Brenda Pei Chui Song, Jing Yi Lai, Yee Siew Choong, Nafiseh Khanbabaei, Andreas Latz, Theam Soon Lim
{"title":"Isolation of anti-Ancylostoma-secreted protein 5 (ASP5) antibody from a naïve antibody phage library.","authors":"Brenda Pei Chui Song, Jing Yi Lai, Yee Siew Choong, Nafiseh Khanbabaei, Andreas Latz, Theam Soon Lim","doi":"10.1016/j.jim.2024.113776","DOIUrl":"https://doi.org/10.1016/j.jim.2024.113776","url":null,"abstract":"<p><p>Ancylostoma species are parasitic nematodes that release a multitude of proteins to manipulate host immune responses to facilitate their survival. Among the released proteins, Ancylostoma-secreted protein 5 (ASP5) plays a pivotal role in mediating host-parasite interactions, making it a promising target for interventions against canine hookworm infections caused by Ancylostoma species. Antibody phage display, a widely used method for generating human monoclonal antibodies was employed in this study. A bacterial expression system was used to produce ASP5 for biopanning. A single-chain fragment variable (scFv) monoclonal antibody against ASP5 was generated from the naïve Human AntibodY LibrarY (HAYLY). The resulting scFv antibody was characterized to elucidate its antigen-binding properties. The identified monoclonal antibody showed good specificity and binding characteristics which highlighting its potential for diagnostic applications in combating hookworm infections.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113776"},"PeriodicalIF":1.6,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142648023","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":"Inducing and regulating human naive CD4<sup>+</sup> t cell proliferation by different antigen presenting cells.","authors":"Fabienne Mazerolles, Frédéric Rieux-Laucat","doi":"10.1016/j.jim.2024.113775","DOIUrl":"https://doi.org/10.1016/j.jim.2024.113775","url":null,"abstract":"<p><p>We have shown in previous studies that naive CD4<sup>+</sup> T cells isolated from human peripheral blood are induced to proliferate by CD4<sup>neg</sup>CD11c<sup>+</sup>CD14<sup>neg</sup>CD16<sup>neg</sup> dendritic cells presenting the superantigen SEE. Since this population is very poorly expressed in blood, we tried to find other antigen presenting cells (APCs) to induce this proliferation. The aim of the previous studies was to investigate the regulation of T cell proliferation in pediatric monogenic autoimmune diseases and the regulation of this proliferation by regulatory T cells (TREGs). Since the blood samples from pediatric patients were very small, it was important to study other APCs that are more commonly present in the blood. In this study we tested different APCs isolated from controls, CD19<sup>+</sup> B cells, CD11c<sup>+</sup>CD14<sup>+</sup> and CD11c<sup>+</sup>CD14<sup>neg</sup> monocytes, CD11c<sup>+</sup>CD14<sup>neg</sup>CD16<sup>+</sup> and CD16<sup>neg</sup> dendritic cells. The different T cell populations, naive effector T cells and regulatory T cells were separated simultaneously from the same sample. We show in these studies that CD19<sup>+</sup> B cells, CD14<sup>neg</sup> and more specifically CD14<sup>neg</sup>CD16<sup>+</sup>, are also able to induce T cell proliferation as previously described with CD14<sup>neg</sup>CD16<sup>neg</sup> DCs, but under different conditions. No proliferation was induced with CD14<sup>+</sup> monocytes. However, these three APCs are less potent than CD16<sup>neg</sup> and inhibition by TREG is more difficult to detect. In addition, when we test the role of CTLA-4 in the regulation of TEFF proliferation, we observe that for some APCs, the inhibition by CTLA-4 was quite different. No inhibition was observed with CD19<sup>+</sup> B cells in contrast to CD11c<sup>+</sup>CD14<sup>neg</sup>CD16<sup>+</sup> and CD11c<sup>+</sup>CD14<sup>neg</sup>CD16<sup>neg</sup> .</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113775"},"PeriodicalIF":1.6,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142639145","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 note of caution for using calmodulin antibodies","authors":"Mads Munk, Martin W. Berchtold","doi":"10.1016/j.jim.2024.113772","DOIUrl":"10.1016/j.jim.2024.113772","url":null,"abstract":"<div><div>Calmodulin (CaM) is a ubiquitous intracellular calcium receptor that regulates a plethora of cellular functions through interactions with target proteins. In mammals, an identical Calmodulin protein is expressed by 3 independent genes (CALM1, CALM2, CALM3). Therefore, antibodies generated against either of the three products (CaM1, CaM2, CaM3) of these genes cannot be distinguished, and conclusions based on the supposedly specific CaM antibodies claiming functions of one of the 3 genes may be misleading. In this paper we present 44 articles, using such antibodies for Western blot, ELISA assay, immunohistochemistry or which are based on proteomics and the use of databases with incorrect annotations, all potentially reaching misleading conclusions. This should be taken as a note of caution for researchers working with Calmodulin antibodies and misleading databases.