Pub Date : 2025-11-01DOI: 10.1016/j.jim.2025.113946
Alexis S. Dadelahi , Lauren Zuromski , Natalia Sigal , Elizabeth Enrico , Patricia Davis , Douglas W. Sborov , David P. Ng
{"title":"High throughput screening and assessment of a bioinformatics pipeline for novel marker discovery in relapsing/refractory multiple myeloma","authors":"Alexis S. Dadelahi , Lauren Zuromski , Natalia Sigal , Elizabeth Enrico , Patricia Davis , Douglas W. Sborov , David P. Ng","doi":"10.1016/j.jim.2025.113946","DOIUrl":"10.1016/j.jim.2025.113946","url":null,"abstract":"","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113946"},"PeriodicalIF":1.6,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145415807","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}
Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9), thereby triggering a diverse range of immunomodulatory responses. Although the immune-stimulating properties of CpG-ODNs have been extensively studied, their potential application as apoptosis-inducing tools remains underexplored, particularly for probiotic-derived sequences. In this study, we established a method for inducing extrinsic apoptosis in mouse splenocytes using MsST, a CpG-ODN identified from the lacZ gene of Streptococcus thermophilus ATCC19258. In genomic DNA fragmentation assays, MsST induced moderate but significant internucleosomal DNA fragmentation. Flow cytometric analysis using annexin V/7-aminoactinomycin D staining confirmed a time-dependent increase in the apoptotic cell population following MsST treatment. Furthermore, Western blot analysis revealed elevated levels of cleaved caspase-8, thus verifying activation of the extrinsic apoptosis pathway. Compared to a representative CpG-ODN and the apoptosis inducer actinomycin D, MsST exhibited distinct apoptosis-inducing characteristics. These findings highlight MsST as a potential tool for controlled apoptosis induction, providing a methodological basis for further exploration of CpG-ODNs in immunological and therapeutic applications, including cancer immunotherapy and the treatment of autoimmune diseases and inflammatory disorders.
{"title":"CpG-ODN from Streptococcus thermophilus as a tool for inducing extrinsic apoptosis via caspase-8 activation in mouse splenocytes","authors":"Md. Rayhan Chowdhury , Takuma Okajima , Aito Murakami , Ariful Islam , Takeshi Shimosato","doi":"10.1016/j.jim.2025.113999","DOIUrl":"10.1016/j.jim.2025.113999","url":null,"abstract":"<div><div>Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9), thereby triggering a diverse range of immunomodulatory responses. Although the immune-stimulating properties of CpG-ODNs have been extensively studied, their potential application as apoptosis-inducing tools remains underexplored, particularly for probiotic-derived sequences. In this study, we established a method for inducing extrinsic apoptosis in mouse splenocytes using MsST, a CpG-ODN identified from the <em>lacZ</em> gene of <em>Streptococcus thermophilus</em> ATCC19258. In genomic DNA fragmentation assays, MsST induced moderate but significant internucleosomal DNA fragmentation. Flow cytometric analysis using annexin V/7-aminoactinomycin D staining confirmed a time-dependent increase in the apoptotic cell population following MsST treatment. Furthermore, Western blot analysis revealed elevated levels of cleaved caspase-8, thus verifying activation of the extrinsic apoptosis pathway. Compared to a representative CpG-ODN and the apoptosis inducer actinomycin D, MsST exhibited distinct apoptosis-inducing characteristics. These findings highlight MsST as a potential tool for controlled apoptosis induction, providing a methodological basis for further exploration of CpG-ODNs in immunological and therapeutic applications, including cancer immunotherapy and the treatment of autoimmune diseases and inflammatory disorders.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 113999"},"PeriodicalIF":1.6,"publicationDate":"2025-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426812","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":"2025-10-29","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}
Chimeric Antigen Receptor T-cells, commonly known as CAR-T cells, have represented a major scientific breakthrough in cancer cell therapy revolutionizing the treatment of haematological disorders. Pharmacokinetic studies highlighted the importance of peripheral blood CAR-T cell monitoring to predict therapeutic outcomes and manage side effects. This study aimed to establish and evaluate on three different flow cytometers (DxFLEX, Navios and Navios EX) the performances of a dual-platform protocol for CAR-T quantitation method using flow cytometry in a context of hospital routine use. Fresh whole blood was stained using a biotinylated CD19 or BCMA recombinant protein and a PE-coupled anti-biotin antibody. CAR-T cells were identified among CD45+ CD3+ cells after doublets elimination, using a control tube to define CAR-T positive expression threshold among T lymphocytes. Results were expressed as percentages of CAR T cells among CD3+ T lymphocytes. Optilyse C and Versalyse red blood cell lysis reagents (Beckman Coulter) gave comparable results in terms of CAR T cell percentage among T lymphocytes as well as using 0.5 μL CAR detection reagent