derived macrophages with and without prior CD14+ magnetic sorting","authors":"Julia Thomé ,&nbsp;Johannes Zeller ,&nbsp;Thierry Christmann ,&nbsp;Balazs Bogner ,&nbsp;Maike Lind ,&nbsp;Marie Dreck ,&nbsp;Teresa Z. Brose ,&nbsp;Laura Schneider ,&nbsp;Franziska Pankratz ,&nbsp;Sheena Kreuzaler ,&nbsp;Steffen U. Eisenhardt","doi":"10.1016/j.jim.2026.114058","DOIUrl":"10.1016/j.jim.2026.114058","url":null,"abstract":"<div><h3>Introduction</h3><div>For in vitro studies involving macrophages, an efficient protocol for isolation and cell culture of monocyte-derived macrophages (MDM) is essential. However, it remains unclear how far isolation steps improve purity or provoke cell changes. Here we compare the characteristics and functionality of macrophages isolated from leukocyte reduction (LRS) chambers by either CD14+ magnetic sorting (sMac) or by seeding PBMC with separation by plastic adhesion and subsequent washing steps (uMac) to evaluate the necessity of magnetic sorting.</div></div><div><h3>Methods</h3><div>PBMC were isolated from LRS chambers using density gradient centrifugation. sMac were enriched from PBMC by magnetic separation removing lymphocytes, uMac were seeded and separated from lymphocytes via plastic adhesion and washing steps. Both groups were plated under matched conditions to allow monocyte-to-macrophage differentiation after adhesion. Morphology, purity, cell viability, surface marker expression, and cytokine production upon stimulation were assessed using flow cytometry, qPCR, brightfield and confocal microscopy.</div></div><div><h3>Results</h3><div>MDM were successfully cultured from both uMac and sMac in over 75%. Cell viability was similar between uMac (54%) and sMac (48%). CD14 expression was significantly higher in sMac (89.48%) compared to uMac (65.91%), the mean fluorescence intensity of CD14 did not differ significantly. No significant differences were found in CD80 and CD163 expression, nor in the production of pro-inflammatory cytokines upon stimulation.</div></div><div><h3>Conclusions</h3><div>LRS chambers serve as a valid source for culturing of MDM with high reliability and yield. <em>Since both methods produced macrophages of comparable phenotype and function, our findings indicate that magnetic CD14+ sorting is not mandatory for most</em> in vitro <em>applications.</em></div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"548 ","pages":"Article 114058"},"PeriodicalIF":1.6,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147816197","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 method to assign weight-based antibody concentration to antigen-specific Ig in sera","authors":"Marta Benincasa,&nbsp;Salvatore Gemmellaro,&nbsp;Federica Boretto,&nbsp;Miren Iturriza,&nbsp;Rocio Canals,&nbsp;Simona Rondini,&nbsp;Omar Rossi,&nbsp;Francesca Mancini","doi":"10.1016/j.jim.2026.114037","DOIUrl":"10.1016/j.jim.2026.114037","url":null,"abstract":"<div><div>The ability to compare results obtained while assessing immune responses to different antigens using different assays is essential. Enzyme-linked Immunosorbent Assay (ELISA) is one of the most widely used assays to detect and quantify antibodies in human and animal samples. However, it carries the limitation of not giving absolute results but relative measures; specifically, ELISA titers are relative to the sample dilutions tested and ELISA Units/mL, assigned to samples, are dependent on the ELISA Units assigned to a standard serum used in the test, posing the question of how to compare results obtained across different ELISA assays. Some comparability limitations between immunoassays can be solved through calibration against International Standards, but they are not always available and they do not enable comparison across antigens. In an attempt to overcome this problem, we have developed a reliable ELISA-based assay that quantifies antigen-specific antibodies in sera with an absolute concentration of μg/mL, thus allowing comparison of results obtained running the assay with different coating antigens. The methodology is based on comparing the signal of the sera of interest against a curve made with quantified IgG. This method has been successfully applied to quantify total IgG in sera from different species: human, rabbit and mouse.