Journal of immunological methods最新文献

{"title":"Functional immunological assays","authors":"Attila Kumánovics , Medhat Askar , Amir A. Sadighi Akha","doi":"10.1016/j.jim.2025.113822","DOIUrl":"10.1016/j.jim.2025.113822","url":null,"abstract":"","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113822"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143074518","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}

{"title":"The Fas-mediated apoptosis assay: From concept to clinical application","authors":"Jack Bleesing","doi":"10.1016/j.jim.2025.113812","DOIUrl":"10.1016/j.jim.2025.113812","url":null,"abstract":"<div><div>Abnormal lymphocyte homeostasis underly several Inborn Errors of Immunity (IEoI). <em>In vitro</em> assessment of lymphocyte homeostasis is achieved by specific apoptosis assays reflective of specific homeostasis programs and pathways that are mediated through specific proteins. This review discusses those programs, pathways and proteins and describes the development and use of the <em>in vitro</em> Fas-mediated apoptosis assay, as it relates to the IEoI Autoimmune Lymphoproliferative Syndrome (ALPS) and describes other disorders of lymphocyte homeostasis in the context of other forms of <em>in vitro</em> apoptosis assessment.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113812"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143059339","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}

{"title":"Regulatory requirements for laboratory developed tests in the United States","authors":"Shannon A. Bennett,&nbsp;Chelsea M. Conn,&nbsp;Hillary E. Gill,&nbsp;Brenda K. Holmen,&nbsp;Tasha M. McDevitt,&nbsp;Corrisa L. Miliander,&nbsp;Benjamin D. Uphoff,&nbsp;Curtis A. Hanson","doi":"10.1016/j.jim.2025.113813","DOIUrl":"10.1016/j.jim.2025.113813","url":null,"abstract":"<div><div>Clinical laboratories in the United States are governed by a variety of required regulatory and optional accreditation bodies. All laboratories must comply with the federal Clinical Laboratory Improvement Amendments (CLIA) or state equivalents, while some laboratories choose additional accreditation partners. While not a regulatory body, International Standards Organization (ISO) is an internationally recognized quality system that includes best practices specific to clinical laboratories. Likewise, the Clinical Laboratory Standards Institute (CLSI) publishes guidance documents that include best practices for test validation. The Food and Drug Administration (FDA) has historically exercised enforcement discretion in regulating laboratory developed tests (LDTs), but that approach has changed with publication of the FDA's LDT final rule.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113813"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143066031","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}

{"title":"Phospho-flow cytometry assays for diagnostic use – A discussion of assay utility and assay development and validation challenges","authors":"Vijaya Knight","doi":"10.1016/j.jim.2025.113818","DOIUrl":"10.1016/j.jim.2025.113818","url":null,"abstract":"<div><div>Detection of changes in phosphorylation of cell signaling molecules using flow cytometry is termed “phosphoflow” or “phospho-flow cytometry”. Phosphoflow has wide application for basic research into the mechanics of cell signaling, for evaluating aberrant signaling in cancerous cells and tissues, for studying efficacy or off-target effects during drug and vaccine development, and for functional assessment of pathogenic variants of genes that are known to play a role in development or function of the immune system. Phosphoflow has not been widely adopted in clinical laboratories owing to the challenges with developing and validating robust assays consistent with clinical laboratory regulatory standards. This review provides a brief overview of the utility of phosphoflow and points of consideration for development and validation of phosphoflow assays for diagnostic use, with a focus on inborn errors of immunity.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113818"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039301","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}

{"title":"Functional assays for the diagnosis of chronic granulomatous disease","authors":"Debra Long Priel,&nbsp;Karen Lau,&nbsp;Danielle L. Fink,&nbsp;Douglas B. Kuhns","doi":"10.1016/j.jim.2025.113820","DOIUrl":"10.1016/j.jim.2025.113820","url":null,"abstract":"<div><div>Chronic granulomatous disease (CGD) is a rare immunodeficiency characterized by recurrent bacterial and fungal infections that are attributed to reduced production of reactive oxygen species (ROS) by a multi-component enzyme complex known as the phagocyte NADPH oxidase or NOX2. Presented in this review are descriptions of several assays that assess the production of ROS as well as assays that characterize the expression of specific proteins of NOX2.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113820"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143066027","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}