compared to the 2 μL recommended by the manufacturer. Using this protocol, we did not observe any cross-reactivity between CD19 and BCMA CAR detection reagents or decreased signal even up to >3000 circulating CAR-T cells/μL. The coefficient of variations for repeatability and intermediate precision were systematically below 10 % whatever the flow cytometer, the type of CAR reagent detection used and the proportion of circulating CAR-T cells. All three flow cytometers returned correlated and comparable results. Finally, concerning whole blood sample and stained cell storage duration, we observed a significant decreased of CAR-T percentages when staining was delayed for more than one day after sampling, and after three days of stained cells storage at 4 °C. In conclusion, we propose a dual-platform protocol for CAR-T cell enumeration which demonstrated consistent and reliable performances for BCMA and CD19 CAR-T cell quantification.
{"title":"Preliminary evaluation of a whole blood dual-platform flow cytometry protocol for CAR-T cell quantitation: Toward accreditation and clinical routine application","authors":"Rémi Pescarmona , Guillaume Monneret , Eleonore Micoud , Christophe Malcus , Emmanuel Bachy , Pierre Sesques , Lionel Karlin , Fabienne Venet , Morgane Gossez","doi":"10.1016/j.jim.2025.113986","DOIUrl":"10.1016/j.jim.2025.113986","url":null,"abstract":"<div><div>Chimeric Antigen Receptor T-cells, commonly known as CAR-T cells, have represented a major scientific breakthrough in cancer cell therapy revolutionizing the treatment of haematological disorders. Pharmacokinetic studies highlighted the importance of peripheral blood CAR-T cell monitoring to predict therapeutic outcomes and manage side effects. This study aimed to establish and evaluate on three different flow cytometers (DxFLEX, Navios and Navios EX) the performances of a dual-platform protocol for CAR-T quantitation method using flow cytometry in a context of hospital routine use. Fresh whole blood was stained using a biotinylated CD19 or BCMA recombinant protein and a PE-coupled anti-biotin antibody. CAR-T cells were identified among CD45+ CD3+ cells after doublets elimination, using a control tube to define CAR-T positive expression threshold among T lymphocytes. Results were expressed as percentages of CAR T cells among CD3+ T lymphocytes. Optilyse C and Versalyse red blood cell lysis reagents (Beckman Coulter) gave comparable results in terms of CAR T cell percentage among T lymphocytes as well as using 0.5 μL CAR detection reagent compared to the 2 μL recommended by the manufacturer. Using this protocol, we did not observe any cross-reactivity between CD19 and BCMA CAR detection reagents or decreased signal even up to >3000 circulating CAR-T cells/μL. The coefficient of variations for repeatability and intermediate precision were systematically below 10 % whatever the flow cytometer, the type of CAR reagent detection used and the proportion of circulating CAR-T cells. All three flow cytometers returned correlated and comparable results. Finally, concerning whole blood sample and stained cell storage duration, we observed a significant decreased of CAR-T percentages when staining was delayed for more than one day after sampling, and after three days of stained cells storage at 4 °C. In conclusion, we propose a dual-platform protocol for CAR-T cell enumeration which demonstrated consistent and reliable performances for BCMA and CD19 CAR-T cell quantification.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"545 ","pages":"Article 113986"},"PeriodicalIF":1.6,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145368141","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}
Ionizing radiation induced DNA DSBs can cause large deletions in the human genome. This property may be utilized for gene disruption to create knockout mutants. The X-linked hypoxanthine–guanine phosphoribosyl transferase (HPRT) gene is commonly used as a reporter to evaluate the frequency and molecular nature of somatic mutations in the human population. Mutations at the HPRT locus are measured through a T lymphocyte cloning assay. This assay relies on lymphoblast feeder cells that support T lymphocyte proliferation via cell-to-cell contact and by helping in the expression of functional interleukin-2 receptors. However, the feeder cells must be HPRT-depleted to prevent feeder cell DNA from serving as substrate during molecular analysis of T lymphocyte mutations. To the best of our knowledge, the human lymphoblastoid TK6 cell line available in Indian cell repositories has an intact HPRT gene (HPRT+). In this study, the parent TK6 cell line was subjected to the clastogenic effects of a high dose gamma radiation to isolate TK6 cells with a total deletion of the HPRT locus. The HPRT-depleted cell line (TK6LLrrL) was characterized by mPCR for the loss of exons and the extent of the deletion by flanking STS markers analysis. Further, the feeder cell function of TK6LLrrL was evaluated by estimating the cloning efficiency of human peripheral blood lymphocytes. Thus, the study demonstrates the potential of low LET gamma radiation to generate gene knockout cell line. The method described here can be applied to various cell types to produce functionally null cell lines for gene-specific studies.
