Journal of immunological methods最新文献

{"title":"A real-time antibody-dependent cellular phagocytosis assay by live cell imaging","authors":"Yongchang Shi ,&nbsp;Yonglian Sun ,&nbsp;Akiko Seki ,&nbsp;Sascha Rutz ,&nbsp;James T. Koerber ,&nbsp;Jianyong Wang","doi":"10.1016/j.jim.2024.113715","DOIUrl":"10.1016/j.jim.2024.113715","url":null,"abstract":"<div><p>Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated <em>in vitro</em>. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113715"},"PeriodicalIF":1.6,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468604","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":"A universal urinary cell gene signature of acute rejection in kidney allografts","authors":"Thalia Salinas ,&nbsp;Carol Li ,&nbsp;Catherine Snopkowski ,&nbsp;Vijay K. Sharma ,&nbsp;Darshana M. Dadhania ,&nbsp;Karsten Suhre ,&nbsp;Thangamani Muthukumar ,&nbsp;Manikkam Suthanthiran","doi":"10.1016/j.jim.2024.113714","DOIUrl":"10.1016/j.jim.2024.113714","url":null,"abstract":"<div><h3>Introduction</h3><p>Acute rejection (AR) undermines the life-extending benefits of kidney transplantation and is diagnosed using the invasive biopsy procedure. T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), or concurrent TCMR + ABMR (Mixed Rejection [MR]) are the three major types of AR. Development of noninvasive biomarkers diagnostic of AR due to any of the three types is a useful addition to the diagnostic armamentarium.</p></div><div><h3>Methods</h3><p>We developed customized RT-qPCR assays and measured urinary cell mRNA copy numbers in 145 biopsy-matched urine samples from 126 kidney allograft recipients. We determined whether the urinary cell three-gene signature diagnostic of TCMR (<span>Suthanthiran et al., 2013</span>) discriminates patients with no rejection biopsies (NR, <em>n</em> = 50) from those with ABMR (<em>n</em> = 28) or MR (<em>n</em> = 20) biopsies.</p></div><div><h3>Results</h3><p>The urinary cell three-gene signature discriminated all three types of rejection biopsies from NR biopsies (<em>P</em> &lt; 0.0001, One-way ANOVA). Dunnett's multiple comparisons test yielded <em>P</em> &lt; 0.0001 for NR vs. TCMR; <em>P</em> &lt; 0.001 for NR vs. ABMR; and <em>P</em> &lt; 0.0001 for NR vs. MR. By bootstrap resampling, optimism-corrected area under the receiver operating characteristic curve (AUC) was 0.749 (bias-corrected 95% confidence interval [CI], 0.638 to 0.840) for NR vs. TCMR (<em>P</em> &lt; 0.0001); 0.780 (95% CI, 0.656 to 0.878) for NR vs. ABMR (<em>P</em> &lt; 0.0001); and 0.857 (95% CI, 0.727 to 0.947) for NR vs. MR (<em>P</em> &lt; 0.0001). All three rejection categories were distinguished from NR biopsies with similar accuracy (all AUC comparisons <em>P</em> &gt; 0.05).</p></div><div><h3>Conclusion</h3><p>The urinary cell three-gene signature score discriminates AR due to TCMR, ABMR or MR from NR biopsies in human kidney allograft recipients.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"532 ","pages":"Article 113714"},"PeriodicalIF":1.6,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468597","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":"ERAMER: A novel in silico tool for prediction of ERAP1 enzyme trimming","authors":"Anas Al-okaily ,&nbsp;Razan Abu Khashabeh ,&nbsp;Osama Alsmadi ,&nbsp;Yazan Ahmad ,&nbsp;Iyad Sultan ,&nbsp;Abdelghani Tbakhi ,&nbsp;Pramod K Srivastava","doi":"10.1016/j.jim.2024.113713","DOIUrl":"10.1016/j.jim.2024.113713","url":null,"abstract":"<div><p>MHC class I pathway consists of four main steps: proteasomal cleavage in the cytosol in which precursor proteins are cleaved into smaller peptides, which are then transported into the endoplasmic reticulum by the transporter associated with antigen processing, TAP, for further processing (trimming) from the N-terminal region by an ER resident aminopeptidases 1 (ERAP1) enzyme, to generate optimal peptides (8–10 amino acids in length) to produce a stable MHCI-peptide complex, that get transited <em>via</em> the Golgi apparatus to the cell surface for presentation to the cellular immune system. Several studies reported specificities related to the ERAP1 trimming process, yet there is no <em>in silico</em> tool for the prediction of the trimming process of the ERAP1 enzyme. In this paper, we provide and implement a prediction model for the trimming process of the ERAP1 enzyme.