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114037"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146035564","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":"Muscarinic 3 receptor antibodies in Sjögren's disease: Evaluating assay variability and detection methods","authors":"Martha Tsaliki ,&nbsp;Devavrat Dave ,&nbsp;Biji T. Kurien ,&nbsp;R. Hal Scofield","doi":"10.1016/j.jim.2026.114038","DOIUrl":"10.1016/j.jim.2026.114038","url":null,"abstract":"<div><div>The most common manifestations characterizing Sjögren's disease (SjD) are extensive ocular and oral dryness due to decreased lacrimal (keratoconjunctivitis sicca) and salivary gland function (xerostomia). Another hallmark of SjD is the presence of autoantibodies. Acinar cells in the exocrine glands express muscarinic-type-3-receptors (M3R), which act as regulators of saliva secretion. Studies have linked M3R-targeting autoantibodies to reduced saliva secretion in SjD. Earlier studies have proposed that anti-M3R antibodies can pose as potential SjD clinical markers and indicators of dryness. Although functional assays confirm that anti-M3R antibodies have inhibitory activity, other assays have shown a wide range of detection sensitivities for these antibodies (0–100%). Differences in assay type, reagents, or peptide conformation likely explain the wide variation in assay sensitivity and specificity. We searched PubMed/PubMed Central (up to 2024) to assess possible reasons for this discrepancy and identify the best assay with which to screen for these antibodies. This review highlights the importance of conformational assays and recommends standardization to improve diagnostic accuracy.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114038"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146125047","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":"Establishment of reference intervals for serum TNF-α, IFN-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17 and IFN-γ in healthy Chinese adults using flow cytometry","authors":"Hui Xie ,&nbsp;Guiting Zhang ,&nbsp;Yongquan Xia ,&nbsp;Jie Pan ,&nbsp;Han Shen ,&nbsp;Mao Xia","doi":"10.1016/j.jim.2026.114034","DOIUrl":"10.1016/j.jim.2026.114034","url":null,"abstract":"<div><h3>Background</h3><div>Cytokines, small protein molecules from immune and nonimmune cells, are crucial for immune regulation, hematopoiesis, cell growth, and tissue repair. This study employed flow cytometry to measure serum levels of tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interleukin-17 (IL-17) and interferon-γ (IFN-γ), establishing reference intervals to aid clinical diagnostics and treatment.</div></div><div><h3>Methods</h3><div>From January to March 2024, 299 participants were selected based on predetermined exclusion and inclusion criteria. The Kolmogorov-Smirnov test checked the distribution of cytokine levels, and reference intervals were set using the 95th percentile method following EP28-A3c and WS/T 402–2012 guidelines.</div></div><div><h3>Results</h3><div>The cytokines were not normally distributed, with notable sex differences in levels (<em>P</em> &lt; 0.05), necessitating separate reference intervals for each sex. For women, these were: TNF-α ≤2.94 pg/mL, IFN-α ≤7.69 pg/mL, IL-1β ≤2.40 pg/mL, IL-2 ≤ 2.40 pg/mL, IL-4 ≤ 3.93 pg/mL, IL-5 ≤ 2.42 pg/mL, IL-6 ≤ 15.96 pg/mL, IL-8 ≤ 23.42 pg/mL, IL-10 ≤ 7.10 pg/mL, IL-12p70 ≤ 7.36 pg/mL, IL-17 ≤ 4.14 pg/mL, IFN-γ ≤4.29 pg/mL. For men: TNF-α ≤ 2.65 pg/mL, IFN-α ≤ 7.50 pg/mL, IL-1β ≤2.40 pg/mL, IL-2 ≤ 2.52 pg/mL, IL-4 ≤ 4.58 pg/mL, IL-5 ≤ 2.40 pg/mL, IL-6 ≤ 14.61 pg/mL, IL-8 ≤ 15.88 pg/mL, IL-10 ≤ 3.94 pg/mL, IL-12p70 ≤ 7.18 pg/mL, IL-17 ≤ 3.63 pg/mL, IFN-γ ≤4.18 pg/mL.</div></div><div><h3>Conclusions</h3><div>We established reference intervals for serum TNF-α, IFN-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17 and IFN-γ in healthy adults from East China. These findings are crucial for assessing human immune status, diagnosing diseases, and advancing personalized medicine, laying the groundwork for studying inflammatory disease mechanisms.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114034"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145928989","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 a versatile cell ELISA for detection of antibodies against membrane protein BCMA","authors":"Petra Kern ,&nbsp;Ana Kolenc ,&nbsp;Valerija Kovač ,&nbsp;Vladka Čurin Šerbec","doi":"10.1016/j.jim.2025.114024","DOIUrl":"10.1016/j.jim.2025.114024","url":null,"abstract":"<div><div>Monoclonal antibodies against membrane proteins are commonly used for diagnostic and therapeutic applications. They are still mostly produced using hybridoma technology or phage display, generating large numbers of antibody candidates that need to be tested in a short period of time. As most membrane proteins do not retain their three-dimensional structure after their isolation from the cell membrane, cell-based high-throughput assays are needed for the primary screening of hundreds of antibody candidates.