{"title":"Isolation of HPRT gene depleted human TK6 lymphoblastoid cell line through gamma irradiation","authors":"P.R. Vivek Kumar , Anila Gopinathan , Deepak Sharma","doi":"10.1016/j.jim.2025.113985","DOIUrl":"10.1016/j.jim.2025.113985","url":null,"abstract":"<div><div>Ionizing radiation induced DNA DSBs can cause large deletions in the human genome. This property may be utilized for gene disruption to create knockout mutants. The X-linked hypoxanthine–guanine phosphoribosyl transferase (<em>HPRT</em>) gene is commonly used as a reporter to evaluate the frequency and molecular nature of somatic mutations in the human population. Mutations at the <em>HPRT</em> locus are measured through a T lymphocyte cloning assay. This assay relies on lymphoblast feeder cells that support T lymphocyte proliferation via cell-to-cell contact and by helping in the expression of functional interleukin-2 receptors. However, the feeder cells must be <em>HPRT</em>-depleted to prevent feeder cell DNA from serving as substrate during molecular analysis of T lymphocyte mutations. To the best of our knowledge, the human lymphoblastoid TK6 cell line available in Indian cell repositories has an intact <em>HPRT</em> gene (<em>HPRT</em>+). In this study, the parent TK6 cell line was subjected to the clastogenic effects of a high dose gamma radiation to isolate TK6 cells with a total deletion of the <em>HPRT</em> locus. The <em>HPRT</em>-depleted cell line (TK6<sub>LLrrL</sub>) was characterized by mPCR for the loss of exons and the extent of the deletion by flanking STS markers analysis. Further, the feeder cell function of TK6<sub>LLrrL</sub> was evaluated by estimating the cloning efficiency of human peripheral blood lymphocytes. Thus, the study demonstrates the potential of low LET gamma radiation to generate gene knockout cell line. The method described here can be applied to various cell types to produce functionally null cell lines for gene-specific studies.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113985"},"PeriodicalIF":1.6,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145280376","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 : 2025-10-08DOI: 10.1016/j.jim.2025.113982
Nicholas J. Jones, Kim A. Appler, Jodie A. Jarvis, Alexander C. Keyel, April D. Davis
Rabies serology is most importantly done to evaluate immune response following vaccine administration in patients who have been exposed to rabies or are highly likely to be. When detecting antibodies after vaccination it is best to target rabies neutralizing antibodies specifically. Clinical laboratories performing this testing are required to uphold a strict set of standards laid out by federal and state guidelines. Sample integrity throughout the process of testing, from collection to results is of the utmost importance. Samples may experience a wide range of temperature variation depending on how each sample is shipped or handled by the submitter. In order to uphold quality clinical testing it is important to study the stability of rabies neutralizing antibodies across a wide range of temperatures.
