Journal of immunological methods最新文献

CHO-S expression of a novel human recombinant IgG1 of anti-ALD antibody isolated by phage mutation display. 用 CHO-S 表达通过噬菌体突变展示分离的新型人重组抗ALD 抗体 IgG1。

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-25 DOI: 10.1016/j.jim.2025.113843

Guilin Li, Jiazhen Liu, Zhenzhen Guan, Sujuan Wang, Songrui Li, Zhiqiang Wang, Yanna Dong, Yamin Cui, Yaya Li, Weitao Zhang, Xiaoping Tian, Qiaohui Zhao

{"title":"CHO-S expression of a novel human recombinant IgG1 of anti-ALD antibody isolated by phage mutation display.","authors":"Guilin Li, Jiazhen Liu, Zhenzhen Guan, Sujuan Wang, Songrui Li, Zhiqiang Wang, Yanna Dong, Yamin Cui, Yaya Li, Weitao Zhang, Xiaoping Tian, Qiaohui Zhao","doi":"10.1016/j.jim.2025.113843","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113843","url":null,"abstract":"<p><p>Aldosterone (ALD) is elevated in the serum and plasma of patients with various metabolism-related diseases, and anti-ALD antibodies are important materials in early diagnostic kits. However, current anti-ALD antibodies for clinical diagnosis are not specific, resulting in high false positives. We have developed an anti-ALD-601# rabbit monoclonal antibody that is commercially available and licensed for diagnosis. To obtain monoclonal antibodies with better stability and affinity, a phage mutation library was constructed by site-specific mutagenesis of the CDR3 region in the V<sub>L</sub> and V<sub>H</sub> domain of the anti-ALD-601# antibody. Anti-ALD3302#, a single-chain variable fragment (scFv), with enhanced affinity, was screened by enzyme-linked immunoassay (ELISA). To improve the less stable and shorter half-lives of scFvs, anti-ALD-scFv-3302# was inserted into a eukaryotic expression vector (pCHO1.0-Fc) and then transfected into CHO-S cells to express anti-ALD-scFv-Fc. Then, cells were cultured by reducing culture temperature (33.5 °C) and application of CuSO<sub>4</sub> (50-200 μM) treatment. The expression-purified antibody was detected with competitive-ELISA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and capillary electrophoresis. Non-reducing SDS-PAGE electrophoresis showed a single band and the percentage of the highest peaks in capillary electrophoresis was more than 90 %. The coefficient of variation in the competitive-ELISA assay was reduced to 2 % from 5 % before optimization. The correlation between the assay using this monoclonal antibody and the HPLC-FLD determination was also greater than 0.99. In conclusion, the designed anti-ALD-specific antibody (3302#) has been successfully developed for use in metabolism-related diseases to detect the concentration of ALD in early diagnostic.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113843"},"PeriodicalIF":1.6,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143523681","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}

引用次数: 0

Preparation and specific identification of monoclonal antibodies targeting the VP2 structural protein of minute virus of canines

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-15 DOI: 10.1016/j.jim.2025.113833

Xiang Ren , Zhiping Hei , Kai Ji , Jianhui Guo , Yan Yan , Yuning Sun

{"title":"Preparation and specific identification of monoclonal antibodies targeting the VP2 structural protein of minute virus of canines","authors":"Xiang Ren , Zhiping Hei , Kai Ji , Jianhui Guo , Yan Yan , Yuning Sun","doi":"10.1016/j.jim.2025.113833","DOIUrl":"10.1016/j.jim.2025.113833","url":null,"abstract":"<div><div>The Minute Virus of Canines (MVC), classified under the genus Bocaparvovirus, causes severe respiratory and gastrointestinal symptoms in neonatal canines worldwide. The structural protein VP2 is essential for the attachment, infection, uncoating, and induction of the host immunological response to MVC. This study aimed to prepare a monoclonal antibody (mAb) against VP2 using the hybridoma technique. AlphaFold and CavityPlus bioinformatics analysis revealed that the N-terminal region of VP2 (amino acids 1–300) possesses structural characteristics that make it the most suitable target for effective antibody generation. The recombinant plasmid pET-32a(+)-VP2(N300) with fused Trx and His tags was successfully constructed. After immunizing mice, nine hybridoma cell lines were obtained, namely 1G5, 1G5–1, 1I24, 1I24–1, 2E6–1, 2 N9, 3C12–1, 4 M1, and 4 M1–1. The ascitic antibody titers of all cell lines were above 1:100,000. Western blot analysis of Walter Reed canine cells infected with MVC indicated the selection of three strains of monoclonal antibodies with strong specificity: 1G5, 3C12–1, and 4 M1. These three strains can be employed in immunofluorescence (IF) and immunoprecipitation (IP) tests for detecting VP2 protein. The monoclonal antibody mAb VP2 prepared in this study may serve as a valuable tool for detecting MVC and beneficial for investigating the mechanisms of MVC infection.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113833"},"PeriodicalIF":1.6,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143437190","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}

引用次数: 0

Comprehensive normalization and binary classification methods for enhanced sensitivity and reproducibility in Luminex assay quantitation

