Immunoassays for serology have advanced from radio-immunoassays to multiplexed micro-array-based technologies over the past years. Comparative studies can assist users in selecting an assay platform based on its applicability and performance characteristics. This study compared a 9-plex commercial VaxArray Coronavirus SeroAssay (InDevR) and a 10-plex semi-custom Coronavirus SeroAssay (MSD) with a panel of human serum samples. The MSD assay showed superior dynamic range and sensitivity across all coronavirus serotypes, while both assays met accuracy (100 ± 20 %) and precision (%CV < 25 %) criteria. Although both platforms were concordant for anti-SARS-CoV2 IgG measurements, the clinical sensitivity and specificity of the MSD assay were marginally higher than those of VaxArray. Despite VaxArray's shorter total assay time and higher potential for multiplexing and re-readability, it requires improvements in dynamic range, sensitivity, specificity, and automation capabilities for regulated use.
{"title":"Comparative assessment of a multiplex micro-chip immunoassay, VaxArray, and meso scale discovery assay for serotype-specific coronavirus IgG quantitation","authors":"Ashvi Sanjay Jain , Thorsten Verch , Gowrisankar Rajam , Ying Homan","doi":"10.1016/j.jim.2025.113907","DOIUrl":"10.1016/j.jim.2025.113907","url":null,"abstract":"<div><div>Immunoassays for serology have advanced from radio-immunoassays to multiplexed micro-array-based technologies over the past years. Comparative studies can assist users in selecting an assay platform based on its applicability and performance characteristics. This study compared a 9-plex commercial VaxArray Coronavirus SeroAssay (InDevR) and a 10-plex semi-custom Coronavirus SeroAssay (MSD) with a panel of human serum samples. The MSD assay showed superior dynamic range and sensitivity across all coronavirus serotypes, while both assays met accuracy (100 ± 20 %) and precision (%CV < 25 %) criteria. Although both platforms were concordant for anti-SARS-CoV2 IgG measurements, the clinical sensitivity and specificity of the MSD assay were marginally higher than those of VaxArray. Despite VaxArray's shorter total assay time and higher potential for multiplexing and re-readability, it requires improvements in dynamic range, sensitivity, specificity, and automation capabilities for regulated use.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113907"},"PeriodicalIF":1.6,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144674961","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 : 2025-09-01Epub Date: 2025-07-19DOI: 10.1016/j.jim.2025.113906
Hope Mataramvura, Patience Ncube, Kerina Duri
Background
Viral infection/exposure and time from phlebotomy to peripheral blood mononuclear cell (PBMC) isolation (TPP) may affect PBMC yield and viability. We investigated the impact of TPP and early-life HIV/ART exposure on PBMC, including NK cells yield and viability.
Methods
Blood in EDTA tubes with varying TPP was used to isolate PBMCs via density-gradient centrifugation, counted under a microscope, and assessed for viability with Trypan-Blue. Antibody staining (CD14, CD3, and CD19) excluded monocytes and T/B lymphocytes, while CD16 and CD56 staining with a LIVE/DEAD marker identified viable NK cells. Comparisons were made among HIV-exposed uninfected (HEU) children (long-term [HEULT] and medium-term [HEUMT] ART exposure), HIV-exposed infected (HEI), and HIV-unexposed uninfected (HUU) children.
Results
We enrolled 112 children (50 % female) with a median age of 5.1 years [interquartile range (IQR):4.8–6.0], categorized as 37-HUU, 36-HEULT, 34-HEUMT, and 5-HEI children. Median blood volume was 7.5 ml (IQR:7.5–8.0), with HEI children showing slightly higher PBMC yields/ml than HUU and HEU (p = 0.092). Median TPP was 90 min (IQR:64.8–112.0), with viability remaining high (>95 %) up to 3 h, after this the yields/ml decreased compared to TPP ≤ 1 h (1.3 vs. 2.8 × 106 cells/ml) (p = 0.010) for all children. Overall, TPP negatively correlated with yields/ml and viability (r = −0.35, p < 0.001; r = −0.47, p < 0.001 respectively). Median frequency of viable CD56 + CD16+/− NK cells was 8.2 %, unaffected by neither TPP nor HIV/ART exposure. Total lymphocytes were lower in males [18.6 % (IQR:11.3–22.2)] than in females [23.8 % (IQR:14.3–29.9)] (p = 0.018).
Conclusion
HIV infection, not exposure, may increase PBMC yields. TPP under 3 h is ideal for optimal yields.