{"title":"Temperature stability of rabies neutralizing antibodies","authors":"Nicholas J. Jones, Kim A. Appler, Jodie A. Jarvis, Alexander C. Keyel, April D. Davis","doi":"10.1016/j.jim.2025.113982","DOIUrl":"10.1016/j.jim.2025.113982","url":null,"abstract":"<div><div>Rabies serology is most importantly done to evaluate immune response following vaccine administration in patients who have been exposed to rabies or are highly likely to be. When detecting antibodies after vaccination it is best to target rabies neutralizing antibodies specifically. Clinical laboratories performing this testing are required to uphold a strict set of standards laid out by federal and state guidelines. Sample integrity throughout the process of testing, from collection to results is of the utmost importance. Samples may experience a wide range of temperature variation depending on how each sample is shipped or handled by the submitter. In order to uphold quality clinical testing it is important to study the stability of rabies neutralizing antibodies across a wide range of temperatures.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113982"},"PeriodicalIF":1.6,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145274753","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 : 2025-10-02DOI: 10.1016/j.jim.2025.113983
Boxiang Tao , Meng Wang , Yanfei Yang , Na Zhao , Meiling Tan , Xiaolei Xun , Chunxiang Zeng , Fan Wang
Anti-drug antibody (ADA) is a critical indicator of the immunogenicity of biotherapeutics. To investigate the immunogenicity of tislelizumab, a humanized anti-PD-1 (programmed cell death 1) antibody, 3815 patients were analyzed for ADA incidence. The incidence of ADA is reported for the first time as 19.2 %. For any immunogenicity study, appropriate cut points are essential to accurately distinguish true ADA-positive samples from varying backgrounds across populations. Apart from the standardized false positive rate (FPR) method, statistical methods may be more efficient and powerful in evaluating the appropriateness of cut points. However, a detailed protocol for this approach is not yet in consensus. Therefore, by utilizing extensive data from tislelizumab clinical trials, this study explored the feasibility of assessing the suitability of validation cut points (VCPs) using statistical methods. ADA response datasets from 21 clinical tumor populations were evaluated for distribution differences in comparison to the validation population using statistical methods. Fourteen datasets exhibited significant differences from the validation dataset in both medians and variances, with FPRs exceeding the 2–11 % range. Seven datasets only differed significantly in medians, with five having out-of-range FPRs and their positive rates narrowly affected by cut point alteration. The results showed that statistical methods can effectively assess the suitability of VCPs: in-study cut points are recommended for datasets with significantly different median and variance, while VCPs may be appropriate when only medians differ.
{"title":"Immunogenicity of tislelizumab: clinical summary of ADA incidence and feasibility of assessing the suitability of validation cut points using statistical methods","authors":"Boxiang Tao , Meng Wang , Yanfei Yang , Na Zhao , Meiling Tan , Xiaolei Xun , Chunxiang Zeng , Fan Wang","doi":"10.1016/j.jim.2025.113983","DOIUrl":"10.1016/j.jim.2025.113983","url":null,"abstract":"<div><div>Anti-drug antibody (ADA) is a critical indicator of the immunogenicity of biotherapeutics. To investigate the immunogenicity of tislelizumab, a humanized anti-PD-1 (programmed cell death 1) antibody, 3815 patients were analyzed for ADA incidence. The incidence of ADA is reported for the first time as 19.2 %. For any immunogenicity study, appropriate cut points are essential to accurately distinguish true ADA-positive samples from varying backgrounds across populations. Apart from the standardized false positive rate (FPR) method, statistical methods may be more efficient and powerful in evaluating the appropriateness of cut points. However, a detailed protocol for this approach is not yet in consensus. Therefore, by utilizing extensive data from tislelizumab clinical trials, this study explored the feasibility of assessing the suitability of validation cut points (VCPs) using statistical methods. ADA response datasets from 21 clinical tumor populations were evaluated for distribution differences in comparison to the validation population using statistical methods. Fourteen datasets exhibited significant differences from the validation dataset in both medians and variances, with FPRs exceeding the 2–11 % range. Seven datasets only differed significantly in medians, with five having out-of-range FPRs and their positive rates narrowly affected by cut point alteration. The results showed that statistical methods can effectively assess the suitability of VCPs: in-study cut points are recommended for datasets with significantly different median and variance, while VCPs may be appropriate when only medians differ.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113983"},"PeriodicalIF":1.6,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145228251","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 : 2025-10-02DOI: 10.1016/j.jim.2025.113984
Mengying Lun , Yumei Chen , Hongliang Liu , Yanhua Qi , Aiping Wang , Jingming Zhou
The production of carbapenemase is the primary mechanism of bacterial resistance to carbapenem antibiotics. This resistance seriously compromises the efficacy of carbapenem antibiotics in treating infections and poses a significant challenge to clinical anti-infective therapy. Oxacillinase-48 (OXA-48) is a class D carbapenemase, whose genes are usually located on transferable elements and can spread between different strains and genera. Therefore, from the perspective of infection control, effective monitoring and prevention of OXA-48 are imperative. In this study, CdSe/ZnS quantum dots (QDs) were coupled with an anti-OXA-48 monoclonal antibody (mAb) as a fluorescent signal probe to establish a quantum dot-based fluorescent immunochromatographic strip. The test strip contains a Test line (T-line) coated with an anti-OXA-48 monoclonal antibody and a Control line (C-line) coated with Staphylococcus protein A (SPA). Based on a double-antibody sandwich detection mode, it can complete highly sensitive detection of OXA-48 within 10 min. The visual detection limit of this test strip for OXA-48 recombinant protein was 3.13 ng/mL, and there was no cross-reaction with other common carbapenemases (IMP-1, NDM-1, KPC-2, VIM-2, OXA-23). It can be positioned as a rapid and reliable OXA-48 detection tool, providing a novel method for the rapid identification of OXA-48-producing resistant bacterial strains in clinical practice.
