Pub Date : 2026-02-03DOI: 10.1016/j.jim.2026.114038
Martha Tsaliki, Devavrat Dave, Biji T Kurien, R Hal Scofield
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.
{"title":"Muscarinic 3 receptor antibodies in Sjögren's disease: Evaluating assay variability and detection methods.","authors":"Martha Tsaliki, Devavrat Dave, Biji T Kurien, R Hal Scofield","doi":"10.1016/j.jim.2026.114038","DOIUrl":"https://doi.org/10.1016/j.jim.2026.114038","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"114038"},"PeriodicalIF":1.6,"publicationDate":"2026-02-03","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}
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.
{"title":"ELISA method to assign weight-based antibody concentration to antigen-specific Ig in sera","authors":"Marta Benincasa, Salvatore Gemmellaro, Federica Boretto, Miren Iturriza, Rocio Canals, Simona Rondini, Omar Rossi, 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":"2026-01-21","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}
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 Shigella flexneri IpaD and StxB, encapsulated in PLGA nanoparticles, and to evaluate its immunogenic potential in a murine model.
Methods
The recombinant IpaD-StxB (IdSb) protein was expressed in E. coli, 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.
Results
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.
Conclusion
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 Shigella spp.
{"title":"PLGA-based nanovaccine delivering chimeric IpaD-StxB protein induces robust immune response against Shigella infection","authors":"Ayoub Fazeli , Hosein Honari , Davoud Sadeghi , Hossein Samiei-Abianeh , 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":"2026-01-09","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}
Pub Date : 2026-01-08DOI: 10.1016/j.jim.2026.114035
Bernhardt Sachs , Leonie Weinhold , Caspar A. Heubach , Diana Dubrall , Philipp Deck , Günther Weindl , Markus Nöthen , Per Hoffmann , Stefanie Röseler , Gerda Wurpts , Amir S. Yazdi , Andreas Glässner
Background
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.
Methods
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.
Results
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.
Conclusion
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.
背景：已经发表了各种方法来定义使用ELISA读取干扰素- (IFN-γ)的淋巴细胞转化试验（LTT）的阳性结果。虽然统计分析不能提高数据质量，但它们可能会影响确定一个积极的结果。因此，本研究的目的是探索不同的统计方法，根据药物刺激和未刺激的外周血单个核细胞在LTT中的IFN-γ浓度，对药物过敏患者和对照组进行最佳区分。方法：采用7种不同的统计方法，对59例药物过敏ltt患者（39例即刻型，20例延迟型）及相应对照进行病例对照研究。性能参数为受试者工作特征（ROC）分析的曲线下面积（AUC），综合敏感性和特异性。对延迟型、立即型反应及不同药物组进行亚组分析。结果：虽然不同入路的auc在数值上存在差异，但这些差异是微小的，没有一种入路表现出明显的优势。在数值上，增加常数的统计方法获得最高的AUC （0.679; 95% CI 0.581-0.776；敏感性：69.0%；特异性：63.8%）。该方法的性能指标在延迟型反应亚组中较高（AUC 0.791; 95% CI 0.643-0.939；敏感性：78.9%；特异性：75.0%）。如果选择个体最佳方法，则观察药物类别之间auc的数值差异。结论：我们鼓励研究人员使用IFN-γ或其他细胞因子读出的LTT，在原始数据上测试不同的统计方法，以探索不同方法的表现。
{"title":"Defining positivity in the lymphocyte transformation test with cytokine read-out: What is the best of bad choices?","authors":"Bernhardt Sachs , Leonie Weinhold , Caspar A. Heubach , Diana Dubrall , Philipp Deck , Günther Weindl , Markus Nöthen , Per Hoffmann , Stefanie Röseler , Gerda Wurpts , Amir S. Yazdi , 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":"2026-01-08","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}
Pub Date : 2026-01-07DOI: 10.1016/j.jim.2026.114034
Hui Xie , Guiting Zhang , Yongquan Xia , Jie Pan , Han Shen , Mao Xia
Background
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.