{"title":"From phlebotomy to peripheral blood mononuclear cell isolation; effect of time and early-life HIV/ART exposure on cell yield and viability in paediatric samples in a high HIV prevalence setting","authors":"Hope Mataramvura, Patience Ncube, Kerina Duri","doi":"10.1016/j.jim.2025.113906","DOIUrl":"10.1016/j.jim.2025.113906","url":null,"abstract":"<div><h3>Background</h3><div>Viral infection/exposure and time from phlebotomy to peripheral blood mononuclear cell (PBMC) isolation (TPP) may affect PBMC yield and viability. We investigated the impact of TPP and early-life HIV/ART exposure on PBMC, including NK cells yield and viability.</div></div><div><h3>Methods</h3><div>Blood in EDTA tubes with varying TPP was used to isolate PBMCs via density-gradient centrifugation, counted under a microscope, and assessed for viability with Trypan-Blue. Antibody staining (CD14, CD3, and CD19) excluded monocytes and T/B lymphocytes, while CD16 and CD56 staining with a LIVE/DEAD marker identified viable NK cells. Comparisons were made among HIV-exposed uninfected (HEU) children (long-term [HEULT] and medium-term [HEUMT] ART exposure), HIV-exposed infected (HEI), and HIV-unexposed uninfected (HUU) children.</div></div><div><h3>Results</h3><div>We enrolled 112 children (50 % female) with a median age of 5.1 years [interquartile range (IQR):4.8–6.0], categorized as 37-HUU, 36-HEULT, 34-HEUMT, and 5-HEI children. Median blood volume was 7.5 ml (IQR:7.5–8.0), with HEI children showing slightly higher PBMC yields/ml than HUU and HEU (<em>p</em> = 0.092). Median TPP was 90 min (IQR:64.8–112.0), with viability remaining high (>95 %) up to 3 h, after this the yields/ml decreased compared to TPP ≤ 1 h (1.3 vs. 2.8 × 10<sup>6</sup> cells/ml) (<em>p</em> = 0.010) for all children. Overall, TPP negatively correlated with yields/ml and viability (<em>r</em> = −0.35, <em>p</em> < 0.001; <em>r</em> = −0.47, p < 0.001 respectively). Median frequency of viable CD56 + CD16+/− NK cells was 8.2 %, unaffected by neither TPP nor HIV/ART exposure. Total lymphocytes were lower in males [18.6 % (IQR:11.3–22.2)] than in females [23.8 % (IQR:14.3–29.9)] (<em>p</em> = 0.018).</div></div><div><h3>Conclusion</h3><div>HIV infection, not exposure, may increase PBMC yields. TPP under 3 h is ideal for optimal yields.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113906"},"PeriodicalIF":1.6,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144682719","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}
Antibody-dependent enhancement (ADE) of infection is a concerning phenomenon in SARS-CoV-2 infection, since it may develop disease severity. Although ADE has been demonstrated in animal models, the pathogenic mechanism has not been fully elucidated. The present study aimed to develop a simple assay system for detecting SARS-CoV-2 ADE activity in any antibody specimen. Vero E6/TMPRSS2/FcγRIIA, a Vero E6 cell strain expressing TMPRSS2 and FcγRIIA, was established. Seventeen plasma samples from convalescent patients and seven commercial antibodies were used as antibody specimens. Vero E6/TMPRSS2/FcγRIIA cells, infected with SARS-CoV-2 in the presence of antibody, were shown to exhibit ADE activity, with the plaque number increasing dramatically compared with the control in the absence of antibody specimens. Most plasmas displayed both ADE and neutralizing activities. Furthermore, 6 commercial antibodies recognizing structural proteins (membrane, nucleocapsid, envelope, and spike [transmembrane and cytoplasmic domains] proteins and non-structural protein ORF7) showed no potential ADE activity, while a commercial antibody recognizing spike RBD displayed ADE activity. These results indicate that antibodies possessing neutralizing and/or enhancing activity might be generated by either viral infection or vaccination. The present ADE assay may help to evaluate the risk of disease severity for individuals upon future reinfection, vaccination and/or immunotherapy.