{"title":"Challenges for complement functional assays in the clinical laboratory: From test validation to clinical interpretation","authors":"Vijayalakshmi Nandakumar ,&nbsp;Karin M.P. Braun ,&nbsp;Maria Alice V. Willrich","doi":"10.1016/j.jim.2025.113824","DOIUrl":"10.1016/j.jim.2025.113824","url":null,"abstract":"<div><div>Complement functional assays are essential first-tier tests for a gamut of disorders spanning from inborn errors of the immune system which lead to recurrent severe infections, to angioedema attacks, presentation of autoimmune disease, thrombotic microangiopathies and rare kidney disorders. These assays evaluate the activity of the three complement pathways and specific complement components, which helps in differential diagnosis and monitoring disease progression. The rising use of complement inhibitors for treating complement-mediated thrombotic microangiopathies has heightened the demand for personalized treatment plans and laboratory assessment of complement blockage. However, conducting these assays is challenging due to the labile nature of complement proteins, which necessitates strict handling protocols—prompt processing, cold centrifugation, and preferable storage at −80 °C. Currently, the only FDA-approved complement functional test is the classical pathway activity assay while other tests are categorized as laboratory developed tests (LDTs). Validation of LDTs requires thorough evaluation of precision, accuracy, reference intervals, clinical reportable ranges, analytical sensitivity, and specificity. Achieving harmonization across laboratories is critical but heavily relies on the methodologies and calibrators used. This article discusses the various challenges and limitations associated with complement functional assays, highlighting the need for standardization and improved practices in clinical laboratories.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113824"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143365088","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}

{"title":"Theoretical and practical considerations for validating antigen-specific B cell ImmunoSpot assays","authors":"Paul V. Lehmann,&nbsp;Alexey Y. Karulin,&nbsp;Noémi Becza,&nbsp;Lingling Yao,&nbsp;Zhigang Liu,&nbsp;Jack Chepke,&nbsp;Andrea Maul-Pavicic,&nbsp;Carla Wolf,&nbsp;Sebastian Köppert,&nbsp;Alexis V. Valente,&nbsp;Anton V. Gorbachev,&nbsp;Magdalena Tary-Lehmann,&nbsp;Greg A. Kirchenbaum","doi":"10.1016/j.jim.2025.113817","DOIUrl":"10.1016/j.jim.2025.113817","url":null,"abstract":"<div><div>Owing to their ability to reliably detect even very rare antigen-specific B cells in cellular isolates such as peripheral blood mononuclear cells (PBMC), and doing so robustly in a high throughput-compatible manner, B cell ELISPOT/FluoroSpot (collectively “B cell ImmunoSpot”) tests have become increasingly attractive for immune monitoring in regulated settings. Presently, there are no guidelines for the qualification and validation of B cell ImmunoSpot assay results. Here, we propose such guidelines, building on the experience acquired from T cell ImmunoSpot testing in an environment adhering to the requirements of regulatory bodies yet taking the unique features of B cell assays into account. A streamlined protocol is proposed that permits the performance of all tests needed for the formal validation of an antigen-specific B cell ImmunoSpot assay in only three experiments, utilizing 2.2 × 10⁷ PBMC per donor. Subsequently, utilizing only 1–2 × 10⁶ PBMC per sample (obtainable from 1 to 2 mL of blood), a validated multiplexed assay enables accurate quantification of the frequency of antigen-specific memory B cell-derived blasts secreting IgM, IgG, IgA or IgE antibodies. Collectively, such multiplexed B cell ImmunoSpot assays offer immense value for B cell immune monitoring programs due to their ease of implementation, scalability, applicability to essentially any antigenic system, economy of PBMC utilization, and last but not least, the high content information gained.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113817"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Complete primer set for amplification and expression of full-length recombinant human monoclonal antibodies from single human B cells","authors":"Sachin Kushwaha ,&nbsp;Varsha Jawahar ,&nbsp;Ajay Kumar ,&nbsp;Lauren Griffin ,&nbsp;Thomas L. Rothstein ,&nbsp;Devinder Sehgal ,&nbsp;Naeem Khan","doi":"10.1016/j.jim.2025.113823","DOIUrl":"10.1016/j.jim.2025.113823","url":null,"abstract":"<div><div>Human monoclonal antibodies (mAbs) are an important segment in precision therapeutics. Various methodologies are available for generating them. Recombinant human mAbs expression from sorted single B cells is preferred for its rapid expression using mammalian vectors while maintaining in vivo immunoglobulin (Ig) pairing. The success rate of generating recombinant mAbs from single sorted human B cells directly relies on Ig heavy (IgH) and light (IgL) gene coverage of the PCR primers. Existing primer sets fail to cover all functional human Ig gene rearrangements, exhibit high degeneracy leading to non-specific amplifications and mutations arising from primer mismatch/degeneracy, and require high amplification cycles. Some existing primer sets have high coverage but are not designed for expression as recombinant mAbs. Here, we have designed a primer set to amplify all functional V(<em>D</em>)J transcripts in human B cell repertoire using a nested RT-PCR approach. The resultant amplicons can be cloned into mammalian vectors for expression of recombinant mAb. Non-specific amplifications were minimized using isotype-specific primers for cDNA synthesis and limiting primer degeneracy. We validated the designed primers on single sorted B cells, bulk sorted B cells and peripheral blood mononuclear cells. We were successfully able to amplify paired heavy and light chain transcripts in 38.46 % (80/208) from naive, memory and B1 B cell subsets sorted as single B cells. Paired Ig transcripts from five single B cells were cloned into expression vectors and purified from mammalian cells as recombinant mAbs. Thus, our new primer set offers significant advantages over existing primers as it allows amplification of all functional V(<em>D</em>)J rearrangements, facilitating rapid generation of antigen-specific recombinant antibodies from diverse human B cell repertoires following vaccinations and infections previously inaccessible due to primer limitations.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113823"},"PeriodicalIF":1.6,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143074516","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}