{"title":"Fluorescence immunochromatographic assay based on CdSe/ZnS quantum dots for the detection of OXA-48","authors":"Mengying Lun , Yumei Chen , Hongliang Liu , Yanhua Qi , Aiping Wang , Jingming Zhou","doi":"10.1016/j.jim.2025.113984","DOIUrl":"10.1016/j.jim.2025.113984","url":null,"abstract":"<div><div>The production of carbapenemase is the primary mechanism of bacterial resistance to carbapenem antibiotics. This resistance seriously compromises the efficacy of carbapenem antibiotics in treating infections and poses a significant challenge to clinical anti-infective therapy. Oxacillinase-48 (OXA-48) is a class D carbapenemase, whose genes are usually located on transferable elements and can spread between different strains and genera. Therefore, from the perspective of infection control, effective monitoring and prevention of OXA-48 are imperative. In this study, CdSe/ZnS quantum dots (QDs) were coupled with an anti-OXA-48 monoclonal antibody (mAb) as a fluorescent signal probe to establish a quantum dot-based fluorescent immunochromatographic strip. The test strip contains a Test line (T-line) coated with an anti-OXA-48 monoclonal antibody and a Control line (C-line) coated with Staphylococcus protein A (SPA). Based on a double-antibody sandwich detection mode, it can complete highly sensitive detection of OXA-48 within 10 min. The visual detection limit of this test strip for OXA-48 recombinant protein was 3.13 ng/mL, and there was no cross-reaction with other common carbapenemases (IMP-1, NDM-1, KPC-2, VIM-2, OXA-23). It can be positioned as a rapid and reliable OXA-48 detection tool, providing a novel method for the rapid identification of OXA-48-producing resistant bacterial strains in clinical practice.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113984"},"PeriodicalIF":1.6,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145228254","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 : 2025-09-30DOI: 10.1016/j.jim.2025.113981
Chiara Suffritti , Roberta Gualtierotti , Andrea Zanichelli , Matteo Vidali , Massimo Cugno
High-molecular-weight kininogen (HK) is a plasma protein cleaved during the activation of contact system by the enzyme kallikrein, releasing the peptide bradykinin, potent angioedema mediator. Plasma measurement of cleaved HK allows evaluating in vivo contact system activation but, when its main natural inhibitor (C1-inhibitor) is deficient, conflicting results have been reported, possibly due to in vitro activation.
We collected blood samples from 18 patients with C1-inhibitor deficiency and 7 healthy controls using five different anticoagulant mixtures to find the one that could completely block in vitro activation of contact system, so that a snapshot of the in vivo scenario could be obtained. The ability to prevent contact system activation in vitro was evaluated in plasma samples measuring levels of cleaved HK by SDS-PAGE with precast gels and immunoblotting. Correlations between cleaved HK and functional C1-inhibitor were evaluated as well as assay reproducibility.
In healthy subjects, no differences in cleaved HK were found for the different anticoagulants. In patients with C1-inhibitor deficiency, we observed higher levels of cleaved HK in sodium citrate samples (median 60.5 %, IQR 51.9–61.6 %) than in samples collected in a mixture of protease inhibitors (median 49.2 %, IQR 48.1–52.0 %) (P = 0.001). Only in the latter, cleaved HK levels correlated with circulating functional C1-inhibitor levels (P = 0.005). Using precast gels, intra- and inter-assay coefficients of variation were < 5 %.
