Journal of immunological methods最新文献

{"title":"From phlebotomy to peripheral blood mononuclear cell isolation; effect of time and early-life HIV/ART exposure on cell yield and viability in paediatric samples in a high HIV prevalence setting.","authors":"Hope Mataramvura, Patience Ncube, Kerina Duri","doi":"10.1016/j.jim.2025.113906","DOIUrl":"https://doi.org/10.1016/j.jim.2025.113906","url":null,"abstract":"<p><strong>Background: </strong>Viral infection/exposure and time from phlebotomy to peripheral blood mononuclear cell (PBMC) isolation (TPP) may affect PBMC yield and viability. We investigated the impact of TPP and early-life HIV/ART exposure on PBMC, including NK cells yield and viability.</p><p><strong>Methods: </strong>Blood in EDTA tubes with varying TPP was used to isolate PBMCs via density-gradient centrifugation, counted under a microscope, and assessed for viability with Trypan-Blue. Antibody staining (CD14, CD3, and CD19) excluded monocytes and T/B lymphocytes, while CD16 and CD56 staining with a LIVE/DEAD marker identified viable NK cells. Comparisons were made among HIV-exposed uninfected (HEU) children (long-term [HEULT] and medium-term [HEUMT] ART exposure), HIV-exposed infected (HEI), and HIV-unexposed uninfected (HUU) children.</p><p><strong>Results: </strong>We enrolled 112 children (50 % female) with a median age of 5.1 years [interquartile range (IQR):4.8-6.0], categorized as 37-HUU, 36-HEULT, 34-HEUMT, and 5-HEI children. Median blood volume was 7.5 ml (IQR:7.5-8.0), with HEI children showing slightly higher PBMC yields/ml than HUU and HEU (p = 0.092). Median TPP was 90 min (IQR:64.8-112.0), with viability remaining high (>95 %) up to 3 h, after this the yields/ml decreased compared to TPP ≤ 1 h (1.3 vs. 2.8 × 10⁶ cells/ml) (p = 0.010) for all children. Overall, TPP negatively correlated with yields/ml and viability (r = -0.35, p < 0.001; r = -0.47, p < 0.001 respectively). Median frequency of viable CD56 + CD16+/- NK cells was 8.2 %, unaffected by neither TPP nor HIV/ART exposure. Total lymphocytes were lower in males [18.6 % (IQR:11.3-22.2)] than in females [23.8 % (IQR:14.3-29.9)] (p = 0.018).</p><p><strong>Conclusion: </strong>HIV infection, not exposure, may increase PBMC yields. TPP under 3 h is ideal for optimal yields.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113906"},"PeriodicalIF":1.6,"publicationDate":"2025-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144682719","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":"Peptide-driven lateral flow platform for detection of classical swine fever viral antigen","authors":"Gyanendra Singh Sengar ,&nbsp;Rajib Deb ,&nbsp;Seema Rani Pegu ,&nbsp;Pranab Jyoti Das ,&nbsp;Archana Hazarika ,&nbsp;Swaraj Rajkhowa ,&nbsp;Vivek Kumar Gupta","doi":"10.1016/j.jim.2025.113908","DOIUrl":"10.1016/j.jim.2025.113908","url":null,"abstract":"<div><div>Classical Swine Fever (CSF) is a highly contagious viral disease that causes substantial economic losses in the swine industry. Timely detection of CSFV infection is essential for effective disease control; however, current antibody-based diagnostic methods are limited due to the delayed host immune response. In this study, we developed a novel peptide-based lateral flow assay (LFA) targeting the E2 antigen of the Classical Swine Fever Virus (CSFV) to enable rapid and field-deployable detection. A synthetic peptide derived from the immunodominant region of the CSFV E2 protein was identified through bioinformatics analysis, synthesized, and conjugated to Keyhole Limpet Hemocyanin (KLH) to enhance its immunogenicity. Polyclonal antibodies were generated in rabbits, purified, and validated via ELISA to confirm specificity to CSFV, showing no cross-reactivity with other swine viruses such as ASFV, PRRSV, PCV2, and PPV. Silver nanoparticles (AgNPs) were biosynthesized using <em>Mangifera indica</em> leaf extract and characterized by UV–vis spectroscopy, FTIR, and dynamic light scattering (DLS). These nanoparticles were then conjugated to the purified anti-E2 antibodies and incorporated into LFA strips. The resulting assay exhibited high sensitivity, capable of detecting CSFV antigen concentrations as low as 20 ng/ml, with excellent specificity and no cross-reactivity. This peptide-based LFA provides a rapid, reliable, and cost-effective diagnostic tool for on-site CSF surveillance and outbreak management.