Methods
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.
Results
The cytokines were not normally distributed, with notable sex differences in levels (P < 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.
Conclusions
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.
{"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 , Guiting Zhang , Yongquan Xia , Jie Pan , Han Shen , 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> < 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":"2026-01-07","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}
Pub Date : 2025-12-23DOI: 10.1016/j.jim.2025.114024
Petra Kern , Ana Kolenc , Valerija Kovač , Vladka Čurin Šerbec
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.
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.
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.
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.
{"title":"Optimization of a versatile cell ELISA for detection of antibodies against membrane protein BCMA","authors":"Petra Kern , Ana Kolenc , Valerija Kovač , 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":"2025-12-23","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}
Pub Date : 2025-12-20DOI: 10.1016/j.jim.2025.114022
Karen Terry , Kyle W. Kroll , Rhianna A. Jones , R. Keith Reeves
Natural killer and cytotoxic CD8+ T cells are essential effectors of innate systemic antiviral and antitumor immune responses. Their effector functions and target cell recognition are partly regulated by a polymorphic family of activating and inhibitory killer-cell immunoglobulin-like receptors (KIRs) that interact with major histocompatibility complex (MHC) class I molecules. In Rhesus macaques (Macaca mulatta) (RM), a well-established model for studying simian immunodeficiency virus (SIV) infection and cellular immunobiology, the KIR gene family is highly diverse but remains poorly characterized across tissue compartments. In this study, we used a branched DNA, amplification-free assay with high specificity and reproducibility to simultaneously measure mRNA expression of six KIR genes (Mamu-KIR1D, Mamu-KIR3DH5, Mamu-KIR3DL2, Mamu-KIR3DL4, Mamu-KIR3DH, and Mamu-KIR3DL0) in mononuclear cells from peripheral blood, lymph nodes, and spleen of both naïve and chronically lentivirus-infected RM. Our findings reveal tissue-specific expression patterns of KIR genes that are differentially affected by chronic lentivirus infection, emphasizing the importance of compartmentalized KIR regulation in viral pathogenesis. This proof-of-concept study presents a reliable and scalable framework for the detailed characterization of the KIR gene repertoire in non-human primate models, providing a valuable alternative to traditional qPCR for profiling gene expression in complex tissues.
{"title":"Application of a novel branched-DNA assay to quantify killer immunoglobulin-like receptor (KIR) mRNA expression identifies tissue compartmentalization in naïve and SIV-infected rhesus macaques","authors":"Karen Terry , Kyle W. Kroll , Rhianna A. Jones , R. Keith Reeves","doi":"10.1016/j.jim.2025.114022","DOIUrl":"10.1016/j.jim.2025.114022","url":null,"abstract":"<div><div>Natural killer and cytotoxic CD8+ T cells are essential effectors of innate systemic antiviral and antitumor immune responses. Their effector functions and target cell recognition are partly regulated by a polymorphic family of activating and inhibitory killer-cell immunoglobulin-like receptors (KIRs) that interact with major histocompatibility complex (MHC) class I molecules. In Rhesus macaques (<em>Macaca mulatta</em>) (RM), a well-established model for studying simian immunodeficiency virus (SIV) infection and cellular immunobiology, the KIR gene family is highly diverse but remains poorly characterized across tissue compartments. In this study, we used a branched DNA, amplification-free assay with high specificity and reproducibility to simultaneously measure mRNA expression of six KIR genes (Mamu-KIR1D, Mamu-KIR3DH5, Mamu-KIR3DL2, Mamu-KIR3DL4, Mamu-KIR3DH, and Mamu-KIR3DL0) in mononuclear cells from peripheral blood, lymph nodes, and spleen of both naïve and chronically lentivirus-infected RM. Our findings reveal tissue-specific expression patterns of KIR genes that are differentially affected by chronic lentivirus infection, emphasizing the importance of compartmentalized KIR regulation in viral pathogenesis. This proof-of-concept study presents a reliable and scalable framework for the detailed characterization of the KIR gene repertoire in non-human primate models, providing a valuable alternative to traditional qPCR for profiling gene expression in complex tissues.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114022"},"PeriodicalIF":1.6,"publicationDate":"2025-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145809891","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}
Pub Date : 2025-12-18DOI: 10.1016/j.jim.2025.114023
Paulin Dettmann, Hans Werner Mages, Martin Skiba, Daniel Stern, Martin B. Dorner, Brigitte G. Dorner
Staphylococcal enterotoxins (SEs) belong to a family of highly potent superantigens produced by Staphylococcus aureus 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 S. aureus' 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 S. aureus 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.