{"title":"Development of in vitro plaque-based assay for assessment of antibody-dependent enhancement in SARS-CoV-2 infection","authors":"Pimploy Rattanaamnuaychai , Surachet Benjathummarak , Pannamthip Pitaksajjakul , Pongrama Ramasoota , Yong Poovorawan , Hiroto Mizushima , Masashi Tatsumi , Yoshiharu Matsuura , Atsushi Yamanaka","doi":"10.1016/j.jim.2025.113919","DOIUrl":"10.1016/j.jim.2025.113919","url":null,"abstract":"<div><div>Antibody-dependent enhancement (ADE) of infection is a concerning phenomenon in SARS-CoV-2 infection, since it may develop disease severity. Although ADE has been demonstrated in animal models, the pathogenic mechanism has not been fully elucidated. The present study aimed to develop a simple assay system for detecting SARS-CoV-2 ADE activity in any antibody specimen. Vero E6/TMPRSS2/FcγRIIA, a Vero E6 cell strain expressing TMPRSS2 and FcγRIIA, was established. Seventeen plasma samples from convalescent patients and seven commercial antibodies were used as antibody specimens. Vero E6/TMPRSS2/FcγRIIA cells, infected with SARS-CoV-2 in the presence of antibody, were shown to exhibit ADE activity, with the plaque number increasing dramatically compared with the control in the absence of antibody specimens. Most plasmas displayed both ADE and neutralizing activities. Furthermore, 6 commercial antibodies recognizing structural proteins (membrane, nucleocapsid, envelope, and spike [transmembrane and cytoplasmic domains] proteins and non-structural protein ORF7) showed no potential ADE activity, while a commercial antibody recognizing spike RBD displayed ADE activity. These results indicate that antibodies possessing neutralizing and/or enhancing activity might be generated by either viral infection or vaccination. The present ADE assay may help to evaluate the risk of disease severity for individuals upon future reinfection, vaccination and/or immunotherapy.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113919"},"PeriodicalIF":1.6,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144781255","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 : 2025-08-01Epub Date: 2025-06-24DOI: 10.1016/j.jim.2025.113901
John Hok Nin Lowe, Jenny Yanhong Li, Mauricio Maia
Anti-drug antibody (ADA) assays are an important element in the suite of bioanalytical methods required for assessment of the safety and efficacy of recombinant-protein therapeutics. As such, and following extensive optimization, there is an expectation that clinical ADA assays be fully validated for multiple performance parameters, including sensitivity, specificity, reagent stability, and robustness. Among critical reagents used in ADA assays, ADA positive controls (PCs) play a crucial role in multiple stages of assay development and validation, including selection of assay format, establishing assay cut-points, estimating assay relative sensitivity, assessing assay precision, as well as ensuring acceptable assay performance during sample testing. This manuscript highlights an unexpected and highly-impactful PC performance inconsistency attribute we encountered prior to validation of a clinical ADA assay. We also describe the investigation that identified the root cause of this problem: the immune complexation between the murine monoclonal antibody used as the surrogate PC and human anti-mouse antibody present in the serum used to prepare the PC. We conclude that murine monoclonal antibodies are not fully appropriate reagents for use as PCs in clinical ADA assays. Finally, potential approaches to circumvent or mitigate this specific problem in clinical ADA assays are discussed.
{"title":"An unexpected bioanalytical challenge caused by positive control antibodies in a clinical immunogenicity assay – A simple solution and broadly applicable recommendations","authors":"John Hok Nin Lowe, Jenny Yanhong Li, Mauricio Maia","doi":"10.1016/j.jim.2025.113901","DOIUrl":"10.1016/j.jim.2025.113901","url":null,"abstract":"<div><div>Anti-drug antibody (ADA) assays are an important element in the suite of bioanalytical methods required for assessment of the safety and efficacy of recombinant-protein therapeutics. As such, and following extensive optimization, there is an expectation that clinical ADA assays be fully validated for multiple performance parameters, including sensitivity, specificity, reagent stability, and robustness. Among critical reagents used in ADA assays, ADA positive controls (PCs) play a crucial role in multiple stages of assay development and validation, including selection of assay format, establishing assay cut-points, estimating assay relative sensitivity, assessing assay precision, as well as ensuring acceptable assay performance during sample testing. This manuscript highlights an unexpected and highly-impactful PC performance inconsistency attribute we encountered prior to validation of a clinical ADA assay. We also describe the investigation that identified the root cause of this problem: the immune complexation between the murine monoclonal antibody used as the surrogate PC and human anti-mouse antibody present in the serum used to prepare the PC. We conclude that murine monoclonal antibodies are not fully appropriate reagents for use as PCs in clinical ADA assays. Finally, potential approaches to circumvent or mitigate this specific problem in clinical ADA assays are discussed.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113901"},"PeriodicalIF":1.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505931","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}
Farmer's lung disease (FLD) is a form of hypersensitivity pneumonitis due to regular exposure to microbial antigens present in moldy hay. Serological diagnosis is usually based on immunoprecipitation techniques using 1 to 12 in-house antigens. To improve standardization, recombinant antigens (r-Ags) have been produced from three etiological agents involved in FLD.
Aim
The aim of this study was to develop a strategy using an ELISA test for the serological diagnosis of FLD.