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113908"},"PeriodicalIF":1.6,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144674962","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 assessment of a multiplex micro-chip immunoassay, VaxArray, and meso scale discovery assay for serotype-specific coronavirus IgG quantitation","authors":"Ashvi Sanjay Jain ,&nbsp;Thorsten Verch ,&nbsp;Gowrisankar Rajam ,&nbsp;Ying Homan","doi":"10.1016/j.jim.2025.113907","DOIUrl":"10.1016/j.jim.2025.113907","url":null,"abstract":"<div><div>Immunoassays for serology have advanced from radio-immunoassays to multiplexed micro-array-based technologies over the past years. Comparative studies can assist users in selecting an assay platform based on its applicability and performance characteristics. This study compared a 9-plex commercial VaxArray Coronavirus SeroAssay (InDevR) and a 10-plex semi-custom Coronavirus SeroAssay (MSD) with a panel of human serum samples. The MSD assay showed superior dynamic range and sensitivity across all coronavirus serotypes, while both assays met accuracy (100 ± 20 %) and precision (%CV &lt; 25 %) criteria. Although both platforms were concordant for anti-SARS-CoV2 IgG measurements, the clinical sensitivity and specificity of the MSD assay were marginally higher than those of VaxArray. Despite VaxArray's shorter total assay time and higher potential for multiplexing and re-readability, it requires improvements in dynamic range, sensitivity, specificity, and automation capabilities for regulated use.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"543 ","pages":"Article 113907"},"PeriodicalIF":1.6,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144674961","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 in-silico-designed multiepitope vaccine candidate can efficiently protect mice against pathogenic species of Shigella","authors":"Amir Namvar Vansofla,&nbsp;Abbas Hajizade,&nbsp;Shahram Nazarian,&nbsp;Yousof Tarverdizadeh,&nbsp;Ahad Shahmaleki","doi":"10.1016/j.jim.2025.113905","DOIUrl":"10.1016/j.jim.2025.113905","url":null,"abstract":"<div><div><em>Shigella</em> species remain a major global health concern, causing gastrointestinal infections with significant morbidity and mortality. The rise of antibiotic-resistant <em>Shigella</em> strains underscores the urgent need for effective vaccines. This study evaluated the immunogenicity and protective efficacy of a novel multi-epitope recombinant protein vaccine against pathogenic <em>Shigella</em> species in a murine model. The multi-epitope antigen was expressed in <em>E. coli</em> and purified for immunization. BALB/c mice were subcutaneously immunized, and immune responses were assessed through serum IgG quantification (ELISA) and challenge studies. Immunized mice exhibited significantly higher antigen-specific IgG titers compared to controls (<em>p &lt; 0.05</em>). Subsequent challenge with 10LD₅₀ of virulent <em>Shigella flexneri</em> and <em>Shigella boydii</em> demonstrated robust protection, with vaccinated mice surviving lethal infections. Our findings indicate that this in silico-designed multi-epitope vaccine elicits potent humoral immunity and confers cross-species protection against clinically relevant <em>Shigella</em> pathogens. These results support its potential as a promising candidate for further vaccine development against shigellosis.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113905"},"PeriodicalIF":1.6,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144663346","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 high-throughput assay using dysfunctional T cells for phenotypic screening immune checkpoint modulators","authors":"Zhiyu Wang ,&nbsp;Junjie Zhao ,&nbsp;William John Huth ,&nbsp;Edwina Naik ,&nbsp;Kaylee Choi","doi":"10.1016/j.jim.2025.113904","DOIUrl":"10.1016/j.jim.2025.113904","url":null,"abstract":"<div><div>During a prolonged response to virus infection or tumorigenesis, effector T cells are persistently stimulated and become dysfunctional. Dysfunctional T cells overexpress inhibitory regulators, exhibit decreased effector