In conclusion, for measuring cleaved HK in plasma from patients with C1-inhibitor deficiency, the use of anticoagulant with protease inhibitors and precast gels allows reliable evaluation of in vivo contact system activation.
{"title":"Measurement of cleaved high-molecular-weight kininogen in patients with hereditary angioedema due to C1-inhibitor deficiency: preanalytical and analytical optimization","authors":"Chiara Suffritti , Roberta Gualtierotti , Andrea Zanichelli , Matteo Vidali , Massimo Cugno","doi":"10.1016/j.jim.2025.113981","DOIUrl":"10.1016/j.jim.2025.113981","url":null,"abstract":"<div><div>High-molecular-weight kininogen (HK) is a plasma protein cleaved during the activation of contact system by the enzyme kallikrein, releasing the peptide bradykinin, potent angioedema mediator. Plasma measurement of cleaved HK allows evaluating <em>in vivo</em> contact system activation but, when its main natural inhibitor (C1-inhibitor) is deficient, conflicting results have been reported, possibly due to <em>in vitro</em> activation.</div><div>We collected blood samples from 18 patients with C1-inhibitor deficiency and 7 healthy controls using five different anticoagulant mixtures to find the one that could completely block <em>in vitro</em> activation of contact system, so that a snapshot of the <em>in vivo</em> scenario could be obtained. The ability to prevent contact system activation <em>in vitro</em> was evaluated in plasma samples measuring levels of cleaved HK by SDS-PAGE with precast gels and immunoblotting. Correlations between cleaved HK and functional C1-inhibitor were evaluated as well as assay reproducibility.</div><div>In healthy subjects, no differences in cleaved HK were found for the different anticoagulants. In patients with C1-inhibitor deficiency, we observed higher levels of cleaved HK in sodium citrate samples (median 60.5 %, IQR 51.9–61.6 %) than in samples collected in a mixture of protease inhibitors (median 49.2 %, IQR 48.1–52.0 %) (<em>P</em> = 0.001). Only in the latter, cleaved HK levels correlated with circulating functional C1-inhibitor levels (<em>P</em> = 0.005). Using precast gels, intra- and inter-assay coefficients of variation were < 5 %.</div><div>In conclusion, for measuring cleaved HK in plasma from patients with C1-inhibitor deficiency, the use of anticoagulant with protease inhibitors and precast gels allows reliable evaluation of <em>in vivo</em> contact system activation.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113981"},"PeriodicalIF":1.6,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145212881","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 : 2025-09-18DOI: 10.1016/j.jim.2025.113980
Ti Lu, Md Shafiullah Parvej, Sean K. Whittier, Suhrid Maiti, Zackary K. Dietz, Debaki R. Howlader, Mst Nusrat Zahan, Satabdi Biswas, William D. Picking, Wendy L. Picking
In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. BLI-ISA provides rapid relative measurements of antigen-specific antibody levels (in nm binding shift), yielding results consistent with ELISA endpoint titers (expressed in ELISA Units), thereby enabling reliable comparison across treatment groups within a fraction of the time required for conventional ELISA.
{"title":"A high-throughput multi-species platform using Biolayer Interferometry Immunosorbent Assay (BLI-ISA) as an alternative to indirect ELISA for vaccine development","authors":"Ti Lu, Md Shafiullah Parvej, Sean K. Whittier, Suhrid Maiti, Zackary K. Dietz, Debaki R. Howlader, Mst Nusrat Zahan, Satabdi Biswas, William D. Picking, Wendy L. Picking","doi":"10.1016/j.jim.2025.113980","DOIUrl":"10.1016/j.jim.2025.113980","url":null,"abstract":"<div><div>In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. BLI-ISA provides rapid relative measurements of antigen-specific antibody levels (in nm binding shift), yielding results consistent with ELISA endpoint titers (expressed in ELISA Units), thereby enabling reliable comparison across treatment groups within a fraction of the time required for conventional ELISA.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"544 ","pages":"Article 113980"},"PeriodicalIF":1.6,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102864","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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