Methods
The ELISA strategy was developed on an initial group of patients and then validated using sera from 29 patients with FLD and 54 exposed but non-FLD patients. Using sera from the second group of patients, the ELISA was simplified by using purified antigens from Saccharopolyspora rectivirgula, Lichtheimia corymbifera, and Eurotium amstelodami, each mixed with a corresponding r-Ag. The performance of the ELISA was evaluated according to the antigens used and also associated with an immunoprecipitation technique.
Results
The ELISA test using three purified antigens (PAgs) mixed with 3 r-Ags showed a sensitivity of 97 % and a specificity of 83 % (AUC: 0.84). The use of ELISA as a screening technique and double diffusion (using 6 somatic antigens) as a confirmatory technique in cases of moderate or positive ELISAs showed a sensitivity of 93 % and a specificity of 84 % (AUC: 0.86).
Conclusion
The use of a mixture of purified protein antigens and highly standardized recombinant antigens from three etiological agents involved in FLD offers an effective and innovative strategy for the serological diagnosis of FLD cases. This strategy has been successfully applied in the routine workflow of our laboratory.
{"title":"Development of an ELISA strategy for the serological diagnosis of farmer's lung disease","authors":"Adeline Rouzet , Eliane Devillers , Coralie Barrera , Emeline Scherer , Laurence Millon , Anne-Pauline Bellanger","doi":"10.1016/j.jim.2025.113902","DOIUrl":"10.1016/j.jim.2025.113902","url":null,"abstract":"<div><h3>Context</h3><div>Farmer's lung disease (FLD) is a form of hypersensitivity pneumonitis due to regular exposure to microbial antigens present in moldy hay. Serological diagnosis is usually based on immunoprecipitation techniques using 1 to 12 in-house antigens. To improve standardization, recombinant antigens (r-Ags) have been produced from three etiological agents involved in FLD.</div></div><div><h3>Aim</h3><div>The aim of this study was to develop a strategy using an ELISA test for the serological diagnosis of FLD.</div></div><div><h3>Methods</h3><div>The ELISA strategy was developed on an initial group of patients and then validated using sera from 29 patients with FLD and 54 exposed but non-FLD patients. Using sera from the second group of patients, the ELISA was simplified by using purified antigens from <em>Saccharopolyspora rectivirgula</em>, <em>Lichtheimia corymbifera</em>, and <em>Eurotium amstelodami</em>, each mixed with a corresponding r-Ag. The performance of the ELISA was evaluated according to the antigens used and also associated with an immunoprecipitation technique.</div></div><div><h3>Results</h3><div>The ELISA test using three purified antigens (PAgs) mixed with 3 r-Ags showed a sensitivity of 97 % and a specificity of 83 % (AUC: 0.84). The use of ELISA as a screening technique and double diffusion (using 6 somatic antigens) as a confirmatory technique in cases of moderate or positive ELISAs showed a sensitivity of 93 % and a specificity of 84 % (AUC: 0.86).</div></div><div><h3>Conclusion</h3><div>The use of a mixture of purified protein antigens and highly standardized recombinant antigens from three etiological agents involved in FLD offers an effective and innovative strategy for the serological diagnosis of FLD cases. This strategy has been successfully applied in the routine workflow of our laboratory.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113902"},"PeriodicalIF":1.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144491070","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 : 2025-08-01Epub Date: 2025-07-13DOI: 10.1016/j.jim.2025.113903
Mingming Zhang , Youtao Zhang , Fangming Cheng , Hong Chen , Yonghong Yang , Li Li , Yuzhang Jiang , Yaping Dai , Fang Qiu , Zhuye Qin , Chongxu Han , Gang Wang , Xingjuan Shi , Chungen Qian , Xiangdong Liu
Background
The quantification of antineutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) is crucial for diagnosing and monitoring granulomatosis with polyangiitis (GPA). However, a standardized and widely available quantitative reference material for PR3-ANCA detection remains lacking.
Objective
This study aims to develop and evaluate the properties of a novel chimeric human-mouse PR3 monoclonal antibody as a potential quantitative reference for PR3-ANCA detection using various in vitro diagnostic methods.
Methods
Mouse monoclonal antibodies were screened from hybridoma cells derived from mice immunized with recombinant human PR3 (rPR3). A high-affinity monoclonal antibody (A07) was sequenced, and a chimeric antibody incorporating human IgG1 constant regions was produced using a mammalian cell expression system. The antibody's binding to PR3 and its applicability in indirect immunofluorescence (IIF) assay, line-blotting, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA) were analyzed.