cytokine production, and lose robust effector functions, leading to the failure of antigen/cancer elimination. In the present study, we established a protocol to generate dysfunctional T cells from human peripheral blood mononuclear cells (PBMCs) and developed a fully automated high-throughput assay in 384-well format to assess immune checkpoint modulators. Specifically, PBMCs were maintained with interleukin-2 (IL-2) and <em>Phaseolus Vulgaris</em> Leucoagglutinin (PHA-L) to induce dysfunctional T cells. Cell populations were subsequently examined and quantified using flow cytometry, and validated T cells were used for the further assay development. To assess compound activity, cells were pre-incubated with testing compounds for 24 h, followed by a stimulation with anti-CD3 and anti-CD28. The cell supernatants were then harvested for cytokine measurements (<em>e.g.</em>, IL-2 and interferon γ (IFN-γ)) using AlphaLISA assays to evaluate the activation of T cell receptor signaling. Cell lysates in the same assay plate were used for a viability assay to assess cytotoxicity. This workflow was implemented for assessing 15 compounds selected based on cellular pathways they modulate, of which 2 compounds promoted IL-2 and IFN-γ production in dysfunctional T cells upon activation, suggesting potential therapeutic values against T cell dysfunction.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113904"},"PeriodicalIF":1.6,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144653396","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 a chimeric monoclonal antibody as a potential diagnostic reference in PR3-ANCA detection","authors":"Mingming Zhang ,&nbsp;Youtao Zhang ,&nbsp;Fangming Cheng ,&nbsp;Hong Chen ,&nbsp;Yonghong Yang ,&nbsp;Li Li ,&nbsp;Yuzhang Jiang ,&nbsp;Yaping Dai ,&nbsp;Fang Qiu ,&nbsp;Zhuye Qin ,&nbsp;Chongxu Han ,&nbsp;Gang Wang ,&nbsp;Xingjuan Shi ,&nbsp;Chungen Qian ,&nbsp;Xiangdong Liu","doi":"10.1016/j.jim.2025.113903","DOIUrl":"10.1016/j.jim.2025.113903","url":null,"abstract":"<div><h3>Background</h3><div>The quantification of antineutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) is crucial for diagnosing and monitoring granulomatosis with polyangiitis (GPA). However, a standardized and widely available quantitative reference material for PR3-ANCA detection remains lacking.</div></div><div><h3>Objective</h3><div>This study aims to develop and evaluate the properties of a novel chimeric human-mouse PR3 monoclonal antibody as a potential quantitative reference for PR3-ANCA detection using various in vitro diagnostic methods.</div></div><div><h3>Methods</h3><div>Mouse monoclonal antibodies were screened from hybridoma cells derived from mice immunized with recombinant human PR3 (rPR3). A high-affinity monoclonal antibody (A07) was sequenced, and a chimeric antibody incorporating human IgG1 constant regions was produced using a mammalian cell expression system. The antibody's binding to PR3 and its applicability in indirect immunofluorescence (IIF) assay, line-blotting, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA) were analyzed.</div></div><div><h3>Results</h3><div>The chimeric A07 antibody recognized the conformational epitopes with high affinity for only non-denatured PR3 and demonstrated the correct cytoplasmic staining pattern in IIF. Line-blotting assays confirmed its specificity and potency. ELISA and CLIA experiments showed well-defined standard curves, with the chimeric antibody displaying consistent reactivity comparable to native PR3-ANCA in serum samples across different antigen conditions.</div></div><div><h3>Conclusions</h3><div>These findings indicate that the chimeric A07 antibody effectively recognizes both recombinant and native PR3 antigens and can be applied across multiple in vitro PR3-ANCA assays. Furthermore, this antibody may serve as a standard reference for determining serum PR3-ANCA titers or as a substitute for PR3-positive serum in diagnostic applications.