Pub Date : 2026-01-01Epub Date: 2025-11-22DOI: 10.1016/j.jim.2025.114006
Lan Mai , Caleb Cornaby , Lee Ann Baxter-Lowe , Neena Kapoor , Maurice R. O'Gorman
This study explores the value of the Dihydrorhodamine-123 (DHR-123) flow cytometry assay to rapidly measure donor neutrophil engraftment (chimerism) in patients with Chronic Granulomatous Disease (CGD) following hematopoietic stem cell transplant (HSCT). Flow cytometry-based chimerism testing was compared with traditional molecular methods including sequence tandem repeats (STR), Quantitative-PCR DNA fingerprinting, and next-generation sequencing (NGS). Results comparing the two technologies in a cohort of CGD patients and healthy control subjects demonstrated that the DHR-123 based flow cytometry chimerism assay is an accurate, inexpensive, and rapid method for assessing functional donor neutrophil engraftment post-transplant, and which demonstrated significant correlation with the results of the molecular chimerism assays (R2 = 0.9965), making it a valuable tool for ongoing chimerism monitoring post-transplant. The flow cytometry-based assay offers a more rapid and inexpensive alternative to traditional molecular chimerism techniques.
{"title":"Evaluation of a rapid flow cytometry assay to assess functional engraftment in CGD patients post-transplant and comparison with molecular chimerism","authors":"Lan Mai , Caleb Cornaby , Lee Ann Baxter-Lowe , Neena Kapoor , Maurice R. O'Gorman","doi":"10.1016/j.jim.2025.114006","DOIUrl":"10.1016/j.jim.2025.114006","url":null,"abstract":"<div><div>This study explores the value of the Dihydrorhodamine-123 (DHR-123) flow cytometry assay to rapidly measure donor neutrophil engraftment (chimerism) in patients with Chronic Granulomatous Disease (CGD) following hematopoietic stem cell transplant (HSCT). Flow cytometry-based chimerism testing was compared with traditional molecular methods including sequence tandem repeats (STR), Quantitative-PCR DNA fingerprinting, and next-generation sequencing (NGS). Results comparing the two technologies in a cohort of CGD patients and healthy control subjects demonstrated that the DHR-123 based flow cytometry chimerism assay is an accurate, inexpensive, and rapid method for assessing functional donor neutrophil engraftment post-transplant, and which demonstrated significant correlation with the results of the molecular chimerism assays (R<sup>2</sup> = 0.9965), making it a valuable tool for ongoing chimerism monitoring post-transplant. The flow cytometry-based assay offers a more rapid and inexpensive alternative to traditional molecular chimerism techniques.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114006"},"PeriodicalIF":1.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145596645","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 : 2026-01-01Epub Date: 2025-12-01DOI: 10.1016/j.jim.2025.114008
Miao Shen , Jielin Yao , Shengkui Xu , Bowen Zhang , Xiang Li , Wenke Ruan
Avian coronavirus, also known as infectious bronchitis virus (IBV), poses a significant threat to the poultry industry, with the IBV QX variant strain now endemic in multiple countries. Traditional vaccines have demonstrated limited effectiveness in providing robust immune protection against this pathogen. In this study, we developed two IBV QX strain S1-mRNA vaccines based on different antigen domains and evaluated their safety and immune efficacy. The S1A mRNA-LNP and S1B mRNA-LNP vaccines were successfully produced based on epitope prediction of the S1 protein of the IBV QX strain.
Both developed mRNA vaccines, along with a conventional inactivated vaccine, were capable of stimulating serum antibody production. ELISPOT assay results showed that the mRNA vaccines were more potent in stimulating lymphocytes to secrete interferon-γ (IFN-γ) than the inactivated vaccine. This enhanced immunostimulatory capacity translated into a significant reduction in viral load within the kidneys. Moreover, the S1A mRNA vaccine conferred immune protection earlier than the inactivated vaccine.
In conclusion, the IBV S1-mRNA vaccines developed in this study can protect against IBV infection by inducing cellular and humoral immune responses. Selecting different S1 regions also affects the immunogenic efficacy of the mRNA vaccine.
