Journal of immunological methods最新文献

{"title":"Establishment of human post-vaccination SARS-CoV-2 standard reference sera","authors":"Jinhua Xiang ,&nbsp;Louis Katz ,&nbsp;Patricia L. Winokur ,&nbsp;Ashok Chaudhary ,&nbsp;Barbara Digmann ,&nbsp;Rebecca Bradford ,&nbsp;Sujatha Rashid ,&nbsp;Sudakshina Ghosh ,&nbsp;Angela Robertson ,&nbsp;Joseph Menetski ,&nbsp;Miao Xu ,&nbsp;Peng Gao ,&nbsp;Catherine Z. Chen ,&nbsp;Taylor Lee ,&nbsp;Brittany Poelaert ,&nbsp;Richard T. Eastman ,&nbsp;Matthew D. Hall ,&nbsp;Jack T. Stapleton","doi":"10.1016/j.jim.2024.113698","DOIUrl":"10.1016/j.jim.2024.113698","url":null,"abstract":"<div><p>There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of reference reagents comprised of post-vaccination sera from recipients of different primary vaccines with or without different vaccine booster regimens in order to allow standardized characterization of SARS-CoV-2 neutralization in vitro. We prepared and pooled serum obtained from donors who received a either primary vaccine series alone, or a vaccination strategy that included primary and boosted immunization using available SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), replication-incompetent adenovirus type 26 vaccine (Ad26.COV2·S, Johnson and Johnson), or recombinant baculovirus-expressed spike protein in a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). No subjects had a history of clinical SARS-CoV-2 infection, and sera were screened with confirmation that there were no nucleocapsid antibodies detected to suggest natural infection. Twice frozen sera were aliquoted, and serum antibodies were characterized for SARS-CoV-2 spike protein binding (estimated WHO antibody binding units/ml), spike protein competition for ACE-2 binding, and SARS-CoV-2 spike protein pseudotyped lentivirus transduction. These reagents are available for distribution to the research community (BEI Resources), and should allow the direct comparison of antibody neutralization results between different laboratories. Further, these sera are an important tool to evaluate the functional neutralization activity of vaccine-induced antibodies against emerging SARS-CoV-2 variants of concern.</p></div><div><h3>Importance</h3><p>The explosion of COVID-19 demonstrated how novel coronaviruses can rapidly spread and evolve following introduction into human hosts. The extent of vaccine- and infection-induced protection against infection and disease severity is reduced over time due to the fall in concentration, and due to emerging variants that have altered antibody binding regions on the viral envelope spike protein. Here, we pooled sera obtained from individuals who were immunized with different SARS-CoV-2 vaccines and who did not have clinical or serologic evidence of prior infection. The sera pools were characterized for direct spike protein binding, blockade of virus-receptor binding, and neutralization of spike protein pseudotyped lentiviruses. These sera pools were aliquoted and are available to allow inter-laboratory comparison of results and to provide a tool to determine the effectiveness of prior vaccines in recognizing and neutralizing emerging variants of concern.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113698"},"PeriodicalIF":2.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000838/pdfft?md5=2f3b18684af42df9ef4d895246933554&pid=1-s2.0-S0022175924000838-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141185791","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":"Sources of variability in Luminex bead-based cytokine assays: Evidence from twelve years of multi-site proficiency testing","authors":"Wes Rountree ,&nbsp;Heather E. Lynch ,&nbsp;Thomas N. Denny ,&nbsp;Gregory D. Sempowski ,&nbsp;Andrew N. Macintyre","doi":"10.1016/j.jim.2024.113699","DOIUrl":"10.1016/j.jim.2024.113699","url":null,"abstract":"<div><p>Bead array assays, such as those sold by Luminex, BD Biosciences, Sartorius, Abcam and other companies, are a well-established platform for multiplexed quantification of cytokines and other biomarkers in both clinical and discovery research environments. In 2011, the National Institute of Allergy and Infectious Diseases (NIAID)-funded External Quality Assurance Program Oversight Laboratory (EQAPOL) established a proficiency assessment program to monitor participating laboratories performing multiplex cytokine measurements using Luminex bead array technology. During every assessment cycle, each site was sent an assay kit, a protocol, and blinded samples of human sera spiked with recombinant cytokines. Site results were then evaluated for performance relative to peer laboratories. After over a decade of biannual assessments, the cumulative dataset