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113903"},"PeriodicalIF":1.6,"publicationDate":"2025-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144642682","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 ELISA strategy for the serological diagnosis of farmer's lung disease","authors":"Adeline Rouzet ,&nbsp;Eliane Devillers ,&nbsp;Coralie Barrera ,&nbsp;Emeline Scherer ,&nbsp;Laurence Millon ,&nbsp;Anne-Pauline Bellanger","doi":"10.1016/j.jim.2025.113902","DOIUrl":"10.1016/j.jim.2025.113902","url":null,"abstract":"<div><h3>Context</h3><div>Farmer's lung disease (FLD) is a form of hypersensitivity pneumonitis due to regular exposure to microbial antigens present in moldy hay. Serological diagnosis is usually based on immunoprecipitation techniques using 1 to 12 in-house antigens. To improve standardization, recombinant antigens (r-Ags) have been produced from three etiological agents involved in FLD.</div></div><div><h3>Aim</h3><div>The aim of this study was to develop a strategy using an ELISA test for the serological diagnosis of FLD.</div></div><div><h3>Methods</h3><div>The ELISA strategy was developed on an initial group of patients and then validated using sera from 29 patients with FLD and 54 exposed but non-FLD patients. Using sera from the second group of patients, the ELISA was simplified by using purified antigens from <em>Saccharopolyspora rectivirgula</em>, <em>Lichtheimia corymbifera</em>, and <em>Eurotium amstelodami</em>, each mixed with a corresponding r-Ag. The performance of the ELISA was evaluated according to the antigens used and also associated with an immunoprecipitation technique.</div></div><div><h3>Results</h3><div>The ELISA test using three purified antigens (PAgs) mixed with 3 r-Ags showed a sensitivity of 97 % and a specificity of 83 % (AUC: 0.84). The use of ELISA as a screening technique and double diffusion (using 6 somatic antigens) as a confirmatory technique in cases of moderate or positive ELISAs showed a sensitivity of 93 % and a specificity of 84 % (AUC: 0.86).</div></div><div><h3>Conclusion</h3><div>The use of a mixture of purified protein antigens and highly standardized recombinant antigens from three etiological agents involved in FLD offers an effective and innovative strategy for the serological diagnosis of FLD cases. This strategy has been successfully applied in the routine workflow of our laboratory.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113902"},"PeriodicalIF":1.6,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144491070","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 unexpected bioanalytical challenge caused by positive control antibodies in a clinical immunogenicity assay – A simple solution and broadly applicable recommendations","authors":"John Hok Nin Lowe,&nbsp;Jenny Yanhong Li,&nbsp;Mauricio Maia","doi":"10.1016/j.jim.2025.113901","DOIUrl":"10.1016/j.jim.2025.113901","url":null,"abstract":"<div><div>Anti-drug antibody (ADA) assays are an important element in the suite of bioanalytical methods required for assessment of the safety and efficacy of recombinant-protein therapeutics. As such, and following extensive optimization, there is an expectation that clinical ADA assays be fully validated for multiple performance parameters, including sensitivity, specificity, reagent stability, and robustness. Among critical reagents used in ADA assays, ADA positive controls (PCs) play a crucial role in multiple stages of assay development and validation, including selection of assay format, establishing assay cut-points, estimating assay relative sensitivity, assessing assay precision, as well as ensuring acceptable assay performance during sample testing. This manuscript highlights an unexpected and highly-impactful PC performance inconsistency attribute we encountered prior to validation of a clinical ADA assay. We also describe the investigation that identified the root cause of this problem: the immune complexation between the murine monoclonal antibody used as the surrogate PC and human anti-mouse antibody present in the serum used to prepare the PC. We conclude that murine monoclonal antibodies are not fully appropriate reagents for use as PCs in clinical ADA assays. Finally, potential approaches to circumvent or mitigate this specific problem in clinical ADA assays are discussed.