{"title":"Subclass optimised antibodies as an effective tool to suppress protein A induced false positives in immunoassays detecting staphylococcal enterotoxin B","authors":"Paulin Dettmann, Hans Werner Mages, Martin Skiba, Daniel Stern, Martin B. Dorner, 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":"2025-12-18","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}
Bovine viral diarrhea virus (BVDV), a globally prevalent immunosuppressive pathogen, continues to challenge the cattle industry, prompting the search for more effective diagnostic and immunogenic targets. The envelope glycoprotein E2, once the central focus of vaccine and diagnostic research, is now considered suboptimal because of its pronounced sequence variability, which limits cross-subtype efficacy. In contrast, the E0 protein, a more conserved outer membrane glycoprotein, has emerged as a promising alternative owing to its immunogenicity and cross-subtype stability. In this study, the E0 gene of BVDV 1b (XZ02) was engineered by removing its membrane anchor region and incorporating heterologous signal peptides (SPs) to enhance secretion efficiency in an HEK293 suspension cell system. Among the evaluated SPs, the tissue plasminogen activator signal peptide yielded the highest level of secreted E0 protein (up to 2.4 mg/mL after purification). Based on the optimized recombinant E0 antigen, an indirect ELISA was developed to detect BVDV-specific antibodies. The assay demonstrated high sensitivity, specificity, and reproducibility, with a diagnostic agreement rate of 96.53 % (κ = 0.9110), when compared with the commercial IDEXX BVDV Total Antibody kit. These results support the potential of the engineered E0 protein as a reliable, scalable antigen for the serological diagnosis of BVDV.
{"title":"Development of an optimized E0-based indirect ELISA for serological detection of bovine viral diarrhea virus","authors":"Chenjun Jiang , Wei Sheng , Zhuoma Gesang , Yanan Zhong , Da Qiong , Jiayan Huang , Hongbo Zhou , Sizhu Suolang","doi":"10.1016/j.jim.2025.114021","DOIUrl":"10.1016/j.jim.2025.114021","url":null,"abstract":"<div><div>Bovine viral diarrhea virus (BVDV), a globally prevalent immunosuppressive pathogen, continues to challenge the cattle industry, prompting the search for more effective diagnostic and immunogenic targets. The envelope glycoprotein E2, once the central focus of vaccine and diagnostic research, is now considered suboptimal because of its pronounced sequence variability, which limits cross-subtype efficacy. In contrast, the E0 protein, a more conserved outer membrane glycoprotein, has emerged as a promising alternative owing to its immunogenicity and cross-subtype stability. In this study, the E0 gene of BVDV 1b (XZ02) was engineered by removing its membrane anchor region and incorporating heterologous signal peptides (SPs) to enhance secretion efficiency in an HEK293 suspension cell system. Among the evaluated SPs, the tissue plasminogen activator signal peptide yielded the highest level of secreted E0 protein (up to 2.4 mg/mL after purification). Based on the optimized recombinant E0 antigen, an indirect ELISA was developed to detect BVDV-specific antibodies. The assay demonstrated high sensitivity, specificity, and reproducibility, with a diagnostic agreement rate of 96.53 % (κ = 0.9110), when compared with the commercial IDEXX BVDV Total Antibody kit. These results support the potential of the engineered E0 protein as a reliable, scalable antigen for the serological diagnosis of BVDV.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114021"},"PeriodicalIF":1.6,"publicationDate":"2025-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145756918","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}
Pub Date : 2025-12-09DOI: 10.1016/j.jim.2025.114020
Alexander Launa, Qiuyue Peng
