Pub 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":"2025-12-02","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 : 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":"2025-12-02","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 : 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":"2025-12-01","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 : 2025-12-01DOI: 10.1016/j.jim.2025.114007
Stephen C. Mayer , Jennifer K. Stevenson , Siva Gandhapudi , Jerold G. Woodward
During the COVID-19 Pandemic in 2020, the need for highly-specific, wide-spread, and rapid serological testing surged. In this study, we tested the utility of the Volumetric Absorptive Microsampling (VAMS) using Mitra home sampling kits by comparing SARS CoV2 spike antibody levels from serum or Mitra dried blood samples from the same individuals with or without prior COVID 19 infection. We showed a very strong correlation between venous blood collection and capillary VAMS for the detection of anti-SARS CoV2 IgGs using a sensitive, semi-quantitative ELISA protocol. Furthermore, we found that the antibody levels detected from the Mitra blood samples were stable at room temperature for several months. This study demonstrates the utility of using at-home, patient-centric testing to enhance the sero-surveillance methods and large scale community testing for viral tracking, monitoring and vaccine studies.
{"title":"Validation of at-home blood sampling for large scale, cost effective serological analysis for anti-viral antibody responses","authors":"Stephen C. Mayer , Jennifer K. Stevenson , Siva Gandhapudi , Jerold G. Woodward","doi":"10.1016/j.jim.2025.114007","DOIUrl":"10.1016/j.jim.2025.114007","url":null,"abstract":"<div><div>During the COVID-19 Pandemic in 2020, the need for highly-specific, wide-spread, and rapid serological testing surged. In this study, we tested the utility of the Volumetric Absorptive Microsampling (VAMS) using Mitra home sampling kits by comparing SARS CoV2 spike antibody levels from serum or Mitra dried blood samples from the same individuals with or without prior COVID 19 infection. We showed a very strong correlation between venous blood collection and capillary VAMS for the detection of anti-SARS CoV2 IgGs using a sensitive, semi-quantitative ELISA protocol. Furthermore, we found that the antibody levels detected from the Mitra blood samples were stable at room temperature for several months. This study demonstrates the utility of using at-home, patient-centric testing to enhance the sero-surveillance methods and large scale community testing for viral tracking, monitoring and vaccine studies.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114007"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145581703","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 : 2025-11-22DOI: 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":"2025-11-22","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 : 2025-11-19DOI: 10.1016/j.jim.2025.114005
Yikeshan Yalikun, Xinyue Qiao, Tyuji Hoshino
Monoclonal antibodies are currently essential biological molecules for immunotherapy and diagnosis. The adaptability of an antibody to humans is crucial for ensuring safety and achieving high therapeutic efficacy as a medicinal drug. A considerable amount of effort has been devoted to predicting the human likeness of recombinant antibody molecules to assess their suitability for clinical use. Many previous studies have utilized a database of antibody sequences. However, a rapid assessment of adaptability is also helpful in the early stage of high-affinity antibody molecular design to a target antigen. To characterize the amino acid sequences of human antibodies, we statistically analyzed 742 antigen-antibody complex structures extracted from the Protein Data Bank. The frequency and position of the appearances of the respective residues are surveyed, and their probabilities were obtained for three complementarity-determining regions of heavy and light chains. In particular, the populations were examined from the viewpoint of which positions the respective residues were likely to appear in each complementary determining region. Based on a statistical analysis, an arithmetic index was proposed to evaluate sequence compatibility with humans and assess antibody models in computational molecular design. An examination using a collection of neutralizing antibodies for viral infectious diseases suggested that the index can distinguish human antibodies from those of mice. An examination of a collection of approved antibody drugs showed a certain degree of correlation between the calculated index and the immunogenicity of the antibody drugs. These evaluations demonstrated the feasibility of the proposed index as a rapid method for evaluating molecular adaptability to medicinal antibodies.