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113713"},"PeriodicalIF":1.6,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141457314","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":"Strategies to determine positive anti-SARS-CoV-2 memory T lymphocyte response during the evolution of an epidemic","authors":"Isabelle Nel ,&nbsp;Ajeeva Ithayakumar ,&nbsp;Noémie Blumenthal ,&nbsp;Charlotte Duneton ,&nbsp;Valérie Guérin-El Khourouj ,&nbsp;Jérôme Viala ,&nbsp;Catherine Dollfus ,&nbsp;Véronique Baudouin ,&nbsp;Sophie Guilmin-Crepon ,&nbsp;Ioannis Theodorou ,&nbsp;Guislaine Carcelain","doi":"10.1016/j.jim.2024.113712","DOIUrl":"10.1016/j.jim.2024.113712","url":null,"abstract":"<div><p>During SARS-CoV-2 pandemic, the assessment of immune protection of people at risk of severe infection was an important goal. The appearance of VOCs (Variant of Concern) highlighted the limits of evaluating immune protection through the humoral response. While the humoral response partly loses its neutralizing activity, the anti-SARS-CoV-2 memory T cell response strongly cross protects against VOCs becoming an indispensable tool to assess immune protection. We compared two techniques available in laboratory to evaluate anti-SARS-CoV-2 memory T cell response in a cohort of infected or vaccinated patients with different levels of risk to develop a severe disease: the ELISpot assay and the T-Cell Lymphocyte Proliferation Assay respectively exploring IFNγ production and cell proliferation. We showed that the ELISpot assay detected more anti-Spike memory T cell response than the Lymphocyte Proliferation Assay. We next observed that the use of two different suppliers as antigenic source in the ELISpot assay did not affect the detection of anti-Spike memory T cell response. Finally, we explored a new approach for defining the positivity threshold, using unsupervised mixed Gaussian modeling, challenging the traditional ROC curve used by the supplier. That will be helpful in endemic situation where it could be difficult to recruit “negative” patients.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113712"},"PeriodicalIF":1.6,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000978/pdfft?md5=9d26956e717f00130e70ab4fbc05bb8e&pid=1-s2.0-S0022175924000978-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141436974","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":"Discrepancies between two total IgE assays and difference in reference intervals in healthy adults","authors":"Mai Elzieny ,&nbsp;Gabriel N. Maine ,&nbsp;Robin A. Carey-Ballough ,&nbsp;Qian Sun","doi":"10.1016/j.jim.2024.113711","DOIUrl":"10.1016/j.jim.2024.113711","url":null,"abstract":"<div><h3>Objective</h3><p>To compare total immunoglobulin (Ig) E assay performance characteristics between Abbott Architect and Siemens Immulite test systems. Reference intervals were also determined for both platforms in an American population of healthy adults.</p></div><div><h3>Methods</h3><p>Agreement of the two total IgE assays was evaluated in a cohort of 331 subjects with normal complete blood count (CBC) and comprehensive metabolic panel (CMP) results. Reference intervals were established in 302 subjects after exclusion of atopic individuals on the Abbott Architect and Siemens Immulite test systems.</p></div><div><h3>Results</h3><p>We demonstrated a 32% positive bias for total IgE quantitation on the Siemens Immulite platform compared to the Abbott Architect, despite both methods calibrated against the same WHO international reference material (75/502), Furthermore, the upper limit of the reference interval (95th percentile) was determined to be higher for the Siemens Immulite assay compared to the Abbott Architect (132 and 102 IU/mL, respectively).</p></div><div><h3>Conclusion</h3><p>Despite the use of a common WHO reference material for total IgE assay calibration, significant differences in quantitation was observed between two FDA-cleared test systems. Given that, it is warranted for clinical laboratories to verify vendor established reference intervals and adjust accordingly based on internal assessment of the normal range.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113711"},"PeriodicalIF":1.6,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141327525","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 an immunoassay for quantification of soluble human CD40L (CD154) in plasma and serum samples","authors":"Kathrine Pedersen ,&nbsp;Nick Stub Laursen ,&nbsp;Annette Gudmann Hansen ,&nbsp;Yaseelan Palarasah ,&nbsp;Steffen Thiel","doi":"10.1016/j.jim.2024.113710","DOIUrl":"10.1016/j.jim.2024.113710","url":null,"abstract":"<div><p>When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation. Dysregulation of the CD40L:CD40 axis is associated with inflammatory and autoimmune diseases. The presence of soluble CD40L (sCD40L) in plasma is implicated in several diseases, from cardiovascular and autoimmune diseases to different types of cancer, and sCD40L has been suggested as a valuable marker of disease. If sCD40L is to be used as a biomarker, being able to precisely measure and quantify the levels of sCD40L in human blood samples is of utmost importance. We demonstrate the development of a sandwich-type time-resolved immunofluorometric assay for quantification of sCD40L in plasma or serum samples. For this, we generate 29 monoclonal anti-CD40L antibodies, and from these, we select the optimal combination of capture antibody and detection antibody. A number of variables were tested: the influence of the type of sample (comparing 3 different blood collection tubes for serum sampling and 4 different types of tubes for plasma sampling), the influence of freeze-thaw cycles, the influence of sampling time during night and day, and the influence of centrifugation of the samples. We found a very similar level of sCD40L in paired EDTA plasma and serum samples. Out of 100 healthy blood donor samples 61 had a level of sCD40L below the detection level of the assay, whereas the remaining 39 samples had ranging levels of sCD40L from 1.14 to 33.14 ng/mL. In summary, we present a time-resolved immunofluorometric assay based on paired monoclonal antibodies, ensuring high specificity, sensitivity, and homogeneity. The Eu³⁺-based assay additionally provides consistent assay readouts due to the extended decay time not seen in standard enzyme-linked immunosorbent assays. The assay paves the way for specific and consistent quantification of sCD40L in human plasma and serum samples.