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113901"},"PeriodicalIF":1.6,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505931","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 microfluidic ELISA for measuring humoral responses to clostridial antigens in vaccinated cattle","authors":"Joo Youn Park ,&nbsp;Amelia Woolums ,&nbsp;Robert Wills ,&nbsp;Rhonda Vann ,&nbsp;Keun Seok Seo","doi":"10.1016/j.jim.2025.113900","DOIUrl":"10.1016/j.jim.2025.113900","url":null,"abstract":"<div><div>Clostridial diseases significantly threaten livestock health, particularly in cattle, underscoring the need for effective vaccination strategies. This study develops and optimizes a microfluidic-based enzyme-linked immunosorbent assay (ELISA) for the rapid (&lt; 1 h) and cost-effective measurement of IgG antibody levels against various clostridial toxins in cattle vaccinated with a multivalent clostridial vaccine. This assay requires only 5 μL of sample and reagent volume, demonstrating high repeatability and reproducibility with coefficient of variation (CV) values ranging from 0.1 % to 8.5 % across all tested clostridial antigens. The Limit of Detection (LOD) of the assay ranged from 1:150 to 1:800, allowing for sensitive detection of antibody levels. For <em>Clostridium perfringens</em> ε toxin, antibody titers were measured using a commercial kit, while microfluidic ELISA was applied to assess titers against tetanus toxoid, <em>Clostridium septicum</em> α toxin, <em>Clostridium novyi</em> type B toxins, and <em>Clostridium sordellii</em> toxins. Significant increases in IgG antibody levels were observed for <em>C. perfringens</em> ε toxin and tetanus toxoid following both primary and booster vaccine doses, peaking by day 42. Antibody titers against <em>C. septicum</em> α toxins and <em>C. novyi</em> type B toxins increased after the primary dose, peaking at day 42, while no booster effect was seen for <em>C. sordellii</em>. These findings highlight the utility of microfluidic ELISAs as a practical and efficient tool for assessing humoral immune responses to clostridial toxins in vaccinated cattle, with potential application for the herd level disease surveillance and vaccine efficacy assessment.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113900"},"PeriodicalIF":1.6,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144497233","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 vectors for reformatting scFv fragments derived from phage display libraries into native IgG1 structures for in vivo imaging and therapeutic applications","authors":"Marton Fogarasi ,&nbsp;Corina Roman ,&nbsp;Simona Dima","doi":"10.1016/j.jim.2025.113899","DOIUrl":"10.1016/j.jim.2025.113899","url":null,"abstract":"<div><div>Monoclonal antibodies are a crucial class of therapeutic agents utilized for the treatment of various disorders. Typically, fully human therapeutic antibodies are created using the antibody fragment phage display from naive libraries and are selected against the antigen in a format known as the single-chain Fv (scFv) fragment. To be employed in immunotherapy, the scFv fragment needs to be reformatted into the complete IgG structure with a native-like conformation and expressed in mammalian cells. In this context, we have established an expression system for reformatting scFv fragments into complete IgG1 molecules, enabling both antibody chains to be cloned within the same vector. These constructs are based on the IgGγ1 heavy chain, with the light chain belonging to either the lambda or kappa isotype. For <em>in vivo</em> imaging, we have designed vectors with specific mutations in the IgG1 lower hinge region and C_H2 domain to impede the antibody's effector function, ensuring that immune cells are not activated. The efficiency of this expression system was evaluated by producing two antibodies: one with the lambda light chain and the other with the kappa light chain. Their expression levels were assessed in HEK-293 and CHO-K1 cell lines. Structurally, the resulting antibodies are properly assembled and folded into their quaternary structure, while functionally, these immunoglobulins specifically recognize the antigens and inhibit cancer cell invasion.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"542 ","pages":"Article 113899"},"PeriodicalIF":1.6,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144366071","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}