Journal of immunological methods最新文献

{"title":"Development of sandwich enzyme-linked immunosorbent assays quantifying mouse urinary megalin, a novel proximal tubular biomarker","authors":"","doi":"10.1016/j.jim.2024.113763","DOIUrl":"10.1016/j.jim.2024.113763","url":null,"abstract":"<div><div>Megalin, a type I transmembrane protein, serves as a multi-ligand endocytic receptor in the apical membrane of proximal tubules. Its ectodomain and full-length forms are excreted into human urine, with the former being more abundant. We previously developed two types of sandwich enzyme-linked immunosorbent assays (ELISAs) utilizing monoclonal antibodies that target the amino-terminal ligand-binding domain-I and the carboxyl-terminal cytoplasmic region of human megalin, respectively. The former, termed “A-megalin” ELISA, primarily identifies ectodomains of megalin, whereas the latter, “C-megalin” ELISA, specifically recognizes full-length megalin originating from urinary extracellular vesicles. This study developed novel sandwich ELISAs to assess mouse urinary A-megalin and C-megalin, thereby facilitating studies involving these biomarkers in mouse disease models. Immunoblotting and immunohistochemistry of monoclonal antibodies against human megalin were performed to assess their compatibility with mouse megalin in novel sandwich ELISAs, which were constructed and validated using human assay protocols. Immunoblot analysis of megalin in urinary extracellular vesicles and supernatant was performed to investigate the ratio of ectodomain to full-length forms in mouse urine. Stable measurements having a precision and accuracy within 15 % were achieved in the measurement of quality control samples. A-megalin and C-megalin were detectable in the urine of C57BL/6 mice, whereas most urine samples from kidney-specific conditional megalin-knockout mice were below detection limits. Ectodomain forms of megalin were at least approximately 70 times more abundant than the full-length form, even in mouse urine. In conclusion, we successfully developed sandwich ELISAs for assessing mouse urinary A-megalin and C-megalin to evaluate primarily ectodomain and full-length forms of megalin, respectively.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391049","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":"A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens","authors":"","doi":"10.1016/j.jim.2024.113758","DOIUrl":"10.1016/j.jim.2024.113758","url":null,"abstract":"<div><h3>Background</h3><div><em>Bordetella pertussis</em> continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination.</div></div><div><h3>Material and methods</h3><div>Longitudinal WB samples were collected from a small set of subjects (<em>n</em> = 38) aged 7–70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated <em>B. pertussis</em> lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay.</div></div><div><h3>Results</h3><div>The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles.</div></div><div><h3>Conclusions</h3><div>The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365506","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":"Highly effective strategy for isolation of mononuclear cells from frozen cord blood","authors":"","doi":"10.1016/j.jim.2024.113762","DOIUrl":"10.1016/j.jim.2024.113762","url":null,"abstract":"<div><h3>Background aims</h3><div>Cord blood mononuclear cells (CBMCs) comprise a variety of single-nucleated cells found in the cord blood, mainly consisting of monocytes and lymphocytes. They also include a smaller proportion of other cell types, such as hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). CBMCs are vital for acquiring HSPCs, MSCs, and other immune cells, like natural killer cells. These cells are essential for supporting subsequent research and clinical applications. Although automated equipment for CBMC enrichment has shown promise, the high cost of these machines and the expense of disposable consumables limit their routine use. Furthermore, limited information is available on manual strategies for isolating CBMCs from cryopreserved cord blood. Therefore, we aimed to optimize the dilution buffer and refine the isolation procedure for CBMCs.</div></div><div><h3>Methods</h3><div>We enhanced the CBMC recovery rate from cryopreserved cord blood using an optimized dilution buffer and a modified isolation procedure.</div></div><div><h3>Results</h3><div>We achieved average recovery rates of 42.4 % and 54.3 % for CBMCs and CD34+ cells, respectively. Notably, all reagents used in the isolation procedure were of GMP-grade or pharmaceutical preparations, underscoring the potential clinical benefits of our strategy.</div></div><div><h3>Discussion</h3><div>We devised an optimized protocol suitable for routine research and clinical applications for enhanced recovery of CBMCs from cryopreserved cord blood units using an optimized dilution buffer and a modified isolation procedure.