</div><div>We designed a simple cell-based ELISA by taking the protocols for indirect cell immunostaining and integrating them into a standard ELISA format. This cell ELISA is conducted in 96-well round bottom plates and consists of two immunostaining and washing steps, and a detection step. We designed the assay on B-cell maturation antigen as a model membrane protein antigen. We used several cell lines and a wide range of antibody concentrations. The assay was validated with imaging flow cytometry.</div><div>Although we obtained comparable results in specificity with cell ELISA and flow cytometry, screening multiple samples with cell ELISA is substantially faster than with flow cytometry, due to its ability to test several samples simultaneously. Using cell ELISA, a single operator can easily test several hundred antibodies per day. Key advantages of the protocol presented here are that it can be used for suspension or adherent cell lines and for cell lines that express the membrane protein in native or recombinant form. As such, it can potentially be translated to diverse antibody–antigen–cell line systems. This was confirmed by successfully detecting anti-CD3 and anti-CD19 antibodies binding to Jurkat and Raji cells, respectively.</div><div>Cell ELISA developed in our laboratory is a high-throughput, inexpensive, and easy-to-perform method for screening hybridoma that produce antibodies targeting membrane proteins. It does not require the use of soluble pure protein and it can be performed in any laboratory that specializes in antibody development and ELISA testing.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114024"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145833712","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":"PLGA-based nanovaccine delivering chimeric IpaD-StxB protein induces robust immune response against Shigella infection","authors":"Ayoub Fazeli ,&nbsp;Hosein Honari ,&nbsp;Davoud Sadeghi ,&nbsp;Hossein Samiei-Abianeh ,&nbsp;Rasoul Rafiei-Foroushani","doi":"10.1016/j.jim.2026.114036","DOIUrl":"10.1016/j.jim.2026.114036","url":null,"abstract":"<div><h3>Background and aim</h3><div>Shigellosis is a major cause of morbidity and mortality worldwide, especially among children under five in low- and middle-income countries. Despite its public health burden, there is no licensed vaccine. Recombinant and nanoparticle-based vaccine approaches offer promising alternatives to conventional methods. This study aimed to develop a chimeric nanovaccine based on the fusion of <em>Shigella flexneri</em> IpaD and StxB, encapsulated in PLGA nanoparticles, and to evaluate its immunogenic potential in a murine model.</div></div><div><h3>Methods</h3><div>The recombinant IpaD-StxB (IdSb) protein was expressed in <em>E. coli</em>, purified under denaturing conditions, and encapsulated into PLGA nanoparticles using a double emulsion solvent evaporation technique. Particle size, morphology, encapsulation efficiency, and in vitro release were characterized. BALB/c mice were immunized with either Nano-IdSb or free IdSb, and serum IgG levels were assessed via ELISA to evaluate the humoral immune response.</div></div><div><h3>Results</h3><div>The 24 kDa recombinant IdSb protein was successfully expressed in a prokaryotic system, yielding 10 mg/mL. PLGA nanoparticles encapsulating IdSb had an average diameter of 166 nm, high encapsulation efficiency (97%), and a loading capacity of 3.5%. They enabled sustained protein release over 40 days. Mice immunized with Nano-IdSb exhibited significantly stronger IgG responses than those receiving the free protein, indicating enhanced immunogenicity and antigen stability.</div></div><div><h3>Conclusion</h3><div>PLGA-encapsulated IdSb nanovaccine elicited a robust humoral immune response and demonstrated favorable physicochemical properties, supporting its potential as a cost-effective and broadly protective vaccine candidate against <em>Shigella</em> spp.