Journal of immunological methods最新文献

{"title":"Overcoming cross-reactivity of antibodies against human lactate dehydrogenase.","authors":"Akira Kondo, Ayumu Nakamura","doi":"10.1016/j.jim.2025.113821","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113821","url":null,"abstract":"<p><p>Lactate dehydrogenase subunit A (LD-A) plays an important role in cancer regulation and therapy. We attempted to develop an enzyme-linked immune-solvent assay (ELISA) for LD-A in human serum. However, commercial antibodies against LD-A exhibited cross-reactivity with an unknown protein. The unknown protein was purified and characterized by protein sequencing and Western blotting. In addition, we attempted to prepare a specific antibody for the ELISA using partially synthesized peptides of LD-A as immunogens. The epitope position in LD-A was carefully selected based on bioinformatics analysis. Peptide sequencer elucidated a ten amino acid sequence of the purified protein at the N-terminal. A BLAST search revealed that this sequence perfectly matched that at the N-terminus of the IgG heavy chain (H-chain). Furthermore, we demonstrated that twelve commercially available antibodies targeting LD-A or LD-B (subunit B) primarily cross-reacted with IgG or its H-chain, with only one specific antibody for each subunit. As the specific antibody against LD-A is no longer commercially accessible, we successfully produced two kinds of specific antibodies using partially synthesized LD-A peptides as immunogens. In conclusion, we have successfully produced specific antibodies against LD-A. Moreover, our findings underscore the utility of bioinformatics tools for determining the optimal positions of immunizing peptides.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113821"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143123061","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 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":" ","pages":"113822"},"PeriodicalIF":1.6,"publicationDate":"2025-01-31","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":"Complete primer set for amplification and expression of full-length recombinant human monoclonal antibodies from single human B cells.","authors":"Sachin Kushwaha, Varsha Jawahar, Ajay Kumar, Lauren Griffin, Thomas L Rothstein, Devinder Sehgal, Naeem Khan","doi":"10.1016/j.jim.2025.113823","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113823","url":null,"abstract":"<p><p>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(D)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(D)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.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113823"},"PeriodicalIF":1.6,"publicationDate":"2025-01-30","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":"Functional assays for the diagnosis of chronic granulomatous disease.","authors":"Debra Long Priel, Karen Lau, Danielle L Fink, Douglas B Kuhns","doi":"10.1016/j.jim.2025.113820","DOIUrl":"10.1016/j.jim.2025.113820","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113820"},"PeriodicalIF":1.6,"publicationDate":"2025-01-28","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":"Regulatory requirements for laboratory developed tests in the United States.","authors":"Shannon A Bennett, Chelsea M Conn, Hillary E Gill, Brenda K Holmen, Tasha M McDevitt, Corrisa L Miliander, Benjamin D Uphoff, Curtis A Hanson","doi":"10.1016/j.jim.2025.113813","DOIUrl":"10.1016/j.jim.2025.113813","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113813"},"PeriodicalIF":1.6,"publicationDate":"2025-01-27","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":"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":"<p><p>Abnormal lymphocyte homeostasis underly several Inborn Errors of Immunity (IEoI). In vitro 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 in vitro 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 in vitro apoptosis assessment.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113812"},"PeriodicalIF":1.6,"publicationDate":"2025-01-26","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":"Theoretical and practical considerations for validating antigen-specific B cell ImmunoSpot assays.","authors":"Paul V Lehmann, Alexey Y Karulin, Noémi Becza, Lingling Yao, Zhigang Liu, Jack Chepke, Andrea Maul-Pavicic, Carla Wolf, Sebastian Köppert, Alexis V Valente, Anton V Gorbachev, Magdalena Tary-Lehmann, Greg A Kirchenbaum","doi":"10.1016/j.jim.2025.113817","DOIUrl":"10.1016/j.jim.2025.113817","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113817"},"PeriodicalIF":1.6,"publicationDate":"2025-01-24","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":"","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":"Basophil activation test; User's manual.","authors":"Soren Ulrik Sonder, Matthew Plassmeyer, Nikhila Schroeder, Steven Peyton, Mikell Paige, Michael Girgis, Hamed Safi, Oral Alpan","doi":"10.1016/j.jim.2025.113815","DOIUrl":"10.1016/j.jim.2025.113815","url":null,"abstract":"<p><p>Immediate allergic responses, orchestrated by basophils and mast cells, are pivotal in severe allergic reactions. The flow cytometry-based Basophil Activation Test (BAT) is a clinically important assay for testing allergic reactions using CD63 and CD203c as endpoints. The test measures the concentration dependent response to the allergens providing a functional readout of the patients' allergies. BAT is presently in clinical use within the Unites States as well as several other countries for the diagnosis and monitoring of allergies, most commonly against food allergens. This article details assay validation, both analytical and clinical with reference to existing regulations/recommendations through CAP, CLIA, NYS and CLSI on issues including accuracy, precision, linearity, reportable range, reference range, analytical sensitivity & specificity, pre-analytical considerations, the utility of the assay in diagnosis or monitoring and interpretation of the results; and the assay's limitations. The BAT plays a crucial role in assessing patient suitability for food challenges and therapies such as oral immunotherapy, sublingual immunotherapy or omalizumab and can aid in predicting treatment outcomes. We further review the current research on advancing the test, focused on improvements in its clinical utility. Continuous efforts are warranted for enhanced regulatory oversight and comprehensive clinical validation, ensuring BAT's seamless integration into diverse clinical settings.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113815"},"PeriodicalIF":1.6,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039084","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":"Clinical utility of the lymphocyte proliferation assay, an in vitro functional readout of the adaptive immune response.","authors":"Eszter Lázár-Molnár, Lisa K Peterson","doi":"10.1016/j.jim.2025.113819","DOIUrl":"10.1016/j.jim.2025.113819","url":null,"abstract":"<p><p>Despite great advancements in the discovery of genetic variants underlying inborn errors of immunity, functional assessment of the immunological profile of patients in routine clinical practice remains challenging. The lymphocyte proliferation assay using ³H-thymidine incorporation has been the gold standard for decades for functional evaluation of T cells in the clinical laboratory, however, recently developed flow cytometric methods allowing for single cell analysis provide non-radioactive alternatives. Understanding the technical and analytical challenges of test development, validation and maintenance is essential for correct interpretation and test utilization, to assure appropriate and timely patient care. This review will discuss the technological aspects, validation and clinical utilization of in vitro lymphocyte proliferation assays performed in the clinical immunology laboratory, providing a diagnostic readout for T lymphocyte function, an essential hallmark of a functional cellular immune response, and allowing for the detection of impaired responses such as in patients with functional T cell defects.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113819"},"PeriodicalIF":1.6,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039243","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}