contained over 15,500 bead array observations collected at more than forty laboratories in twelve countries. These data were evaluated alongside post-assessment survey results to empirically test factors that may contribute to variability and accuracy in Luminex bead-based cytokine assays. Bead material, individual technical ability, analyte, analyte concentration, and assay kit vendor were identified as significant contributors to assay performance. In contrast, the bead reader instrument model and the use of automated plate washers were found not to contribute to variability or accuracy, and sample results were found to be highly-consistent between assay kit-manufacturing lots and over time. In addition to these statistical analyses, subjective evaluations identified technical ability, instrument failure, protocol adherence, and data transcription errors as the most common causes of poor performance in the proficiency program. The findings from the EQAPOL multiplex program were then used to develop recommended best practices for bead array monitoring of human cytokines. These included collecting samples to assay as a single batch, centralizing analysis, participating in a quality assurance program, and testing samples using paramagnetic-bead kits from a single manufacturer using a standardized protocol.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113699"},"PeriodicalIF":2.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141185839","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":"Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population","authors":"Ishita Gupta ,&nbsp;Himani Dhanze ,&nbsp;Megha Gupta ,&nbsp;Praveen Singh ,&nbsp;Deepa Mehta ,&nbsp;Mithilesh K. Singh ,&nbsp;Abhishek ,&nbsp;M. Suman Kumar ,&nbsp;K.N. Bhilegaonkar","doi":"10.1016/j.jim.2024.113695","DOIUrl":"10.1016/j.jim.2024.113695","url":null,"abstract":"<div><p>Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2–3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 μg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 μg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 μl of GNP conjugate and 50 μl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113695"},"PeriodicalIF":2.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141133835","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":"Repurposing discarded leukodepletion filters as a source of mononuclear cells for advanced in vitro research","authors":"Joyce Alessandra Lima ,&nbsp;Bruna Pereira Sorroche ,&nbsp;Katiane Tostes ,&nbsp;Tauana Christina Dias ,&nbsp;Nathália de Carvalho Rodrigues ,&nbsp;Aline Tansini ,&nbsp;Renato José da Silva Oliveira ,&nbsp;Lidia Maria Rebolho Batista Arantes","doi":"10.1016/j.jim.2024.113694","DOIUrl":"10.1016/j.jim.2024.113694","url":null,"abstract":"<div><p>In light of advancements in the field of immuno-oncology, the demand for obtaining mononuclear cells for in vitro assays has surged. However, obtaining these cells from healthy donors remains a challenging task due to difficulties in donor recruitment and the requirement for substantial blood volumes. Here, we present a protocol for isolating peripheral blood mononuclear cells (PBMCs) from leukodepletion filters used in whole blood and erythrocytes by apheresis donations at the Hemonucleus of the Barretos Cancer Hospital, Brazil. The method involves rinsing the leukodepletion filters and subsequent centrifugation using a Ficoll-Paque concentration gradient. The isolated PBMCs were analyzed by flow cytometry, which allowed the identification of various subpopulations, including CD4⁺ T lymphocytes (CD45⁺CD4⁺), CD8⁺ T lymphocytes (CD45⁺CD8⁺), B lymphocytes (CD45⁺CD20⁺CD19⁺), non-classical monocytes (CD45⁺CD64⁺CD14⁻), classical monocytes (CD45⁺CD64⁺CD14⁺), and granulocytes (CD45⁺CD15⁺CD14^⁻). In our comparative analysis of filters, we observed a higher yield of PBMCs from whole blood filters than those obtained from erythrocytes through apheresis. Additionally, fresh samples exhibited superior viability when compared to cryopreserved ones. Given this, leukodepletion filters provide a practical and cost-effective means to isolate large quantities of pure PBMCs, making it a feasible source for obtaining mononuclear cells for in vitro experiments.</p></div><div><h3>Summary</h3><p>Here, we provide a detailed protocol for the isolation of mononuclear cells from leukodepletion filters, which are routinely discarded at the Barretos Cancer Hospital's Hemonucleus.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113694"},"PeriodicalIF":2.