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114036"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145952200","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":"Two technologies, one goal: Developing and evaluating anti-Zika virus NS1 antibodies using phage display and hybridoma technologies for diagnostic applications","authors":"Romain Pantigny ,&nbsp;Maxime Combe ,&nbsp;Céline Roesch ,&nbsp;Gilles Gadea ,&nbsp;Audrey Miltcheff ,&nbsp;Soizic Daniel ,&nbsp;Pierre Martineau","doi":"10.1016/j.jim.2026.114041","DOIUrl":"10.1016/j.jim.2026.114041","url":null,"abstract":"<div><div>Currently, the phage display technology is probably one of the most mature animal-free alternatives to the hybridoma technology for developing antibodies. However, the performances of these two technologies have been rarely compared. To address this question, hybridoma and phage display approaches were used to generate antibodies against Zika virus (ZIKV) non-structural protein 1 (NS1), a biomarker of the acute phase of flavivirus infection that displays high sequence homology among flaviviruses. Phage display-derived antibodies showed enhanced specificity towards ZIKV recombinant NS1 (rNS1). Conversely, hybridoma-derived antibodies cross-reacted with other flavivirus rNS1. This likely reflects differences in selection strategies rather than in the intrinsic properties of the technologies. Despite the moderate binding affinity of paired antibodies obtained by phage display, a limit of detection of 25 ng/mL was achieved in human serum spiked with ZIKV rNS1. This sensitivity dropped to 1 ng/mL with antibodies obtained by mice immunization. Then, the high specificity of phage display-derived antibodies and sensitivity of hybridoma-derived antibodies were combined in a hybrid antibody format immunoassay. In this assay, a very specific phage display-derived antibody was used for capture and a hybridoma-derived antibody with a low dissociation rate constant (k_off) was used for detection, maintaining sensitivity as low as 1 ng/mL. This hybrid assay format also preserved high specificity when tested using serum samples from patients with acute Dengue fever that contain high levels of Dengue virus NS1. Our findings highlight the complementary strengths of both antibody generation strategies for the sensitive and specific detection of ZIKV NS1.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114041"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146258482","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":"Immune cell separation for cell therapy: A comprehensive review of techniques, and challenges","authors":"Masoud Hassanzadeh Makoui ,&nbsp;Amir Bayat Bodaghi ,&nbsp;Mohammad Amin Razavi ,&nbsp;Arman Taran ,&nbsp;Amir Pooya Akbari ,&nbsp;Mohammad Bahrami ,&nbsp;Abdolreza Esmaeilzadeh","doi":"10.1016/j.jim.2026.114040","DOIUrl":"10.1016/j.jim.2026.114040","url":null,"abstract":"<div><div>Cell-based immunotherapies, such as CAR-T and TIL therapies, represent a significant shift in the treatment of cancers and immune disorders. A critical first step in manufacturing these therapeutics is the efficient and selective isolation of specific immune cell populations (e.g., T cells, NK cells, and monocytes) from complex biological sources like peripheral blood or leukapheresis products. This comprehensive review systematically examines the current landscape of immune cell separation technologies that are essential for cell therapy manufacturing. We detail established methods, including density gradient centrifugation, magnetic-activated cell sorting (MACS), and fluorescence-activated cell sorting (FACS), as well as emerging techniques like microfluidics, acoustophoresis, and affinity ligand-based platforms. The review evaluates each technique based on key parameters: purity, yield, viability, processing time, scalability, cost-effectiveness, and compatibility with Good Manufacturing Practice (GMP) requirements. Furthermore, we emphasize the crucial role of rigorous post-separation characterization, which includes phenotypic analysis (flow cytometry), functional assessment (proliferation, cytokine release, cytotoxicity), and sterility testing to ensure product quality and safety. We thoroughly analyze significant challenges, such as the need for gentle processing to preserve cell function, achieving sufficient scalability for clinical and commercial production, minimizing process-induced cellular stress, reducing reagent costs (especially for antibody-based methods), navigating complex regulatory pathways, and establishing robust release criteria. This synthesis underscores that while considerable progress has been made, advancements in separation technologies, integrated characterization strategies, and standardized approaches are vital for overcoming existing bottlenecks and fully realizing the transformative potential of cell-based immunotherapies.