{"title":"An index for deviation distance among amino acid sequences of antibody complementarity determining regions","authors":"Yikeshan Yalikun, Xinyue Qiao, Tyuji Hoshino","doi":"10.1016/j.jim.2025.114005","DOIUrl":"10.1016/j.jim.2025.114005","url":null,"abstract":"<div><div>Monoclonal antibodies are currently essential biological molecules for immunotherapy and diagnosis. The adaptability of an antibody to humans is crucial for ensuring safety and achieving high therapeutic efficacy as a medicinal drug. A considerable amount of effort has been devoted to predicting the human likeness of recombinant antibody molecules to assess their suitability for clinical use. Many previous studies have utilized a database of antibody sequences. However, a rapid assessment of adaptability is also helpful in the early stage of high-affinity antibody molecular design to a target antigen. To characterize the amino acid sequences of human antibodies, we statistically analyzed 742 antigen-antibody complex structures extracted from the Protein Data Bank. The frequency and position of the appearances of the respective residues are surveyed, and their probabilities were obtained for three complementarity-determining regions of heavy and light chains. In particular, the populations were examined from the viewpoint of which positions the respective residues were likely to appear in each complementary determining region. Based on a statistical analysis, an arithmetic index was proposed to evaluate sequence compatibility with humans and assess antibody models in computational molecular design. An examination using a collection of neutralizing antibodies for viral infectious diseases suggested that the index can distinguish human antibodies from those of mice. An examination of a collection of approved antibody drugs showed a certain degree of correlation between the calculated index and the immunogenicity of the antibody drugs. These evaluations demonstrated the feasibility of the proposed index as a rapid method for evaluating molecular adaptability to medicinal antibodies.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114005"},"PeriodicalIF":1.6,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145573687","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 : 2025-11-13DOI: 10.1016/j.jim.2025.114003
Yvonne Dube , Wina Hasang , Andrew Teo , Mwayiwawo Madanitsa , Morten A. Nielsen , Feiko O. Ter Kuile , Elizabeth H. Aitken , Stephen J. Rogerson
Placental malaria due to Plasmodium falciparum (Pf) is associated with adverse pregnancy outcomes. Infected erythrocytes (IEs) bind to placental chondroitin sulfate A (CSA) and antibodies that inhibit this adhesion are a potential correlate of protection. We developed a simplified adhesion inhibition assay that uses diaminofluorene to measure haemoglobin release from IEs bound to CSA.
Using hyperimmune plasma, the assay demonstrated concentration-dependent inhibition of CSA adhesion. Assay performance was consistent over time with strong reproducibility (r = 0.82), and results correlated with a published assay (r = 0.53). In 466 Malawian pregnant women (321 P. falciparum-infected and 145 uninfected at first antenatal visit), adhesion inhibitory antibodies were significantly higher in mid-pregnancy in infected multigravidae (46.3 % IQR 23.2 %, 74.8 %) compared to infected primigravidae (9.7 % IQR 0 %, 29.3 %, p < 0.001) and in uninfected multigravidae (29.9 % IQR 8.6 %, 54.5 %) than uninfected primigravidae (15.6 % IQR 0 %, 36.4 %, p = 0.04). Similar, significant gravidity-dependent differences were observed at delivery in both infected and uninfected women. Between enrolment and delivery, changes in antibodies were similar in infected and uninfected women.
Adhesion inhibitory antibodies protected against placental malaria. Of 162 women with infection at mid-pregnancy, 88 with no placental malaria had more adhesion inhibitory antibody (33.8 %, IQR 1.9 %, 63.2 %) than 74 with past placental malaria (12.5 %, IQR 0 %, 37.8 %; p = 0.002). This low cost, reproducible, and rapid high-throughput adhesion inhibition assay shows promise as a correlate of protection against placental malaria.
{"title":"A high-throughput assay to measure antibodies that block adhesion of Plasmodium falciparum infected erythrocytes to chondroitin sulfate A.","authors":"Yvonne Dube , Wina Hasang , Andrew Teo , Mwayiwawo Madanitsa , Morten A. Nielsen , Feiko O. Ter Kuile , Elizabeth H. Aitken , Stephen J. Rogerson","doi":"10.1016/j.jim.2025.114003","DOIUrl":"10.1016/j.jim.2025.114003","url":null,"abstract":"<div><div>Placental malaria due to <em>Plasmodium falciparum</em> (<em>Pf</em>) is associated with adverse pregnancy outcomes. Infected erythrocytes (IEs) bind to placental chondroitin sulfate A (CSA) and antibodies that inhibit this adhesion are a potential correlate of protection. We developed a simplified adhesion inhibition assay that uses diaminofluorene to measure haemoglobin release from IEs bound to CSA.</div><div>Using hyperimmune plasma, the assay demonstrated concentration-dependent inhibition of CSA adhesion. Assay performance was consistent over time with strong reproducibility (<em>r</em> = 0.82), and results correlated with a published assay (<em>r</em> = 0.53). In 466 Malawian pregnant women (321 <em>P. falciparum</em>-infected and 145 uninfected at first antenatal visit), adhesion inhibitory antibodies were significantly higher in mid-pregnancy in infected multigravidae (46.3 % IQR 23.2 %, 74.8 %) compared to infected primigravidae (9.7 % IQR 0 %, 29.3 %, <em>p</em> < 0.001) and in uninfected multigravidae (29.9 % IQR 8.6 %, 54.5 %) than uninfected primigravidae (15.6 % IQR 0 %, 36.4 %, <em>p</em> = 0.04). Similar, significant gravidity-dependent differences were observed at delivery in both infected and uninfected women. Between enrolment and delivery, changes in antibodies were similar in infected and uninfected women.