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141135107","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":"An easy assay to detect autoantibodies neutralizing cytokines in subjects with critical infections","authors":"Nicola Donadel ,&nbsp;Alessandra Tesser ,&nbsp;Erica Valencic ,&nbsp;Eleonora De Martino ,&nbsp;Valentina Boz ,&nbsp;Alessia Pin ,&nbsp;Francesca Zorat ,&nbsp;Gabriele Pozzato ,&nbsp;Alberto Tommasini","doi":"10.1016/j.jim.2024.113696","DOIUrl":"10.1016/j.jim.2024.113696","url":null,"abstract":"<div><p>Autoantibodies against type I interferon (IFN) are associated with a worse outcome in COVID-19. The measurement of cytokine-neutralizing autoantibodies has been limited, hindering understanding of their role in clinical practice. We showed that an easy and reliable assay can be reproduced and validated to measure the neutralizing potency of autoantibodies directed to type I or type II IFN. Identifying of anti-cytokine autoantibodies might reflect on early treatments for subsequent infections, such as with antivirals or virus-neutralizing monoclonal antibodies.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113696"},"PeriodicalIF":2.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000814/pdfft?md5=ea29b4ef77abb484a15e128eeabfa4a2&pid=1-s2.0-S0022175924000814-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141139060","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":"How to verify the analytical and clinical performance of ELISA immunoanalysis in the real laboratory practice. PCSK9 as an example","authors":"Tereza Vacková ,&nbsp;Antonín Jabor ,&nbsp;Zdenek Kubíček ,&nbsp;Janka Franeková","doi":"10.1016/j.jim.2024.113693","DOIUrl":"10.1016/j.jim.2024.113693","url":null,"abstract":"<div><h3>Background</h3><p>Manufacturers and diagnostic companies often recommend on-site verification of analytical performance in the clinical laboratory. The validation process used by manufacturers is rarely described in detail, and certain information on analytical performance is missing from the product sheet, especially for immunoanalytical methods. We describe an approach to the detailed validation of an ELISA method for the measurement of proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma concentrations. We compared manufacturers' claims of analytical performance with data obtained in the field laboratory using several approaches.</p></div><div><h3>Methods</h3><p>We used the Human Proprotein Convertase 9/PCSK9 Quantikine ELISA diagnostic kit (R&amp;D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) and three levels of quality control solution Quantikine Immunoassay Control Group 235 (R&amp;D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) to verify precision. We measured the concentration of PCSK9 using the DS2 ELISA Reader (Dynex Technologies GmbH, Denkendorf, Germany). We used analysis of variance (ANOVA) and the R statistical package (R core team, version 1.4.5). Statistical analysis and terminology were performed according to protocol CLSI EP15-A3, and the reference interval was checked according to CLSI/IFCC C28-A3c.</p></div><div><h3>Results</h3><p>We found a significant difference between the manufacturer's claims of analytical performance and real data measured in the routine clinical laboratory. The calculated CV (%) for repeatability (calculated by simple estimation of the mean and SD, as used by the manufacturer) was between 5.5% and 7.4%, but the manufacturer's claim was between 4.1% and 6.5%. Using ANOVA, the true repeatability was between 5.0% and 6.9%. Similarly, ANOVA revealed values of CV (%) for within-laboratory imprecision between 6.5% and 9.1%, while the manufacturer's claims were between 4.1% and 5.9%. The recovery ranged from 105.5% to 121.8%. The manufacturer's recommended reference interval was checked and we didn't find any significant difference between men and women.</p></div><div><h3>Conclusions</h3><p>We describe a comprehensive approach to verify the analytical performance of an ELISA method using the measurement of PCSK9 plasma concentration as an example. We found differences between the results of this approach based on the CLSI EP15-A3 protocol and data provided by the manufacturer. We recommend the verification of analytical performance by more complex statistical tools in laboratory practice.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113693"},"PeriodicalIF":2.