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114040"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147276386","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":"Defining positivity in the lymphocyte transformation test with cytokine read-out: What is the best of bad choices?","authors":"Bernhardt Sachs ,&nbsp;Leonie Weinhold ,&nbsp;Caspar A. Heubach ,&nbsp;Diana Dubrall ,&nbsp;Philipp Deck ,&nbsp;Günther Weindl ,&nbsp;Markus Nöthen ,&nbsp;Per Hoffmann ,&nbsp;Stefanie Röseler ,&nbsp;Gerda Wurpts ,&nbsp;Amir S. Yazdi ,&nbsp;Andreas Glässner","doi":"10.1016/j.jim.2026.114035","DOIUrl":"10.1016/j.jim.2026.114035","url":null,"abstract":"<div><h3>Background</h3><div>Various approaches have been published to define a positive result in the lymphocyte transformation test (LTT) with interferon-γ (IFN-γ) read-out using ELISA. While statistical analyses cannot improve data quality, they may impact on defining a positive result. The objective of this study was thus to explore different statistical approaches for the optimal discrimination of drug-allergic patients and controls based on the IFN-γ concentrations of their drug-stimulated and unstimulated peripheral blood mononuclear cells in the LTT.</div></div><div><h3>Methods</h3><div>The IFN-γ concentrations of 59 LTTs with drug-allergic patients (39 immediate-type, 20 delayed-type) and corresponding controls obtained in a case-control study design were applied in seven different statistical approaches. The performance parameter was the area under the curve (AUC) of the receiver operating characteristics (ROC) analysis, integrating sensitivity and specificity. Subgroup analyses were performed for delayed and immediate-type reactions and different drug groups.</div></div><div><h3>Results</h3><div>Although there were numerical differences between the AUCs of the different approaches, these differences were marginal, and no approach demonstrated clear superiority. Numerically, the statistical approach with an added constant yielded the highest AUC (0.679; 95% CI 0.581–0.776; sensitivity: 69.0%; specificity: 63.8%). The performance indicators for this approach were higher in the subgroup of delayed-type reactions (AUC 0.791; 95% CI 0.643–0.939; sensitivity: 78.9%; specificity: 75.0%). If the individual best approach was selected, numerical differences of the AUCs between the drug classes were observed.</div></div><div><h3>Conclusion</h3><div>We encourage researchers working with the LTT with IFN-γ or another cytokine read-out to test different statistical approaches on their raw data to explore how the different approaches perform.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114035"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145948939","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, validation and stability study of an ELISA method to quantify the major allergen Jun a 1 in Juniperus ashei extracts","authors":"M. Angeles López-Matas,&nbsp;Javier Álvarez,&nbsp;Tamara Aranda,&nbsp;Jerónimo Carnés","doi":"10.1016/j.jim.2026.114039","DOIUrl":"10.1016/j.jim.2026.114039","url":null,"abstract":"<div><div>A functional allergen quantification kit requires two key components: a pair of monoclonal antibodies (mAbs) that do not interfere with each other at the protein binding site, and a reference standard with sufficient purity and stability. Despite the clinical relevance of Jun a 1 in patients with Cupressaceae pollinosis, no commercial kits are currently available for its quantification in allergenic extracts. The aim of this study was to develop and validate a sandwich ELISA kit for the quantification of Jun a 1 in <em>Juniperus ashei</em> extracts, using appropriately selected mAbs and a stable standard. To this end, Jun a 1 was purified to &gt;95% purity, as confirmed by SDS-PAGE, Western blot, and mass spectrometry analysis. Its stability was demonstrated after 48 months of storage. The linear B-cell epitopes recognized by the mAbs were identified and mapped onto the Jun a 1 structure, confirming that they are non-overlapping. Following validation, the ELISA method proved to be specific, linear, accurate, and precise. In conclusion, we successfully developed and validated an ELISA assay for the quantification of Jun a 1 in <em>J. ashei</em> extracts, supported by the generation of suitable monoclonal antibodies and a stable reference standard.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"547 ","pages":"Article 114039"},"PeriodicalIF":1.6,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146157408","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}