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113710"},"PeriodicalIF":2.2,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141317526","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 role of gamma globulin, complement component 1q, factor B, properdin, body temperature, C-reactive protein and serum amyloid alpha to the activity and the function of the human complement system and its pathways","authors":"Janne Atosuo ,&nbsp;Outi Karhuvaara ,&nbsp;Eetu Suominen ,&nbsp;Julia Virtanen ,&nbsp;Liisa Vilén ,&nbsp;Jari Nuutila","doi":"10.1016/j.jim.2024.113709","DOIUrl":"10.1016/j.jim.2024.113709","url":null,"abstract":"<div><p>The complement system plays a crucial role in orchestrating the activation and regulation of inflammation within the human immune system. Three distinct activation pathways—classical, lectin, and alternative—converge to form the common lytic pathway, culminating in the formation of the membrane-attacking complex that disrupts the structure of pathogens. Dysregulated complement system activity can lead to tissue damage, autoimmune diseases, or immune deficiencies.</p><p>In this study, the antimicrobial activity of human serum was investigated by using a bioluminescent microbe probe, <em>Escherichia coli</em> (pEGFPluxABCDEamp). This probe has previously been used to determine the antimicrobial activity of complement system and the polymorphonuclear neutrophils. In this study, blocking antibodies against key serum activators and components, including IgG, complement component 1q, factor B, and properdin, were utilized. The influence of body temperature and acute phase proteins, such as C reactive protein (CRP) and serum amyloid alpha (SAA), on the complement system was also examined.</p><p>The study reveals the critical factors influencing complement system activity and pathway function. Alongside crucial factors like C1q and IgG, alternative pathway components factor B and properdin played pivotal roles. Results indicated that the alternative pathway accounted for approximately one third of the overall serum antimicrobial activity, and blocking this pathway disrupted the entire complement system. Contrary to expectations, elevated body temperature during inflammation did not enhance the antimicrobial activity of human serum.</p><p>CRP demonstrated complement activation properties, but at higher physiological concentrations, it exhibited antagonistic tendencies, dampening the response. On the other hand, SAA enhanced the serum's activity.</p><p>Overall, this study sheds a light on the critical factors affecting both complement system activity and pathway functionality, emphasizing the importance of a balanced immune response.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113709"},"PeriodicalIF":1.6,"publicationDate":"2024-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000942/pdfft?md5=7763d9d644b054f3918f8f75abc630b9&pid=1-s2.0-S0022175924000942-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141306126","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":"Antisera against flagellin A or B inhibits Pseudomonas aeruginosa motility as measured by novel video microscopy assay","authors":"Ethel Apolinario ,&nbsp;James Sinclair ,&nbsp;Myeongjin Choi ,&nbsp;Kun Luo ,&nbsp;Surekha Shridhar ,&nbsp;Sharon M. Tennant ,&nbsp;Raphael Simon ,&nbsp;Erik Lillehoj ,&nbsp;Alan Cross","doi":"10.1016/j.jim.2024.113701","DOIUrl":"10.1016/j.jim.2024.113701","url":null,"abstract":"<div><p>Flagellum-mediated motility is essential to <em>Pseudomonas aeruginosa (P. aeruginosa)</em> virulence. Antibody against flagellin reduces motility and inhibits the spread of the bacteria from the infection site. The standard soft-agar assay to demonstrate anti-flagella motility inhibition requires long incubation times, is difficult to interpret, and requires large amounts of antibody. We have developed a time-lapse video microscopy method to analyze anti-flagellin <em>P. aeruginosa</em> motility inhibition that has several advantages over the soft agar assay. Antisera from mice immunized with flagellin type A or B were incubated with Green Fluorescent Protein (GFP)-expressing <em>P. aeruginosa</em> strain PAO1 (FlaB+) and GFP-expressing <em>P. aeruginosa</em> strain PAK (FlaA+). We analyzed the motion of the bacteria in video taken in ten second time intervals. An easily measurable decrease in bacterial locomotion was observed microscopically within minutes after the addition of small volumes of flagellin antiserum. From data analysis, we were able to quantify the efficacy of anti-flagellin antibodies in the test serum that decreased <em>P. aeruginosa</em> motility. This new video microscopy method to assess functional activity of anti-flagellin antibodies required less serum, less time, and had more robust and reproducible endpoints than the standard soft agar motility inhibition assay.