</div><div>Adhesion inhibitory antibodies protected against placental malaria. Of 162 women with infection at mid-pregnancy, 88 with no placental malaria had more adhesion inhibitory antibody (33.8 %, IQR 1.9 %, 63.2 %) than 74 with past placental malaria (12.5 %, IQR 0 %, 37.8 %; <em>p</em> = 0.002). This low cost, reproducible, and rapid high-throughput adhesion inhibition assay shows promise as a correlate of protection against placental malaria.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114003"},"PeriodicalIF":1.6,"publicationDate":"2025-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145530547","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 : 2025-11-12DOI: 10.1016/j.jim.2025.114004
Tania L. Weiss , Justine Paniagua , Tonny Johnson , Timothy P. Cripe , Peter Ralph
A cytotoxicity assay was developed to measure the potency of GP101, a single chain diabody. GP101 is comprised of two linked Fv domains, with one that binds CD3 (expressed on T cells), and the other that binds CD19 (expressed on B cells). GP101 directs CD3 positive T lymphocytes to CD19 positive B lymphocytes. This immunotherapy redirects CD3+ T cells to CD19-expressing B-cell malignancies, enabling T-cell-mediated tumor cell lysis. We developed a GMP cell-based potency assay to satisfy the FDA requirements. The potency assay uses a cytotoxic T cell line, and a B cell lymphoma cell line as the target. The target lymphoma B cell line is prelabeled with a fluorescent dye, and upon T cell mediated killing, the fluorescence dye is released and detected using a fluorometer. The emitted fluorescence is proportional to the dose of GP101. Greater than 90 % of the target B cells were killed within two hours exposure in vitro with the lowest amount of detectable killing at 60 pg/mL GP101. The assay is suitable for measuring purified GP101, or GP101 expressed by cells transduced by GP101 plasmid or AAV preparations, and bioactivity in animal or human blood. This novel assay met GMP/GLP compliance, allowing a quantifiable and reproducible measure of efficacy, ensuring batch-to-batch consistency, and met safety and effectiveness regulatory requirements. This potency assay may be applicable for testing other CD3-CD19 T cell engagers and suitable for developing other diabody mediated potency assays with appropriate antigens.
{"title":"Cell-based potency assay for anti-CD3-anti-CD19 diabody","authors":"Tania L. Weiss , Justine Paniagua , Tonny Johnson , Timothy P. Cripe , Peter Ralph","doi":"10.1016/j.jim.2025.114004","DOIUrl":"10.1016/j.jim.2025.114004","url":null,"abstract":"<div><div>A cytotoxicity assay was developed to measure the potency of GP101, a single chain diabody. GP101 is comprised of two linked Fv domains, with one that binds CD3 (expressed on T cells), and the other that binds CD19 (expressed on B cells). GP101 directs CD3 positive T lymphocytes to CD19 positive B lymphocytes. This immunotherapy redirects CD3+ T cells to CD19-expressing B-cell malignancies, enabling T-cell-mediated tumor cell lysis. We developed a GMP cell-based potency assay to satisfy the FDA requirements. The potency assay uses a cytotoxic T cell line, and a B cell lymphoma cell line as the target. The target lymphoma B cell line is prelabeled with a fluorescent dye, and upon T cell mediated killing, the fluorescence dye is released and detected using a fluorometer. The emitted fluorescence is proportional to the dose of GP101. Greater than 90 % of the target B cells were killed within two hours exposure in vitro with the lowest amount of detectable killing at 60 pg/mL GP101. The assay is suitable for measuring purified GP101, or GP101 expressed by cells transduced by GP101 plasmid or AAV preparations, and bioactivity in animal or human blood. This novel assay met GMP/GLP compliance, allowing a quantifiable and reproducible measure of efficacy, ensuring batch-to-batch consistency, and met safety and effectiveness regulatory requirements. This potency assay may be applicable for testing other CD3-CD19 T cell engagers and suitable for developing other diabody mediated potency assays with appropriate antigens.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114004"},"PeriodicalIF":1.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145512989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-07DOI: 10.1016/j.jim.2025.114002
Marcelo S. Conzentino , Thais A. Silva , Ana Carolina A. Gonçalves , Fabiane G.M. Rego , Vera J.L. Chicora , Stephanie M.S. Lo , Fernanda L. Martiny , Fabio O. Pedrosa , Luciano F. Huergo
Here we describe a magnetic bead-based indirect ELISA that enables the rapid and scalable detection of human IgM and IgG antibodies reactive to dengue virus (DENV). The assay can be performed in 12 min in the chromogenic format to IgM or IgG detection. The system can also handle simultaneous detection of IgM and IgG within less than 8 min when combined with fluorescent-labelled secondary conjugated IgM and IgG detection antibodies. The sensitivity and specificity of the assay are in the same range as described for commercially available ELISA kits. This simple and ultrafast assay for dengue seroconversion detection is an efficient alternative for the rapid track dengue cases.