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348168","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":"Indirect ELISAs with sucrose subcellular fractions of Neospora caninum as antigens for diagnosis of neosporosis in cattle","authors":"","doi":"10.1016/j.jim.2024.113760","DOIUrl":"10.1016/j.jim.2024.113760","url":null,"abstract":"<div><div>Neosporosis is one of the major causes of abortion in cattle, and it is responsible for significant economic losses in those animals. Thus, this study aimed to evaluate indirect ELISA using subcellular fractions of <em>Neospora caninum</em> obtained via sucrose gradient separation. Eighty-five sera from dairy cattle previously tested using indirect immunofluorescence assay (IFA) were used. Three distinct bands were separated at 1.0 M, 1.4 M, 1.6 M, and the pellet at 1.8 M, which were identified as fractions one (F1), two (F2), three (F3), and four (F4), respectively. These fractions showed parasite membranes in the F1, rhoptry and conoids in the F2, mitochondria in the F3, and tachyzoite ghosts remain in F4. Indirect ELISAs for IgM, and IgG were performed. Additionally, sensitivity, specificity, and kappa values were defined considering the IFA as the gold standard. The highest and lowest specificities were observed for F1 (76 %) and F3 (16 %), respectively. F2 and F4 showed the highest sensitivity (93.3 %), kappa agreement (0.46), and Negative Preventive Value (NPV) (73 %) respectively. It was possible to standardize indirect ELISAs using whole soluble antigen and subcellular fractions of <em>N. caninum</em>, and F2 and F4 showed higher sensitivity (93.3 %), kappa (0.41), and NPV values (75 %) than F1, and F3, which could be used for epidemiology studies such as screening.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348169","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":"Candidate antibody reference reagents for Chlamydia trachomatis serology","authors":"","doi":"10.1016/j.jim.2024.113761","DOIUrl":"10.1016/j.jim.2024.113761","url":null,"abstract":"<div><div><em>Chlamydia trachomatis</em> (Ct) serology is an important tool for monitoring infection and disease burden but there are currently no formal reference reagents to harmonize results reporting. Our objective was to develop a panel of candidate reference reagents with reactivity against the major outer membrane protein (MOMP) and virulence factor (pgp3) antigens. Plasma packs from females (20–40 years old) were screened against MOMP and pgp3 antigens and selected positive and negative samples pooled to create a panel of candidate antibody reference reagents that were tested in two laboratories. Antigen specificity and internal quality assurance were also evaluated. Suitable candidate materials have been selected to produce Ct reference reagents.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142323085","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":"Isolation of phage-antibodies against Eimeria species that infect chickens","authors":"","doi":"10.1016/j.jim.2024.113759","DOIUrl":"10.1016/j.jim.2024.113759","url":null,"abstract":"<div><div><em>Eimeria</em> is one of the most economically important pathogens in poultry production. Diagnosis of infection has the potential to inform treatment and prevention strategies. Here, phage display technology was used to isolate single chain antibodies (scFvs) that had a broad specificity against oocysts from the seven pathogenic species of <em>Eimeria</em> found in poultry. Three such scFvs, representing 2 scFv HCDR3 motifs, were isolated by random picks of clones isolated after five rounds of iterative enrichment (panning) of phage against the seven <em>Eimeria</em> species. Phage-antibody binding to <em>Eimeria</em> oocysts was also interrogated using next generation sequencing of the HCDR3 region of scFv genes contained with phage particles. This analysis demonstrated that the most abundant scFv found after 5 rounds of panning accounted for over &gt;90 % of scFvs. Furthermore, the three scFvs isolated from random picks of clones were the only antibodies that were enriched through each round of panning. They were also seen to be enriched through the stages of phage panning that included binding to the <em>Eimeria</em> oocysts (selection phase) and to be selected against during the stages that consisted solely of phage propagation (growth only phase). The NGS data was further analysed to identify an additional scFv that demonstrated specific enrichment against 3 <em>Eimeria</em> species at the third round of panning and had the same pattern of enrichment during the selection and growth phases of panning. Rescue and analysis of this phage-scFv demonstrated a binder with broad specificity for Eimeria species. The four antibodies with broad specificity detected all seven <em>Eimeria</em> species in immunoassays. The binding of one such scFv that recognised all species was further validated by fluorescent microscopy.