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-08 DOI: 10.1016/j.jim.2025.113826

B.A. Burns , C.A. Shaw , M. Chandra , C.S. Forconi , A.M. Moormann , V. Konduri , V.N. Tubman , W.K. Decker

{"title":"Comprehensive normalization and binary classification methods for enhanced sensitivity and reproducibility in Luminex assay quantitation","authors":"B.A. Burns , C.A. Shaw , M. Chandra , C.S. Forconi , A.M. Moormann , V. Konduri , V.N. Tubman , W.K. Decker","doi":"10.1016/j.jim.2025.113826","DOIUrl":"10.1016/j.jim.2025.113826","url":null,"abstract":"<div><div>The Luminex assay is a powerful tool for large-scale quantitation of antibody levels and cytokines, but its utility can be limited by issues of specificity, sensitivity, and reproducibility. The corrections for background fluorescence and machine drift are essential steps in the normalization process. However, traditional methods often oversimplify these steps, failing to account for the complexity of the data, leading to the introduction of error and decreasing the sensitivity and reproducibility of the analysis. Furthermore, conventional methods to determine cut-points in binary measures do not consider the true distribution of the data, leading to arbitrary cut-points that compromise the integrity of the analysis. Here, we present a novel approach to normalize Luminex data and split the normalized bimodal data. Our method uses orthogonal regression of the measured fluorescence of a negative control bead and a blank bead to correct for background fluorescence, enhancing accuracy by preventing overcorrection due to cross-reactivity. To account for machine drift, we use a generalized additive model (GAM) on the standard curves to calculate a plate correction, thus reducing error and improving reproducibility. To distinguish between positive and negative results in bimodal measures, we use a clustering analysis to accurately split the data based on distribution. Finally, we developed a web application to easily carry out the developed method. These methods collectively increase sensitivity, specificity, and reproducibility of Luminex assay data analysis by effectively addressing the limitations of current normalization techniques, correcting for background fluorescence and machine drift, and improving the specificity and accuracy in splitting bimodal data.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113826"},"PeriodicalIF":1.6,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143391098","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}

引用次数: 0

Complement function and activation in human serum and plasma collected in different blood collection tubes

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-05 DOI: 10.1016/j.jim.2025.113825

Linnea I. Andersson , Dick J. Sjöström , Ricardo J.M.G.E. Brandwijk , Erik J.M. Toonen , Tom Eirik Mollnes , Per H. Nilsson

{"title":"Complement function and activation in human serum and plasma collected in different blood collection tubes","authors":"Linnea I. Andersson , Dick J. Sjöström , Ricardo J.M.G.E. Brandwijk , Erik J.M. Toonen , Tom Eirik Mollnes , Per H. Nilsson","doi":"10.1016/j.jim.2025.113825","DOIUrl":"10.1016/j.jim.2025.113825","url":null,"abstract":"<div><div>Complement analysis necessitates strict control of pre-analytical blood handling, including time, temperature, and additives. Here, we compared complement function and activation status across five different serum preparations and two plasma preparations. Serum was collected from ten healthy volunteers using glass tubes without additives, tubes with a silica clot activator (with or without a gel separator), and tubes containing thrombin (with or without a gel separator). Plasma was collected in the presence of EDTA or the thrombin inhibitor lepirudin. Serum and plasma aliquots were snap-frozen in liquid nitrogen and stored at −80 °C. Complement functional analysis was performed using Wieslab and Hycult Biotech pathway-specific assays. Complement activation was determined by quantifying specific activation markers: C1s/C1-INH, MASP-1/C1-INH, C3bc, C3bBbP, and sC5b-9. All serum samples exhibited increased complement activation compared to EDTA and lepirudin plasma, with serum tubes containing thrombin and gel separators showing the highest levels of complement activation. However, normal complement function was observed in all serum preparations, indicating that the complement activation and consumption that occurred did not affect complement functional analysis. While all tested serum tubes provided accurate functional activity, the type of tube and the presence of additives like thrombin and gel separators significantly influenced the degree of complement activation. We recommend preparing functionally active serum either in glass tubes or in silica clot activator tubes, and avoiding gel separators. For complement activation studies, lepirudin plasma is preferable over serum due to its complement functional capacity, low level of complement activation, and lack of excessive hemostatic activation.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113825"},"PeriodicalIF":1.6,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143374013","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}

引用次数: 0

Basophil activation test; User's manual

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-01 DOI: 10.1016/j.jim.2025.113815

Soren Ulrik Sonder , Matthew Plassmeyer , Nikhila Schroeder , Steven Peyton , Mikell Paige , Michael Girgis , Hamed Safi , Oral Alpan