{"title":"Detection of human IgM and IgG antibodies reactive to Dengue virus using magnetic bead immunoassay","authors":"Marcelo S. Conzentino , Thais A. Silva , Ana Carolina A. Gonçalves , Fabiane G.M. Rego , Vera J.L. Chicora , Stephanie M.S. Lo , Fernanda L. Martiny , Fabio O. Pedrosa , Luciano F. Huergo","doi":"10.1016/j.jim.2025.114002","DOIUrl":"10.1016/j.jim.2025.114002","url":null,"abstract":"<div><div>Here we describe a magnetic bead-based indirect ELISA that enables the rapid and scalable detection of human IgM and IgG antibodies reactive to dengue virus (DENV). The assay can be performed in 12 min in the chromogenic format to IgM or IgG detection. The system can also handle simultaneous detection of IgM and IgG within less than 8 min when combined with fluorescent-labelled secondary conjugated IgM and IgG detection antibodies. The sensitivity and specificity of the assay are in the same range as described for commercially available ELISA kits. This simple and ultrafast assay for dengue seroconversion detection is an efficient alternative for the rapid track dengue cases.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114002"},"PeriodicalIF":1.6,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145482327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
RAW 264.7, a well-established cell line, is widely used in research; however, it is easily polarized upon subtle changes in environmental conditions or repeated passages. As the effects of cell storage conditions and duration on their phenotypic changes remain unknown, we investigated the effects of long-term storage at −80 °C on M2 marker expression in RAW 264.7 cells after stimulation with IL-4. Cells, stored in a − 80 °C deep freezer for a few years (defined as “long-stored cells”) and newly purchased cells (referred to as “new cells”), were compared through flow cytometry for CD68-positive naïve macrophages (M0) and the expression of three types of M2 markers, CD206-FITC, CD206-PE, and Arginase-1-Alexa Fluor 488, in the M2 state. CD68 positivity was similar between the M0 macrophage types. After stimulation with IL-4, the proportion of CD206-FITC-positive cells was 5.0 ± 0.7 % and 61.5 ± 3.1 % in long-stored cells and new cells, respectively (p < 0.0001), while that of CD206-PE-positive cells was 9.2 ± 0.2 % and 69.8 ± 0.6 %. Similarly, the proportion of Arginase-1- Alexa Fluor 488-positive cells was 3.0 ± 0.1 and 9.0 ± 9.6 %, respectively (p = 0.0006). Overall, these results demonstrate that long-term storage of RAW 264.7 in a deep freezer affects their M2 marker expression. These results highlight the need to consider the effect of cell storage conditions and duration on experimental results.
{"title":"Long-term cell storage of RAW 264.7 cells in a deep freezer dampens M2 marker expression","authors":"Tomonori Makiguchi , Akio Nakane , Sadatomo Tasaka","doi":"10.1016/j.jim.2025.114001","DOIUrl":"10.1016/j.jim.2025.114001","url":null,"abstract":"<div><div>RAW 264.7, a well-established cell line, is widely used in research; however, it is easily polarized upon subtle changes in environmental conditions or repeated passages. As the effects of cell storage conditions and duration on their phenotypic changes remain unknown, we investigated the effects of long-term storage at −80 °C on M2 marker expression in RAW 264.7 cells after stimulation with IL-4. Cells, stored in a − 80 °C deep freezer for a few years (defined as “long-stored cells”) and newly purchased cells (referred to as “new cells”), were compared through flow cytometry for CD68-positive naïve macrophages (M0) and the expression of three types of M2 markers, CD206-FITC, CD206-PE, and Arginase-1-Alexa Fluor 488, in the M2 state. CD68 positivity was similar between the M0 macrophage types. After stimulation with IL-4, the proportion of CD206-FITC-positive cells was 5.0 ± 0.7 % and 61.5 ± 3.1 % in long-stored cells and new cells, respectively (<em>p</em> < 0.0001), while that of CD206-PE-positive cells was 9.2 ± 0.2 % and 69.8 ± 0.6 %. Similarly, the proportion of Arginase-1- Alexa Fluor 488-positive cells was 3.0 ± 0.1 and 9.0 ± 9.6 %, respectively (<em>p</em> = 0.0006). Overall, these results demonstrate that long-term storage of RAW 264.7 in a deep freezer affects their M2 marker expression. These results highlight the need to consider the effect of cell storage conditions and duration on experimental results.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 114001"},"PeriodicalIF":1.6,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471167","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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