Journal of immunological methods最新文献

{"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}

{"title":"Expression of bluetongue virus full-length VP7 protein in insect cells and its diagnostic utility for detection of antibodies to the virus infection","authors":"Sanchay Kumar Biswas ,&nbsp;Madhusudan Hosamani ,&nbsp;Karam Chand ,&nbsp;Ankita Chauhan ,&nbsp;Kurat Ul Ain ,&nbsp;Vanitha Selvarajan ,&nbsp;Sushmita Nautiyal ,&nbsp;Muzamil Bashir ,&nbsp;Divakar Hemadri ,&nbsp;Gaurav Kumar Sharma ,&nbsp;B.P. Sreenivasa","doi":"10.1016/j.jim.2025.113801","DOIUrl":"10.1016/j.jim.2025.113801","url":null,"abstract":"<div><div>Bluetongue (BT) is a vector-borne viral disease of multiple domestic and wild ruminants across the globe. The VP7 protein of bluetongue virus (BTV) is the major immune-dominant structural protein that is conserved across the BTV serotypes and therefore, targeted for the development of immuno-diagnostics for BT. In this study, full-length recombinant VP7 protein (rVP7) of BTV-1 was expressed in <em>Trochoplusia ni</em> derived insect cells (Tn5) using codon-optimized synthetic gene construct through baculovirus expression system. The seed stock of recombinant baculovirus was amplified to a high titre (&gt;1 × 10⁸ pfu/ml) at P2 and P3 in sf9 cells. The rVP7 was successfully produced and purified from infected Tn5 culture lysate for evaluation of the immuno-reactivity and its diagnostic potential. The purified protein showed strong reactivity in western blot analysis with the polyclonal immune serum produced against BTV core antigen in guinea pigs. An indirect ELISA (iELISA) was optimized by using the purified rVP7 for the detection of the group-specific antibodies to BTV in sheep and goats. The iELISA was found to be highly sensitive (98.9 %), specific (98.1 %), and reproducible (CV &lt; 10 %) for detection of the antibodies to BTV in sheep and goat serum. The iELISA could detect the specific antibodies in naturally infected goat serum containing type-specific neutralizing antibodies to different BTV serotypes indicating the potential of the rVP7 for the development of the group-specific sero-diagnostics for BT.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113801"},"PeriodicalIF":1.6,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006745","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":"Evaluation of self-collected dried blood spots for detection of SARS-CoV-2 nucleocapsid antibodies shows low sensitivity","authors":"Thomas Egger ,&nbsp;Tamara Dörr ,&nbsp;Reto Thoma ,&nbsp;Susanne Nigg ,&nbsp;Lorenz Risch ,&nbsp;Alessio Cremonesi ,&nbsp;Pietro Vernazza ,&nbsp;Philipp Kohler ,&nbsp;Christian R. Kahlert","doi":"10.1016/j.jim.2024.113800","DOIUrl":"10.1016/j.jim.2024.113800","url":null,"abstract":"<div><h3>Background and aims</h3><div>Dried blood spots (DBS) have been proposed as a cost-effective surveillance method for population-wide screening of SARS-CoV-2 immunity but sensitivity of DBS based on self-collected DBS samples is unknown. To evaluate the success of vaccination strategies, it is necessary to differentiate vaccination from natural infection. Therefore, a test for antibodies against the viral nucleocapsid protein (anti-N) is desirable.</div></div><div><h3>Materials and methods</h3><div>In our prospectively followed cohort of healthcare workers (HCW) in eastern Switzerland, we assessed SARS-CoV-2-anti-N-seroprevalence using DBS on a biweekly basis from March to September 2020. Phlebotomy samples were collected in March and September and tested for anti-N-seropositivity, as well as SARS-CoV-2 spike antibodies for quantitative validation. Venous antibody testing was compared with DBS results for anti-N using the Roche Elecsys electro-chemiluminescence immunoassay.</div></div><div><h3>Results</h3><div>792 HCW (median age 38.3 years) were included, 35 (4.4 %) were SARS-CoV-2-anti-N-seropositive. Of 43 matching DBS, 25 tested positive for anti-N, accounting for a sensitivity of 58.1 % (95 %CI 43.3–71.6 %). We found a significant correlation of anti-N from DBS with results from phlebotomy samples (<em>r</em> = 0.77;<em>p</em> &lt; 0.0001), with higher levels being associated with a higher true-positive rate. Anti-N in DBS correlated significantly with quantitatively validated anti-S obtained from serum (<em>r</em> = 0.67;<em>p</em> &lt; 0.0001).</div></div><div><h3>Conclusion</h3><div>Although home DBS collection was feasible in a larger cohort and we found a high correlation between anti-N detection in DBS and phlebotomy samples, the sensitivity of self-collected DBS samples was significantly impaired for the Roche Elecsys anti-N assay. Therefore, we cannot recommend this method for DBS when testing from venous blood is possible.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"536 ","pages":"Article 113800"},"PeriodicalIF":1.6,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142950012","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":"Adaptation of the in vivo respiratory burst assay for fathead minnow larvae (Pimephales promelas)","authors":"Nicole Kooij,&nbsp;Tisha C. King-Heiden","doi":"10.1016/j.jim.2024.113797","DOIUrl":"10.1016/j.jim.2024.113797","url":null,"abstract":"<div><div>Initial innate immune responses such as the respiratory burst response of phagocytes present the first line of defense in response to exposure to pathogens. Several respiratory burst assays have been developed in mammals, cell cultures, and whole zebrafish embryos as a reliable indicator of the innate immune response of a host, and these assays are being used to screen various environmental contaminants for their immunotoxic potential. While zebrafish are a common laboratory fish used in toxicology studies geared towards human health effects, fathead minnows are commonly used as an ecotoxicological indicator species for North America. In this technical note, we describe how we adapted the zebrafish <em>in vivo</em> respiratory burst assay for use in fathead minnow larvae. This assay provides promising expansion of using <em>in vivo</em> respiratory burst responses in different species of larval fish for future comparative immunotoxicity assays, as well as laying the groundwork for studies that can better define the development of the innate and adaptive immune responses of fathead minnow larvae.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"536 ","pages":"Article 113797"},"PeriodicalIF":1.6,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854332","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}