Results
The chimeric A07 antibody recognized the conformational epitopes with high affinity for only non-denatured PR3 and demonstrated the correct cytoplasmic staining pattern in IIF. Line-blotting assays confirmed its specificity and potency. ELISA and CLIA experiments showed well-defined standard curves, with the chimeric antibody displaying consistent reactivity comparable to native PR3-ANCA in serum samples across different antigen conditions.
Conclusions
These findings indicate that the chimeric A07 antibody effectively recognizes both recombinant and native PR3 antigens and can be applied across multiple in vitro PR3-ANCA assays. Furthermore, this antibody may serve as a standard reference for determining serum PR3-ANCA titers or as a substitute for PR3-positive serum in diagnostic applications.
{"title":"Development of a chimeric monoclonal antibody as a potential diagnostic reference in PR3-ANCA detection","authors":"Mingming Zhang , Youtao Zhang , Fangming Cheng , Hong Chen , Yonghong Yang , Li Li , Yuzhang Jiang , Yaping Dai , Fang Qiu , Zhuye Qin , Chongxu Han , Gang Wang , Xingjuan Shi , Chungen Qian , Xiangdong Liu","doi":"10.1016/j.jim.2025.113903","DOIUrl":"10.1016/j.jim.2025.113903","url":null,"abstract":"<div><h3>Background</h3><div>The quantification of antineutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) is crucial for diagnosing and monitoring granulomatosis with polyangiitis (GPA). However, a standardized and widely available quantitative reference material for PR3-ANCA detection remains lacking.</div></div><div><h3>Objective</h3><div>This study aims to develop and evaluate the properties of a novel chimeric human-mouse PR3 monoclonal antibody as a potential quantitative reference for PR3-ANCA detection using various in vitro diagnostic methods.</div></div><div><h3>Methods</h3><div>Mouse monoclonal antibodies were screened from hybridoma cells derived from mice immunized with recombinant human PR3 (rPR3). A high-affinity monoclonal antibody (A07) was sequenced, and a chimeric antibody incorporating human IgG1 constant regions was produced using a mammalian cell expression system. The antibody's binding to PR3 and its applicability in indirect immunofluorescence (IIF) assay, line-blotting, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA) were analyzed.</div></div><div><h3>Results</h3><div>The chimeric A07 antibody recognized the conformational epitopes with high affinity for only non-denatured PR3 and demonstrated the correct cytoplasmic staining pattern in IIF. Line-blotting assays confirmed its specificity and potency. ELISA and CLIA experiments showed well-defined standard curves, with the chimeric antibody displaying consistent reactivity comparable to native PR3-ANCA in serum samples across different antigen conditions.</div></div><div><h3>Conclusions</h3><div>These findings indicate that the chimeric A07 antibody effectively recognizes both recombinant and native PR3 antigens and can be applied across multiple in vitro PR3-ANCA assays. Furthermore, this antibody may serve as a standard reference for determining serum PR3-ANCA titers or as a substitute for PR3-positive serum in diagnostic applications.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113903"},"PeriodicalIF":1.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144642682","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 : 2025-08-01Epub Date: 2025-05-31DOI: 10.1016/j.jim.2025.113883
Maitreyi Bharath , Anh Le , Vaishnavi Konda , Shivika Srivastava , Mark Beliaev , Mengbing Chen , Anand George , Andy Zhang , Sophie Tan , Eben Ginto , Aaditya Awatiger , Kushagra Bahuguna , Vienna Li , Suditi Kedambadi , Xiaolin Cao , Hao Zeng , Guofei Xiong , Mihika Prabhu , Ayana Modi , Yanlan Wang , John Wang
Quantitative flow cytometry (qFCM) is a powerful approach for the precise measurement of cellular and molecular characteristics, offering significant advantages in biomedical research, clinical diagnostics, and therapeutic applications. However, current qFCM beads, relying on chemical conjugation face several limitations. This study explored the use of streptavidin-coated beads with a defined quantity of Protein G-biotin for quantitative flow cytometry, demonstrating that these quantitative streptavidin-Protein G-biotin beads (qBeads) can be used for qFCM analysis through both direct and indirect methods, enabling the development of standardized assays for a wide range of applications.