The THP-1 monocyte cell line can be differentiated into macrophage-like cells upon stimulation with phorbol 12-myristate 13-acetate (PMA) and therefore widely used in inflammatory research. However, there is a lack of evidence-based protocols for cell culture and differentiation. In this study, we highlight how culture conditions influence THP-1 cells and their response to PMA stimulation. After thawing, THP-1 cells were cultured for up to 4 weeks and monitored by phase-contrast microscopy. Stressed cells (>1 × 106 cells/mL for 72 h without medium replenishment) were assessed for CD80 and MHC-II expression and the subset distribution by flow cytometry. For PMA stimulation (10 μg/mL), standard cells under normal nutrient conditions (5 × 105 cells/mL), low (2 × 105 cells/mL), and high (1 × 106 cells/mL) cell densities, along with stressed cells at 1 × 106 cells/mL density, were seeded and observed at 24 and 48 h. Cells formed aggregates containing more than 10 cells during the first week in suspension but gradually transitioned to single cells with continuous culture. Metabolically stressed cells developed protrusions, showed increased adherence, and expressed higher levels of MHC-II compared to standard cells (p = 0.02). These cells also displayed signs of partial differentiation, with fewer MHC-II−CD80− cells (p < 0.001) and more MHC-II+CD80− cells (p < 0.001) than non-stressed cells. Optimal and uniform differentiation was observed after 3–4 weeks of recovery, at seeding densities below 1 × 106 cells/mL, and under non-stressed conditions. These findings show that THP-1 culture conditions, including recovery time post-thawing, stress status and seeding density, have a major impact on their responsiveness to PMA.
{"title":"Effects of culture conditions on cell morphology and differentiation responses to PMA in THP-1 cells","authors":"Alexander Launa, Qiuyue Peng","doi":"10.1016/j.jim.2025.114020","DOIUrl":"10.1016/j.jim.2025.114020","url":null,"abstract":"<div><div>The THP-1 monocyte cell line can be differentiated into macrophage-like cells upon stimulation with phorbol 12-myristate 13-acetate (PMA) and therefore widely used in inflammatory research. However, there is a lack of evidence-based protocols for cell culture and differentiation. In this study, we highlight how culture conditions influence THP-1 cells and their response to PMA stimulation. After thawing, THP-1 cells were cultured for up to 4 weeks and monitored by phase-contrast microscopy. Stressed cells (>1 × 10<sup>6</sup> cells/mL for 72 h without medium replenishment) were assessed for CD80 and MHC-II expression and the subset distribution by flow cytometry. For PMA stimulation (10 μg/mL), standard cells under normal nutrient conditions (5 × 10<sup>5</sup> cells/mL), low (2 × 10<sup>5</sup> cells/mL), and high (1 × 10<sup>6</sup> cells/mL) cell densities, along with stressed cells at 1 × 10<sup>6</sup> cells/mL density, were seeded and observed at 24 and 48 h. Cells formed aggregates containing more than 10 cells during the first week in suspension but gradually transitioned to single cells with continuous culture. Metabolically stressed cells developed protrusions, showed increased adherence, and expressed higher levels of MHC-II compared to standard cells (<em>p</em> = 0.02). These cells also displayed signs of partial differentiation, with fewer MHC-II<sup>−</sup>CD80<sup>−</sup> cells (<em>p</em> < 0.001) and more MHC-II<sup>+</sup>CD80<sup>−</sup> cells (<em>p</em> < 0.001) than non-stressed cells. Optimal and uniform differentiation was observed after 3–4 weeks of recovery, at seeding densities below 1 × 10<sup>6</sup> cells/mL, and under non-stressed conditions. These findings show that THP-1 culture conditions, including recovery time post-thawing, stress status and seeding density, have a major impact on their responsiveness to PMA.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114020"},"PeriodicalIF":1.6,"publicationDate":"2025-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145734852","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}
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