Journal of immunological methods最新文献

{"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}

{"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}