{"title":"Quantitative flow cytometry using quantitative streptavidin-protein G-biotin beads (qBeads)","authors":"Maitreyi Bharath , Anh Le , Vaishnavi Konda , Shivika Srivastava , Mark Beliaev , Mengbing Chen , Anand George , Andy Zhang , Sophie Tan , Eben Ginto , Aaditya Awatiger , Kushagra Bahuguna , Vienna Li , Suditi Kedambadi , Xiaolin Cao , Hao Zeng , Guofei Xiong , Mihika Prabhu , Ayana Modi , Yanlan Wang , John Wang","doi":"10.1016/j.jim.2025.113883","DOIUrl":"10.1016/j.jim.2025.113883","url":null,"abstract":"<div><div>Quantitative flow cytometry (qFCM) is a powerful approach for the precise measurement of cellular and molecular characteristics, offering significant advantages in biomedical research, clinical diagnostics, and therapeutic applications. However, current qFCM beads, relying on chemical conjugation face several limitations. This study explored the use of streptavidin-coated beads with a defined quantity of Protein G-biotin for quantitative flow cytometry, demonstrating that these quantitative streptavidin-Protein G-biotin beads (qBeads) can be used for qFCM analysis through both direct and indirect methods, enabling the development of standardized assays for a wide range of applications.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113883"},"PeriodicalIF":1.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144190358","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 : 2025-08-01Epub Date: 2025-06-09DOI: 10.1016/j.jim.2025.113896
Harun Kaya Kesik , Figen Celik , Seyma Gunyakti Kilinc , Muhammet Uslug , Sami Simsek
Cystic Echinococcosis (CE) is one of the most common helminth infections in many parts of the world. Antigen B (AgB) is a key molecule secreted by both the germinal membrane and protoscoleces during the larval stages of Echinococcus granulosus. Characterizing polymorphisms in the genes encoding AgB can improve the interpretation of serological diagnostic tests. This study aimed to determine the polymorphism in the AgB subunit 2 (AgB2) gene in sheep isolates of E. granulosus sensu lato and to investigate its effect on the serological response. Germinal membranes from 41 sheep hydatid cysts and corresponding blood serum samples were collected from slaughterhouses in Elazig and Bingol provinces of Türkiye. Total genomic DNA was isolated, and PCR amplification of the AgB2 gene region was followed by DNA sequencing to evaluate genetic diversity. Western Blot (WB) analysis was performed using partially purified cyst fluid antigen rich in AgB. Sequence analysis revealed that the 663 bp AgB2 gene region exhibited high polymorphism. A total of 33 polymorphic sequences were identified and classified into 10 different haplotypes (AgB2.Hap_01 to AgB2.Hap_10). Among these, 31 (93.9 %) samples were WB-positive, while 2 (6.1 %) were negative. The WB test demonstrated a sensitivity of 80.4 % and a specificity of 100 %. These results suggest a relationship between the polymorphism in the AgB2 gene and variations in the serological response.
囊性包虫病（CE）是世界上许多地区最常见的寄生虫感染之一。抗原B （Antigen B, AgB）是细粒棘球绦虫幼虫期生发膜和原头节分泌的关键分子。表征编码AgB基因的多态性可以改善血清学诊断测试的解释。本研究旨在检测绵羊分离物中AgB亚基2 （AgB2）基因的多态性，并探讨其对血清反应的影响。在塔吉克斯坦埃拉兹格省和Bingol省的屠宰场采集了41只羊包虫的生发膜和相应的血清样本。分离总基因组DNA，对AgB2基因区域进行PCR扩增，并进行DNA测序，评估遗传多样性。使用部分纯化的富含AgB的囊液抗原进行Western Blot （WB）分析。序列分析显示，663 bp的AgB2基因区具有较高的多态性。共鉴定出33个多态性序列，并将其划分为10个不同的单倍型(AgB2；Hap_01到AgB2.Hap_10)。其中wb阳性31份（93.9%），阴性2份（6.1%）。WB试验的敏感性为80.4%，特异性为100%。这些结果表明，AgB2基因多态性与血清学反应的变化之间存在关系。
{"title":"Characterization of antigen B subunit 2 (AgB2) gene polymorphism in sheep isolates of Echinococcus granulosus sensu lato and effect on serologic response","authors":"Harun Kaya Kesik , Figen Celik , Seyma Gunyakti Kilinc , Muhammet Uslug , Sami Simsek","doi":"10.1016/j.jim.2025.113896","DOIUrl":"10.1016/j.jim.2025.113896","url":null,"abstract":"<div><div>Cystic Echinococcosis (CE) is one of the most common helminth infections in many parts of the world. Antigen B (AgB) is a key molecule secreted by both the germinal membrane and protoscoleces during the larval stages of <em>Echinococcus granulosus</em>. Characterizing polymorphisms in the genes encoding AgB can improve the interpretation of serological diagnostic tests. This study aimed to determine the polymorphism in the AgB subunit 2 (AgB2) gene in sheep isolates of <em>E. granulosus</em> sensu lato and to investigate its effect on the serological response. Germinal membranes from 41 sheep hydatid cysts and corresponding blood serum samples were collected from slaughterhouses in Elazig and Bingol provinces of Türkiye. Total genomic DNA was isolated, and PCR amplification of the AgB2 gene region was followed by DNA sequencing to evaluate genetic diversity. Western Blot (WB) analysis was performed using partially purified cyst fluid antigen rich in AgB. Sequence analysis revealed that the 663 bp AgB2 gene region exhibited high polymorphism. A total of 33 polymorphic sequences were identified and classified into 10 different haplotypes (AgB2.Hap_01 to AgB2.Hap_10). Among these, 31 (93.9 %) samples were WB-positive, while 2 (6.1 %) were negative. The WB test demonstrated a sensitivity of 80.4 % and a specificity of 100 %. These results suggest a relationship between the polymorphism in the AgB2 gene and variations in the serological response.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113896"},"PeriodicalIF":1.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144254802","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 : 2025-08-01Epub Date: 2025-07-16DOI: 10.1016/j.jim.2025.113905