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141131776","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":"Assessment of an in-house IgG ELISA targeting SARS-CoV-2 RBD: Applications in infected and vaccinated individuals","authors":"Hernan Hermes Monteiro da Costa ,&nbsp;Valeria Oliveira Silva ,&nbsp;Gustavo Carvalho Amorim ,&nbsp;Marcia Grando Guereschi ,&nbsp;Luciana Marciano Sergio ,&nbsp;Carlos Henrique Rodrigues Gomes ,&nbsp;Marisa Ailin Hong ,&nbsp;Elaine Lopes de Oliveira ,&nbsp;Luis Fernando de Macedo Brígido ,&nbsp;Jose Angelo Lauletta Lindoso ,&nbsp;Carlos Roberto Prudencio","doi":"10.1016/j.jim.2024.113683","DOIUrl":"10.1016/j.jim.2024.113683","url":null,"abstract":"<div><p>The study evoluated an in-house Spike Receptor Binding Domain Enzyme-Linked Immunosorbent Assay (RBD-IgG-ELISA) for detecting SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. The assay demonstrated a sensitivity of 91%, specificity of 99.25%, and accuracy of 95.13%. Precision and reproducibility were highly consistent. The RBD-IgG-ELISA was able to detect 96.25% of Polymerase chain reaction (PCR) confirmed cases for SARS-CoV-2 infection, demonstrating positive and negative predictive values of 99,18% and 91,69%, respectively. In an epidemiological survey, ELISA, lateral flow immunochromatographic assay (LFIA), and electrochemiluminescence immunoassay (ECLIA) exhibited diagnostic sensitivities of 68.29%, 63.41%, and 70.73%, respectively, along with specificities of 82.93%, 80.49%, and 80.49%, respectively. Agreement between RBD-IgG-ELISA/PCR was moderate (k index 0.512). However, good agreement between different assays (RBD-IgG-ELISA/LFIA k index 0.875, RBD-IgG-ELISA/ECLIA k index 0.901). Test performance on individuals' samples were inferior due to seroconversion time and chronicity. The IgG-RBD-ELISA assay demonstrated its effectiveness in monitoring antibody levels among healthcare professionals, revealing significant differences both before and after the administration of the third vaccine dose, with heightened protection levels observed following the third dose in five Coronavirus disease (COVID-19) vaccine regimens. In conclusion, the RBD-IgG-ELISA exhibits high reproducibility, specificity, and sensitivity, making it a suitable assay validated for serosurveillance and for obtaining information about COVID-19 infections or vaccinations.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113683"},"PeriodicalIF":2.2,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957218","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":"Simultaneous multiple target detection platform based on vertical flow immunoassay","authors":"Taek Yong ,&nbsp;Dami Kim ,&nbsp;Sanghyo Kim","doi":"10.1016/j.jim.2024.113690","DOIUrl":"10.1016/j.jim.2024.113690","url":null,"abstract":"<div><p>In general, vertical flow assay (VFA) has a disadvantage of requiring a complex analysis process that involves manually injecting various reagents (target analyte, washing buffer, detection conjugate, etc.) sequentially. However, in this study, we have developed an innovative paper-based VFA device that replaces the complex analysis process with one-step and enables the detection of multiple targets. The fabrication process of the multi-target detection VFA device is as follows: preparation and pre-treatment of the strip materials, design of strip cartridge, design of the multiple detection VFA device, optimization experiments for strip sample flow rates, determination of device analysis time, determination of device limit of detection (LOD), multiple target signal uniformity experiment, immunoglobulin G (IgG) and C-reactive protein (CRP) antigen-antibody multiple detection experiment, and data extraction and analysis method. The use of paper-based materials enables the device to be produced at cost-effective, and cartridge production allowed for uniform array formation. IgG and CRP are used to evaluate the performance of the device as common biomarkers. The device proposed in this study is currently under research. To validate multiple target detection capability of the VFA device proposed in this study, two types of antigens-antibodies (Human IgG and Human CRP) were employed. The detection limit is 0.15 μg/mL for IgG and 0.19 μg/mL for CRP in naked eye. Furthermore, it was confirmed that there is no cross-reactivity caused by the device structure through IgG and CRP antigens. In conclusion, the VFA device proposed in this study consists of a one-step analysis process, and it has been confirmed that it can detect multiple targets simultaneously.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113690"},"PeriodicalIF":2.2,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957228","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":"Novel IgE crosslinking-induced luciferase expression method using human-rat chimeric IgE receptor-carrying mast cells","authors":"Haruyo Akiyama ,&nbsp;Chisato Kurisaka ,&nbsp;Kenichi Kumasaka ,&nbsp;Ryosuke Nakamura","doi":"10.1016/j.jim.2024.113682","DOIUrl":"10.1016/j.jim.2024.113682","url":null,"abstract":"<div><h3>Background</h3><p>The measurement of antigen-specific serum IgE is common in clinical assessments of type I allergies. However, the interaction between antigens and IgE won't invariably trigger mast cell activation. We previously developed the IgE crosslinking-induced luciferase expression (EXiLE) method using the RS-ATL8 mast cell line; however, the method may not be sensitive enough in some cases.