{"title":"Challenges for complement functional assays in the clinical laboratory: From test validation to clinical interpretation","authors":"Vijayalakshmi Nandakumar ,&nbsp;Karin M.P. Braun ,&nbsp;Maria Alice V. Willrich","doi":"10.1016/j.jim.2025.113814","DOIUrl":"10.1016/j.jim.2025.113814","url":null,"abstract":"<div><div>Complement functional assays are essential first-tier tests for a gamut of disorders spanning from inborn errors of the immune system which lead to recurrent severe infections, to angioedema attacks, presentation of autoimmune disease, thrombotic microangiopathies and rare kidney disorders. These assays evaluate the activity of the three complement pathways and specific complement components, which helps in differential diagnosis and monitoring disease progression. The rising use of complement inhibitors for treating complement-mediated thrombotic microangiopathies has heightened the demand for personalized treatment plans and laboratory assessment of complement blockage. However, conducting these assays is challenging due to the labile nature of complement proteins, which necessitates strict handling protocols—prompt processing, cold centrifugation, and preferable storage at −80 °C. Currently, the only FDA-approved complement functional test is the classical pathway activity assay while other tests are categorized as laboratory developed tests (LDTs). Validation of LDTs requires thorough evaluation of precision, accuracy, reference intervals, clinical reportable ranges, analytical sensitivity, and specificity. Achieving harmonization across laboratories is critical but heavily relies on the methodologies and calibrators used. This article discusses the various challenges and limitations associated with complement functional assays, highlighting the need for standardization and improved practices in clinical laboratories.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113814"},"PeriodicalIF":1.6,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039050","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}

{"title":"Peptide fibrils as a vaccine: Proof of concept","authors":"Yana Zabrodskaya ,&nbsp;Konstantin Sivak ,&nbsp;Maria Sergeeva ,&nbsp;Andrey Aleksandrov ,&nbsp;Elena Kalinina ,&nbsp;Aleksandr Taraskin ,&nbsp;Mikhail Eropkin ,&nbsp;Elena Eropkina ,&nbsp;Vladimir Egorov","doi":"10.1016/j.jim.2025.113811","DOIUrl":"10.1016/j.jim.2025.113811","url":null,"abstract":"<div><h3>Background</h3><div>Rapid vaccine platforms development is crucial for responding to epidemics and pandemics of emerging infectious diseases, such as Ebola. This study explores the potential of peptide vaccines that self-organize into amyloid-like fibrils, aiming to enhance immunogenicity while considering safety and cross-reactivity.</div></div><div><h3>Methods</h3><div>We synthesized two peptides, G33 and G31, corresponding to a segment of the Ebola virus GP2 protein, with G33 known to form amyloid-like fibrils. Their toxicity was assessed in vitro using an MTT assay on MDCK and A549 cell cultures. For in vivo studies, balb/c mice were immunized with these peptides. Immunogenicity was gauged by ELISA for specific antibodies against the recombinant eGP protein. Hematological parameters were determined, and histopathological changes in organs were documented post-euthanasia.</div></div><div><h3>Results</h3><div>Both peptides exhibited no cytotoxicity up to 500 μg/mL. G33-immunized mice developed higher antibody titers than those receiving G31 or control, without significant alterations in hematological profiles. However, histological analysis showed periportal infiltration and lymphoid-macrophage clusters in the liver and kidneys, suggesting immune response activation. The homology between the G33 peptide and mammalian proteins poses risks of cross-reactivity.</div></div><div><h3>Conclusion</h3><div>The amyloid-like fibrils formed by the G33 peptide elicited a stronger immune response without significant hematological changes, underlining the feasibility of fibril-based peptide vaccines. However, the potential for autoimmune responses due to molecular mimicry warrants further investigation before clinical applications can be considered.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113811"},"PeriodicalIF":1.6,"publicationDate":"2025-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006747","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}