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348170","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":"Description of a non-competitive ELISA based on time course analysis of ligand binding at saturation, and a direct method for calculating the affinity of monoclonal antibodies","authors":"","doi":"10.1016/j.jim.2024.113756","DOIUrl":"10.1016/j.jim.2024.113756","url":null,"abstract":"<div><p>We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the K_t (= K_s) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format. In this kinetics, the inhibition constant K_i (= K_d) is the time required to double the slope or halve the V_max of the Lineweaver-Burk plot. The K_t values of the time courses were doubled (2 x K_t) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the K_i. The affinity constant of mAb SIM 28 was determined from ELISA data (<em>n</em> = 16) by three methods: i) doubling of K_t, ii) Beatty equation (K_aff = (n-1)/2 (n [HRP’]_t - [HRP]_t), and iii) SPR (Biacore) analysis. The calculated affinities (mean ± 95 % confidence limits) were i) 4.6 ± 0.67 × 10⁻⁹ M, ii) K_aff = 0.23 ± 0.03 × 10⁹ M⁻¹ (K_d = 4.8 ± 0.81 × 10⁻⁹ M), and iii) 4.3 ± 0.57 × 10⁻⁹ M, respectively. The similar results obtained with the three different techniques indicate that this time-course saturation ELISA, combined with the double K_t method, is a repeatable and direct approach to mAb affinity determination.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142229820","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":"Comparative analysis of LAG3 antibodies shows differential binding patterns by flow cytometry","authors":"","doi":"10.1016/j.jim.2024.113757","DOIUrl":"10.1016/j.jim.2024.113757","url":null,"abstract":"<div><h3>Background</h3><p>LAG3 is an immune checkpoint molecule with emerging therapeutic use. Expression of LAG3 is well studied on T cells, but the proportion of LAG3-expressing cells varies greatly by study and its comparative expression between non-T cells is lacking.</p></div><div><h3>Methods/objectives</h3><p>This study uses flow cytometry to compare surface LAG3 expression between T cells, NK cells, B cells, pDCs and monocytes of healthy donors. This study also compares three monoclonal LAG3 antibodies to a commonly used polyclonal LAG3 antibody on ex vivo and PHA-blasts from healthy donors and LAG3+ and LAG3- cell lines.</p></div><div><h3>Results</h3><p>LAG3 was most highly expressed on classical and intermediate monocytes (25 % and 32 %, respectively), while LAG3 expression on B cells, NK cells and iNKT cells was negligible. Notably, the polyclonal antibody stained a higher proportion of all cell types than the monoclonal antibodies, which had similar staining patterns to one another. Further study using LAG3+ and LAG3- cell lines showed greater specificity and similar sensitivity of the monoclonal antibody T47–530 than the polyclonal antibody.</p></div><div><h3>Conclusion</h3><p>Monocytes may represent an unappreciated source of LAG3 and target of LAG3 checkpoint inhibitors. Furthermore, the discrepancies between monoclonal and polyclonal LAG3 antibodies warrants consideration when designing future studies and interpreting past studies, and may explain discrepancies in the literature.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S002217592400142X/pdfft?md5=eb0d17b7b319a48b3b3cee2e6878ed81&pid=1-s2.0-S002217592400142X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142239151","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":"Editorial – Immunobiophysics: Advances and techniques","authors":"","doi":"10.1016/j.jim.2024.113755","DOIUrl":"10.1016/j.jim.2024.113755","url":null,"abstract":"","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142229819","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":"Modified radioimmunoassay versus ELISA to quantify anti-acetylcholine receptor antibodies in a mouse model of myasthenia gravis","authors":"","doi":"10.1016/j.jim.2024.113748","DOIUrl":"10.1016/j.jim.2024.113748","url":null,"abstract":"<div><p>In mouse models of myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies can be quantified to monitor disease progression and treatment response. In mice, enzyme-linked immunosorbent assay (ELISA) is the gold standard to quantify these antibodies. However, this method requires antigen purification, which is both time-consuming and expensive. In humans, radioimmunoassay (RIA)—which is more sensitive than ELISA—is commonly used to quantify AChR antibodies. At present, however, no commercial RIA kits are available to quantify these antibodies in mice. The aim of this study was to compare a modified commercial human RIA kit to two ELISA methods to detect AChR antibodies in an experimental autoimmune mouse model of MG (EAMG). C57BL/6 J mice were immunized with purified AChR from <em>Tetronarce californica</em> (T-AChR). Serum samples were analyzed by RIA and two ELISAs (T-AChR and purified mouse AChR peptide [m-AChR]). The modified RIA showed excellent sensitivity (84.1 %) and specificity (100 %) for the detection of AChR antibodies. RIA showed a good agreement with T-AChR ELISA (κ = 0.69) but only moderate agreement with m-AChR ELISA (κ = 0.49). These results demonstrate the feasibility of modifying a commercially-available RIA kit to quantify AChR antibodies in EAMG. The advantage of this technique is that it eliminates the need to develop the entire methodology in-house and reduces inter and intra-laboratory variability.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145768","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}