{"title":"Basophil activation test; User's manual","authors":"Soren Ulrik Sonder , Matthew Plassmeyer , Nikhila Schroeder , Steven Peyton , Mikell Paige , Michael Girgis , Hamed Safi , Oral Alpan","doi":"10.1016/j.jim.2025.113815","DOIUrl":"10.1016/j.jim.2025.113815","url":null,"abstract":"<div><div>Immediate allergic responses, orchestrated by basophils and mast cells, are pivotal in severe allergic reactions. The flow cytometry-based Basophil Activation Test (BAT) is a clinically important assay for testing allergic reactions using CD63 and CD203c as endpoints. The test measures the concentration dependent response to the allergens providing a functional readout of the patients' allergies. BAT is presently in clinical use within the Unites States as well as several other countries for the diagnosis and monitoring of allergies, most commonly against food allergens. This article details assay validation, both analytical and clinical with reference to existing regulations/recommendations through CAP, CLIA, NYS and CLSI on issues including accuracy, precision, linearity, reportable range, reference range, analytical sensitivity & specificity, pre-analytical considerations, the utility of the assay in diagnosis or monitoring and interpretation of the results; and the assay's limitations.</div><div>The BAT plays a crucial role in assessing patient suitability for food challenges and therapies such as oral immunotherapy, sublingual immunotherapy or omalizumab and can aid in predicting treatment outcomes. We further review the current research on advancing the test, focused on improvements in its clinical utility. Continuous efforts are warranted for enhanced regulatory oversight and comprehensive clinical validation, ensuring BAT's seamless integration into diverse clinical settings.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113815"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039084","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}

引用次数: 0

Clinical utility of the lymphocyte proliferation assay, an in vitro functional readout of the adaptive immune response

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-01 DOI: 10.1016/j.jim.2025.113819

Eszter Lázár-Molnár , Lisa K. Peterson

{"title":"Clinical utility of the lymphocyte proliferation assay, an in vitro functional readout of the adaptive immune response","authors":"Eszter Lázár-Molnár , Lisa K. Peterson","doi":"10.1016/j.jim.2025.113819","DOIUrl":"10.1016/j.jim.2025.113819","url":null,"abstract":"<div><div>Despite great advancements in the discovery of genetic variants underlying inborn errors of immunity, functional assessment of the immunological profile of patients in routine clinical practice remains challenging.</div><div>The lymphocyte proliferation assay using <sup>3</sup>H-thymidine incorporation has been the gold standard for decades for functional evaluation of T cells in the clinical laboratory, however, recently developed flow cytometric methods allowing for single cell analysis provide non-radioactive alternatives. Understanding the technical and analytical challenges of test development, validation and maintenance is essential for correct interpretation and test utilization, to assure appropriate and timely patient care. This review will discuss the technological aspects, validation and clinical utilization of <em>in vitro</em> lymphocyte proliferation assays performed in the clinical immunology laboratory, providing a diagnostic readout for T lymphocyte function, an essential hallmark of a functional cellular immune response, and allowing for the detection of impaired responses such as in patients with functional T cell defects.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"537 ","pages":"Article 113819"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039243","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}

引用次数: 0

Overcoming cross-reactivity of antibodies against human lactate dehydrogenase

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-01 DOI: 10.1016/j.jim.2025.113821

Akira Kondo, Ayumu Nakamura

{"title":"Overcoming cross-reactivity of antibodies against human lactate dehydrogenase","authors":"Akira Kondo, Ayumu Nakamura","doi":"10.1016/j.jim.2025.113821","DOIUrl":"10.1016/j.jim.2025.113821","url":null,"abstract":"<div><div>Lactate dehydrogenase subunit A (LD-A) plays an important role in cancer regulation and therapy. We attempted to develop an enzyme-linked immune-solvent assay (ELISA) for LD-A in human serum.</div><div>However, commercial antibodies against LD-A exhibited cross-reactivity with an unknown protein. The unknown protein was purified and characterized by protein sequencing and Western blotting. In addition, we attempted to prepare a specific antibody for the ELISA using partially synthesized peptides of LD-A as immunogens. The epitope position in LD-A was carefully selected based on bioinformatics analysis.</div><div>Peptide sequencer elucidated a ten amino acid sequence of the purified protein at the N-terminal. A BLAST search revealed that this sequence perfectly matched that at the N-terminus of the IgG heavy chain (H-chain). Furthermore, we demonstrated that twelve commercially available antibodies targeting LD-A or LD-B (subunit B) primarily cross-reacted with IgG or its H-chain, with only one specific antibody for each subunit. As the specific antibody against LD-A is no longer commercially accessible, we successfully produced two kinds of specific antibodies using partially synthesized LD-A peptides as immunogens.</div><div>In conclusion, we have successfully produced specific antibodies against LD-A. Moreover, our findings underscore the utility of bioinformatics tools for determining the optimal positions of immunizing peptides.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"538 ","pages":"Article 113821"},"PeriodicalIF":1.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143123061","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}

引用次数: 0

Diagnostic testing for hemophagocytic lymphohistiocytosis

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-01 DOI: 10.1016/j.jim.2025.113816

Rebecca A. Marsh , Jack J. Bleesing , Samuel Cern Cher Chiang

引用次数: 0

Functional immunological assays

IF 1.6 4区医学

Journal of immunological methods Pub Date : 2025-02-01 DOI: 10.1016/j.jim.2025.113822

Attila Kumánovics , Medhat Askar , Amir A. Sadighi Akha

引用次数: 0

The Fas-mediated apoptosis assay: From concept to clinical application