{"title":"Novel mRNA vaccines targeting epitope-rich regions of avian coronavirus enhance immunogenic efficacy","authors":"Miao Shen , Jielin Yao , Shengkui Xu , Bowen Zhang , Xiang Li , Wenke Ruan","doi":"10.1016/j.jim.2025.114008","DOIUrl":"10.1016/j.jim.2025.114008","url":null,"abstract":"<div><div>Avian coronavirus, also known as infectious bronchitis virus (IBV), poses a significant threat to the poultry industry, with the IBV QX variant strain now endemic in multiple countries. Traditional vaccines have demonstrated limited effectiveness in providing robust immune protection against this pathogen. In this study, we developed two IBV QX strain S1-mRNA vaccines based on different antigen domains and evaluated their safety and immune efficacy. The S1A mRNA-LNP and S1B mRNA-LNP vaccines were successfully produced based on epitope prediction of the S1 protein of the IBV QX strain.</div><div>Both developed mRNA vaccines, along with a conventional inactivated vaccine, were capable of stimulating serum antibody production. ELISPOT assay results showed that the mRNA vaccines were more potent in stimulating lymphocytes to secrete interferon-γ (IFN-γ) than the inactivated vaccine. This enhanced immunostimulatory capacity translated into a significant reduction in viral load within the kidneys. Moreover, the S1A mRNA vaccine conferred immune protection earlier than the inactivated vaccine.</div><div>In conclusion, the IBV S1-mRNA vaccines developed in this study can protect against IBV infection by inducing cellular and humoral immune responses. Selecting different S1 regions also affects the immunogenic efficacy of the mRNA vaccine.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114008"},"PeriodicalIF":1.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145667898","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 : 2026-01-01Epub Date: 2025-12-02DOI: 10.1016/j.jim.2025.114011
Jonathan D. Coker, Mindy C. Kohlhagen, Maria A.V. Willrich, David L. Murray
Modern mass spectrometry-based methods for the isotyping and quantitation of monoclonal proteins (M-proteins) are now supplanting traditional gel-based methods such as serum protein electrophoresis (SPEP). The resultant improvement in resolution poses new signal processing challenges in the separation of M-protein from normal variations in polyclonal immunoglobulin background. We model normal variations in this background by means of a singular value decomposition and apply a coupled alternating-projection algorithm to separate normal and abnormal spectral components, both of which are variable. Ion simulation results guide our algorithm details and highlight limitations that can exist when testing M-proteins of high concentration. We show experimental quantitation results on clinical submitted sera with acceptable results. Finally, we outline enhancements to the fundamental method which have resulted in an enhanced analytical measuring range for clinical care of patients with plasma cell disorders.
{"title":"Identifying immunoglobulin abnormalities in the presence of variable polyclonal background","authors":"Jonathan D. Coker, Mindy C. Kohlhagen, Maria A.V. Willrich, David L. Murray","doi":"10.1016/j.jim.2025.114011","DOIUrl":"10.1016/j.jim.2025.114011","url":null,"abstract":"<div><div>Modern mass spectrometry-based methods for the isotyping and quantitation of monoclonal proteins (M-proteins) are now supplanting traditional gel-based methods such as serum protein electrophoresis (SPEP). The resultant improvement in resolution poses new signal processing challenges in the separation of M-protein from normal variations in polyclonal immunoglobulin background. We model normal variations in this background by means of a singular value decomposition and apply a coupled alternating-projection algorithm to separate normal and abnormal spectral components, both of which are variable. Ion simulation results guide our algorithm details and highlight limitations that can exist when testing M-proteins of high concentration. We show experimental quantitation results on clinical submitted sera with acceptable results. Finally, we outline enhancements to the fundamental method which have resulted in an enhanced analytical measuring range for clinical care of patients with plasma cell disorders.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114011"},"PeriodicalIF":1.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145677910","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 : 2026-01-01Epub 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":"2026-01-01","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}
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":"2026-01-01","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 : 2026-01-01Epub Date: 2025-12-02DOI: 10.1016/j.jim.2025.114010