Amir Namvar Vansofla, Abbas Hajizade, Shahram Nazarian, Yousof Tarverdizadeh, Ahad Shahmaleki
Shigella species remain a major global health concern, causing gastrointestinal infections with significant morbidity and mortality. The rise of antibiotic-resistant Shigella strains underscores the urgent need for effective vaccines. This study evaluated the immunogenicity and protective efficacy of a novel multi-epitope recombinant protein vaccine against pathogenic Shigella species in a murine model. The multi-epitope antigen was expressed in E. coli and purified for immunization. BALB/c mice were subcutaneously immunized, and immune responses were assessed through serum IgG quantification (ELISA) and challenge studies. Immunized mice exhibited significantly higher antigen-specific IgG titers compared to controls (p < 0.05). Subsequent challenge with 10LD50 of virulent Shigella flexneri and Shigella boydii demonstrated robust protection, with vaccinated mice surviving lethal infections. Our findings indicate that this in silico-designed multi-epitope vaccine elicits potent humoral immunity and confers cross-species protection against clinically relevant Shigella pathogens. These results support its potential as a promising candidate for further vaccine development against shigellosis.
{"title":"An in-silico-designed multiepitope vaccine candidate can efficiently protect mice against pathogenic species of Shigella","authors":"Amir Namvar Vansofla, Abbas Hajizade, Shahram Nazarian, Yousof Tarverdizadeh, Ahad Shahmaleki","doi":"10.1016/j.jim.2025.113905","DOIUrl":"10.1016/j.jim.2025.113905","url":null,"abstract":"<div><div><em>Shigella</em> species remain a major global health concern, causing gastrointestinal infections with significant morbidity and mortality. The rise of antibiotic-resistant <em>Shigella</em> strains underscores the urgent need for effective vaccines. This study evaluated the immunogenicity and protective efficacy of a novel multi-epitope recombinant protein vaccine against pathogenic <em>Shigella</em> species in a murine model. The multi-epitope antigen was expressed in <em>E. coli</em> and purified for immunization. BALB/c mice were subcutaneously immunized, and immune responses were assessed through serum IgG quantification (ELISA) and challenge studies. Immunized mice exhibited significantly higher antigen-specific IgG titers compared to controls (<em>p < 0.05</em>). Subsequent challenge with 10LD<sub>50</sub> of virulent <em>Shigella flexneri</em> and <em>Shigella boydii</em> demonstrated robust protection, with vaccinated mice surviving lethal infections. Our findings indicate that this in silico-designed multi-epitope vaccine elicits potent humoral immunity and confers cross-species protection against clinically relevant <em>Shigella</em> pathogens. These results support its potential as a promising candidate for further vaccine development against shigellosis.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113905"},"PeriodicalIF":1.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144663346","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 : 2025-08-01Epub Date: 2025-05-28DOI: 10.1016/j.jim.2025.113887
Juan Ignacio Marfía , Ignacio Smith , Alexandra Marisa Targovnik , Federico Alejandro Di Lello , Federico Javier Wolman , Diego Martín Flichman , María Victoria Miranda , Silvina Noemí Valdez
<div><h3>Introduction</h3><div>The global incidence of Dengue virus (DENV) infections has significantly increased in recent decades, becoming a public health emergency of global concern. Diagnostic tools for DENV infection include detection of the virus, viral RNA, or viral antigens, which are usually cumbersome or require sophisticated medical facilities and trained personnel. Serological tests are the most widely used methods to detect dengue infection due to low cost and operational simplicity. The detection of antibodies (IgM and IgG) in the blood of an infected individual is an indirect method of diagnosing DENV and IgG can be used as long-term detection. The aim of this work was to develop and validate an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-DENV IgG antibodies, and to determine its usefulness in the diagnosis and seroepidemiological survey of DENV.