</p></div><div><h3>Methods</h3><p>In this study, we introduced an NF-AT-regulated luciferase reporter gene into the RBL-2H3 rat mast cell line and expressed a chimeric high-affinity IgE receptor (FcεRI) α chain gene, comprising an extracellular domain from humans and transmembrane/intracellular domains from rats.</p></div><div><h3>Results</h3><p>We generated multiple clones expressing the chimeric receptor. Based on their responsiveness and proliferation, we selected the HuRa-40 clone. This cell line exhibited significantly elevated human α chain expression compared to RS-ATL8 cells, demonstrating a 10-fold enhancement of antigen-specific reactivity. Reproducibility across different batches and operators was excellent. Moreover, we observed a detectable response inhibition by an anti-allergy drugs (omalizumab and cyclosporin A).</p></div><div><h3>Conclusions</h3><p>HuRa-40 cells—which carry the human-rat chimeric IgE receptor—comprise a valuable reporter cell line for the EXiLE method. Their versatility extends to various applications and facilitates high-throughput screening of anti-allergy drugs.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"529 ","pages":"Article 113682"},"PeriodicalIF":2.2,"publicationDate":"2024-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140863262","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":"SARS-CoV-2 spike protein expression as an identification in quality control testing for Adenovector based COVID-19 vaccine","authors":"Harit Kasana ,&nbsp;Ajay Kumar Ade ,&nbsp;Jaipal Meena,&nbsp;Archana Sayal,&nbsp;Faraz Sheikh,&nbsp;Anupkumar R. Anvikar,&nbsp;Harish Chander","doi":"10.1016/j.jim.2024.113680","DOIUrl":"10.1016/j.jim.2024.113680","url":null,"abstract":"<div><h3>Aim</h3><p>Quality control testing of the vaccine for lot release is of paramount importance in public health. A recent pandemic caused by the SARS-CoV-2 virus brought together all spheres of vaccine to combat the virus. The scientific advancement in the development of vaccines facilitated the scientists to develop the vaccine against SARS-CoV-2 in a record time. Thus, these vaccines should be stringently monitored for their safety and efficacy as per the latest WHO and national regulatory guidelines, and quality control evaluation of the product should be done at national control laboratories before releasing the product into the market as it assures the quality and safety of the vaccine.</p></div><div><h3>Methods</h3><p>The SARS-CoV-2 exploited the ACE2 (Angiotensin Converting Enzyme 2) receptor, a surface protein on mammalian cells to gain entry into the host cells. The viral surface protein that interacted with the ACE2 receptor is the Spike protein of SARS-CoV-2. Thus, in the development of the vaccine and assessing its quality, the Spike protein of SARS-CoV-2 became an attractive immunodominant antigen. In National Institute of Biologicals, an apex body in the testing of biologicals in India, received the Adenovector (Adenovirus + vector) based COVID-19 vaccine, a finished product for quality evaluation. Due to the lack of a pharmacopeial monograph, the testing of the vaccine was done as per the manufacturer's specifications and methods. The routine assays of identification employed by the manufacturer do not reflect the expression of Spike protein which is required for the immune system to get activated. In this report, we showed the determination of Spike protein expression by immunoblotting and immunofluorescence for identification parameters in the quality testing of the COVID-19 vaccine. We determined the translation of the SARS-CoV-2 Spike gene cloned into an Adenovector.</p></div><div><h3>Results</h3><p>The results from these experiments indicated the expression of Spike protein upon infection of mammalian cells with viral particles suggested that the expression of immunodominant Spike protein of SARS-CoV-2 may be employed by quality control laboratories as a parameter for identification.</p></div><div><h3>Conclusion</h3><p>The study suggested that the determination of the expression of Spike protein is pertinent to identifying the Adenovector based vaccines against COVID-19.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"529 ","pages":"Article 113680"},"PeriodicalIF":2.2,"publicationDate":"2024-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140864000","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}