Erdem Şanal , Julia Drylewicz , Kiki Tesselaar , Becca Asquith , José A.M. Borghans , Rob J. de Boer
Heavy water () labeling is the state-of-the-art technique to track the dynamics of circulating cells in vivo. labels dividing cells through incorporation of deuterium into newly synthesized DNA, which is measured using GC/MS. The labeling rate depends on (1) the level of body water enrichment, (2) cell kinetics, and (3) an amplification factor quantifying the deoxyribose enrichment relative to the body water enrichment. This amplification factor is typically estimated using a reference population undergoing rapid turnover (such as granulocytes), and is larger than one because deoxyribose contains seven hydrogens that can be replaced by deuterium. In a meta-analysis, we found that individuals differ markedly in this amplification factor. Since the amplification factor also depends on the level of body water enrichment, we use conventional binomial expressions to describe the fractions of deoxyribose incorporating zero, one, two, or more deuterium atoms. We extend this classic binomial model with a new parameter, , describing the relative contribution of hydrogens from body water during deoxyribose synthesis. While for most studies, our ‘novel Binomial’ model reasonably explains the slope with which the amplification factor declines with the level of body water enrichment, we find that some individual amplification factors differ considerably from their expected values Re-fitting deuterium labeling data of granulocytes with the Binomial model reveals that the actual decrease is steeper than expected. We speculate that this residual variation depends on differences in diet, metabolism, and/or life style, which apparently correlate with daily fluid intake.
{"title":"Dependence of the amplification factor on the body water deuterium enrichment","authors":"Erdem Şanal , Julia Drylewicz , Kiki Tesselaar , Becca Asquith , José A.M. Borghans , Rob J. de Boer","doi":"10.1016/j.jim.2025.114010","DOIUrl":"10.1016/j.jim.2025.114010","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 is the state-of-the-art technique 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> labels dividing cells through incorporation of deuterium into newly synthesized DNA, which is measured using GC/MS. The labeling rate depends on (1) the level of body water enrichment, (2) cell kinetics, and (3) an amplification factor quantifying the deoxyribose enrichment relative to the body water enrichment. This amplification factor is typically estimated using a reference population undergoing rapid turnover (such as granulocytes), and is larger than one because deoxyribose contains seven hydrogens that can be replaced by deuterium. In a meta-analysis, we found that individuals differ markedly in this amplification factor. Since the amplification factor also depends on the level of body water enrichment, we use conventional binomial expressions to describe the fractions of deoxyribose incorporating zero, one, two, or more deuterium atoms. We extend this classic binomial model with a new parameter, <span><math><mrow><mn>0</mn><mo><</mo><mi>γ</mi><mo><</mo><mn>1</mn></mrow></math></span>, describing the relative contribution of hydrogens from body water during deoxyribose synthesis. While for most studies, our ‘novel Binomial’ model reasonably explains the slope with which the amplification factor declines with the level of body water enrichment, we find that some individual amplification factors differ considerably from their expected values Re-fitting deuterium labeling data of granulocytes with the Binomial model reveals that the actual decrease is steeper than expected. We speculate that this residual variation depends on differences in diet, metabolism, and/or life style, which apparently correlate with daily fluid intake.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114010"},"PeriodicalIF":1.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145677908","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 : 2026-01-01Epub Date: 2025-12-03DOI: 10.1016/j.jim.2025.114019
Qinqin Liu , Yuanmin Sun , Huiqiang Li , Xueyan Wang
Objective
Developed a rapid, sensitive, and homogeneous immunoassay to characterize specific immunoglobulin E (sIgE) against House Dust Mite (HDM) components.
Methods
Initially, the reaction conditions were optimized, and the levels of sIgE to the major HDM components -Der p 1, Der p 2, and Der p 23- were measured using the Light-initiated Chemiluminescence Assay (LiCA) assay. The performance of this assay was evaluated in accordance with established clinical guidelines. Subsequently, the sIgE reactivities to these components were analyzed among 90 children with various allergic diseases.