</div></div><div><h3>Materials and methods</h3><div>The production of the antigen was carried out using the tetravalent DENV E protein-based provided by Trebe Biotech. The recombinant baculovirus was obtained using the Bac to Bac® baculovirus expression system. The recombinant tetravalent dengue antigen was expressed in insect larvae, purified by IMAC and identified by SDS-PAGE and western blot analysis. The system used in this work has the advantage of using insect pests as biofactories. Recombinant protein production is high-yield and production times are short. Furthermore, it has no product size limit and is highly flexible to sequence changes or the emergence of new serotypes. An indirect ELISA was developed using purified tetravalent DENV antigen immobilized on polystyrene microplates; human sera samples from individuals with no history of DENV infection (<em>n</em> = 22) and human sera samples from individuals with clinical history and diagnosed of DENV infection (<em>n</em> = 23) were then incubated. Immune complexes were revealed with anti-human IgG-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. In order to establish DENV-IgG seroprevalence, the developed ELISA was applied to analyze samples from blood donor centers from different geographic regions of the country (<em>n</em> = 504) and normal human sera (<em>n</em> = 17).</div></div><div><h3>Results</h3><div>The efficiently produced recombinant dengue antigen was used for the development and validation of a high sensitivity (91.30 %) and specificity (90.91 %) indirect ELISA for the detection of anti-DENV IgG antibodies. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from normal control individuals and patients with DENV (AUC = 0.9901); the cut-off value was established at 2 SDs. In the assessment of seroprevalence levels of anti-DENV antibodies, 33 out of 504 analyzed samples (6.55 %) tested positive using our developed ELISA.</div></div><div><h3>Conclusions</h3><div>This technique is rap
{"title":"Development and validation of dengue virus envelope protein domain III IgG antibody enzyme-linked immunosorbent assay","authors":"Juan Ignacio Marfía , Ignacio Smith , Alexandra Marisa Targovnik , Federico Alejandro Di Lello , Federico Javier Wolman , Diego Martín Flichman , María Victoria Miranda , Silvina Noemí Valdez","doi":"10.1016/j.jim.2025.113887","DOIUrl":"10.1016/j.jim.2025.113887","url":null,"abstract":"<div><h3>Introduction</h3><div>The global incidence of Dengue virus (DENV) infections has significantly increased in recent decades, becoming a public health emergency of global concern. Diagnostic tools for DENV infection include detection of the virus, viral RNA, or viral antigens, which are usually cumbersome or require sophisticated medical facilities and trained personnel. Serological tests are the most widely used methods to detect dengue infection due to low cost and operational simplicity. The detection of antibodies (IgM and IgG) in the blood of an infected individual is an indirect method of diagnosing DENV and IgG can be used as long-term detection. The aim of this work was to develop and validate an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-DENV IgG antibodies, and to determine its usefulness in the diagnosis and seroepidemiological survey of DENV.</div></div><div><h3>Materials and methods</h3><div>The production of the antigen was carried out using the tetravalent DENV E protein-based provided by Trebe Biotech. The recombinant baculovirus was obtained using the Bac to Bac® baculovirus expression system. The recombinant tetravalent dengue antigen was expressed in insect larvae, purified by IMAC and identified by SDS-PAGE and western blot analysis. The system used in this work has the advantage of using insect pests as biofactories. Recombinant protein production is high-yield and production times are short. Furthermore, it has no product size limit and is highly flexible to sequence changes or the emergence of new serotypes. An indirect ELISA was developed using purified tetravalent DENV antigen immobilized on polystyrene microplates; human sera samples from individuals with no history of DENV infection (<em>n</em> = 22) and human sera samples from individuals with clinical history and diagnosed of DENV infection (<em>n</em> = 23) were then incubated. Immune complexes were revealed with anti-human IgG-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. In order to establish DENV-IgG seroprevalence, the developed ELISA was applied to analyze samples from blood donor centers from different geographic regions of the country (<em>n</em> = 504) and normal human sera (<em>n</em> = 17).</div></div><div><h3>Results</h3><div>The efficiently produced recombinant dengue antigen was used for the development and validation of a high sensitivity (91.30 %) and specificity (90.91 %) indirect ELISA for the detection of anti-DENV IgG antibodies. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from normal control individuals and patients with DENV (AUC = 0.9901); the cut-off value was established at 2 SDs. In the assessment of seroprevalence levels of anti-DENV antibodies, 33 out of 504 analyzed samples (6.55 %) tested positive using our developed ELISA.</div></div><div><h3>Conclusions</h3><div>This technique is rap","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113887"},"PeriodicalIF":1.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187132","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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