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113701"},"PeriodicalIF":2.2,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000863/pdfft?md5=701924041c37408f92375874d9085620&pid=1-s2.0-S0022175924000863-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141296209","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":"Retention of E-selectin functionalized liposome fanny packs on Jurkat cells following invasion through collagen","authors":"Simon M. King ,&nbsp;Ismael Ortiz ,&nbsp;Nicole S. Sarna ,&nbsp;Wenjun Wang ,&nbsp;Maria Lopez-Cavestany ,&nbsp;Zhenjiang Zhang","doi":"10.1016/j.jim.2024.113700","DOIUrl":"10.1016/j.jim.2024.113700","url":null,"abstract":"<div><p>Circulating immune cells are an appealing candidate to serve as carriers of therapeutic cargo via nanoparticles conjugated to their surface, for several reasons: these cells are highly migratory and can squeeze through small pores of diameter smaller than their resting size; they are easily accessible in the peripheral blood via minimally invasive IV injection of particles, or can be harvested, processed ex vivo, and reintroduced to the body; they are adept at traveling through the circulation with minimal destruction and thus have access to various tissue beds of the body; and immune cells have built-in signal transduction machinery which allows them to actively engage in chemotaxis and home to regions of the tissue containing tumors, invading microorganisms, or injuries in need of wound healing. In this study, we sought to examine and quantify the degree to which nanoscale liposomes, functionalized with <em>E</em>-selectin adhesion receptor, could bind to a model T cell line and remain on the surface of the cells as they migrate through collagen gels of varying density in a transwell cell migration chamber. It is demonstrated that physiological levels of fluid shear stress are necessary to achieve optimal binding of the <em>E</em>-selectin liposomes to the cell surface as expected, and that CD3/CD28 antibody activation of the T cells was not necessary for effective liposome binding. Nanoscale liposomes were successfully conveyed by the migrating cells across a layer of rat tail type 1 collagen gel ranging in composition from 1 to 3 mg/mL. The relative fraction of liposomes carried through the collagen decreased at higher collagen density, likely due to the expected decrease in average pore size, and increased fiber content in the gels. Taken together, these results support the idea that T cells could be an effective cellular carrier of therapeutic molecules either attached to the surface of nanoscale liposomes or encapsulated within their interior.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113700"},"PeriodicalIF":2.2,"publicationDate":"2024-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141283847","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 an enzyme-linked immunosorbent assay using a monoclonal antibody to a dominant epitope in non-structural protein 4 of porcine reproductive and respiratory syndrome virus","authors":"Chaolun Fu ,&nbsp;Qingyuan Shao ,&nbsp;Lei Zhang ,&nbsp;Xinyu Cui ,&nbsp;Ting Chen ,&nbsp;Chang Tian ,&nbsp;Fenglu Qian ,&nbsp;Xuefei Chu ,&nbsp;Yingchao Li ,&nbsp;Pingping Yang ,&nbsp;Yanmeng Hou ,&nbsp;Yihong Xiao","doi":"10.1016/j.jim.2024.113697","DOIUrl":"10.1016/j.jim.2024.113697","url":null,"abstract":"<div><p>Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe swine diseases causing great economic losses for the international swine industry. Non-structural protein 4 (NSP4) is critical to the life cycle of PRRSV and contains dominant B cell epitopes. This study prepared a monoclonal antibody against Nsp4, and 2D11, which contained the sequence ¹³⁸KQGGGIVTRPSGQFCN¹⁵³, was confirmed as the epitope. A 2D11-based double antibody sandwich enzyme-linked immunosorbent assay (dasELISA) was next developed with a cut value of 0.1987. A total of 1354 pig serum samples were detected by dasELISA and compared to a commercial ELISA kit (N-coated iELISA), resulting in a positive coincidence rate of 98.8% and negative coincidence rate of 96.9%. A total of 119 sera were positive by dasELISA while negative by iELISA. Higher positive rates by dasELISA were found in pig farms where PRRSV antibody levels varied widely. These results indicated that the dasELISA was a useful tool to detect PRRSV antibody in clinical samples.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"530 ","pages":"Article 113697"},"PeriodicalIF":2.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141185789","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}