Results
We established an assay for HDM component sIgE using LiCA technology. The coefficient of variation for repeatability ranged from 2.64 % to 9.36 %, while the intermediate precision varied from 5.77 % to 9.89 %. Component-resolved diagnosis (CRD) indicated that Der p 2 was the most frequently recognized component (75.56 %), followed by Der p 1 at 64.44 %. The highest co-sensitization rate was observed for Der p 1 and Der p 2 (35.56 %). Der p 23 sIgE levels were significantly elevated in patients with asthma (P < 0.001). Additionally, a greater complexity in allergic symptoms was associated with an increased positive rate of Der p 23 (P = 0.007).
Conclusion
The HDM assay developed here is the first attempt to characterize HDM components sIgE by LiCA. This method is easy to operate, faster and has high detection accuracy. Der p 2 was most frequently recognized among the HDM components. Furthermore, our findings indicate a significant correlation between the complexity of allergic symptoms and elevated levels of Der p 23 sIgE.
{"title":"Development and clinical application of component-resolved diagnostic using light-initiated chemiluminescence assay to characterize house dust mite components-specific IgE","authors":"Qinqin Liu , Yuanmin Sun , Huiqiang Li , Xueyan Wang","doi":"10.1016/j.jim.2025.114019","DOIUrl":"10.1016/j.jim.2025.114019","url":null,"abstract":"<div><h3>Objective</h3><div>Developed a rapid, sensitive, and homogeneous immunoassay to characterize specific immunoglobulin E (sIgE) against House Dust Mite (HDM) components.</div></div><div><h3>Methods</h3><div>Initially, the reaction conditions were optimized, and the levels of sIgE to the major HDM components -Der p 1, Der p 2, and Der p 23- were measured using the Light-initiated Chemiluminescence Assay (LiCA) assay. The performance of this assay was evaluated in accordance with established clinical guidelines. Subsequently, the sIgE reactivities to these components were analyzed among 90 children with various allergic diseases.</div></div><div><h3>Results</h3><div>We established an assay for HDM component sIgE using LiCA technology. The coefficient of variation for repeatability ranged from 2.64 % to 9.36 %, while the intermediate precision varied from 5.77 % to 9.89 %. Component-resolved diagnosis (CRD) indicated that Der p 2 was the most frequently recognized component (75.56 %), followed by Der p 1 at 64.44 %. The highest co-sensitization rate was observed for Der p 1 and Der p 2 (35.56 %). Der p 23 sIgE levels were significantly elevated in patients with asthma (<em>P</em> < 0.001). Additionally, a greater complexity in allergic symptoms was associated with an increased positive rate of Der p 23 (<em>P</em> = 0.007).</div></div><div><h3>Conclusion</h3><div>The HDM assay developed here is the first attempt to characterize HDM components sIgE by LiCA. This method is easy to operate, faster and has high detection accuracy. Der p 2 was most frequently recognized among the HDM components. Furthermore, our findings indicate a significant correlation between the complexity of allergic symptoms and elevated levels of Der p 23 sIgE.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"546 ","pages":"Article 114019"},"PeriodicalIF":1.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145683916","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 : 2026-01-01Epub Date: 2025-12-04DOI: 10.1016/j.jim.2025.114009
Jessica Chavez, Evan W. McConnell, Rogger Alcalde, Ajay Grover, Deborah Boles, Christopher M. Shuford, Russell P. Grant, Andre Valcour, Saintedym Wills
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 (r > 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.
{"title":"Automation and performance validation of the pooled donor basophil activation test for chronic urticaria using the Hamilton STAR system","authors":"Jessica Chavez, Evan W. McConnell, Rogger Alcalde, Ajay Grover, Deborah Boles, Christopher M. Shuford, Russell P. Grant, Andre Valcour, 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> > 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}
Pub Date : 2026-01-01Epub 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":"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}
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.
{"title":"Advancing cervical cancer screening: A novel in-house ELISA assay for the simultaneous detection of p16 and Ki-67","authors":"Sallam Al-Madhagi , Patricia Khashayar , Lieselotte Volckaert , Jan Vanfleteren , 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}
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