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113772"},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567997","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}
Juliana Pascarelli Compan Boechat , Felipe Rodrigues Semcovici Ramos , Felipe Betoni Saraiva , Vinicius de Lima Gonçalves , Franklin Souza da Silva , Carlos Roberto Alves , José Procópio Moreno Senna , Haroldo Cid da Silva Júnior
{"title":"Generation and characterization of a monoclonal antibody Fab fragment targeting PBP2a in methicillin-resistant Staphylococcus aureus","authors":"Juliana Pascarelli Compan Boechat , Felipe Rodrigues Semcovici Ramos , Felipe Betoni Saraiva , Vinicius de Lima Gonçalves , Franklin Souza da Silva , Carlos Roberto Alves , José Procópio Moreno Senna , Haroldo Cid da Silva Júnior","doi":"10.1016/j.jim.2024.113774","DOIUrl":"10.1016/j.jim.2024.113774","url":null,"abstract":"<div><div>Methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) is one of the main pathogens associated with nosocomial and community infections that are difficult to treat owing to its resistance to all β-lactams and other classes of antibiotics. Reports of MRSA demonstrate the pathogen relevance and urgency for developing innovative diagnostic and treatment strategies against this microorganism. In this context, monoclonal antibodies (mAbs) represent a powerful tool for such purposes. Beta-lactam resistance in MRSA is caused by penicillin-binding protein 2a (PBP2a). The characteristics of PBP2a make this protein a potential target for immunobiologicals to combat this pathogen. This study describes the development of a recombinant Fab fragment from a mAb directed against the PBP2a protein, designed to identify and treat MRSA infections. The Fd and light chain coding sequences for Fab expression were amplified and ligated into the mammalian cell expression vector. Recombinant DNA constructs were used to transfect Expi293F cells expressing anti-PBP2a Fab. A purification based on ion-exchange chromatography was used for Fab separation, followed by analysis of antigen target recognition and interaction, either with the isolated antigen or with the antigen on the MRSA cell surface. The experimental approach allowed us to obtain significant Fab expression levels in the Expi293F system when transfecting the cells with the genetic constructs developed in pCDNA3.4 vector. Antigen target interaction assays revealed the capacity of Fab to recognize and interact with the PBP2a protein. Biodistribution analysis indicated serum Fab presence, in the serum, kidneys, lungs, and spleen, and a plasma half-life averaging 6–8 h.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113774"},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142621924","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}
Debbie Bruno , Cathy du Plessis , Cathy von Rooyen , Shafiq Abdallah , Bilal Abdallah , Erik P. Erdal , Lori Apoll
{"title":"Workflow improvement and financial gain after integration of high-throughput sample processing system with flow cytometer in a high-volume pathology laboratory: Results from a prospective comparative study using Lean principles","authors":"Debbie Bruno , Cathy du Plessis , Cathy von Rooyen , Shafiq Abdallah , Bilal Abdallah , Erik P. Erdal , Lori Apoll","doi":"10.1016/j.jim.2024.113767","DOIUrl":"10.1016/j.jim.2024.113767","url":null,"abstract":"<div><h3>Background</h3><div>Highly efficient clinical laboratories are essential for monitoring many human illnesses. Ampath Laboratory Services, the largest pathology lab in South Africa, analyzes large numbers of peripheral blood samples for CD4 levels yearly.</div></div><div><h3>Objective</h3><div>To assess productivity and quality of a newer integrated automated solution, the BD FACSDuet™ Sample Preparation System/BD FACSLyric™ Flow Cytometer using conventional assessment methods and Lean concepts.</div></div><div><h3>Materials and methods</h3><div>This prospective study compared the performance of the BD FACS™ Sample Preparation Assistant [SPA] III and BD FACSCanto™ II Flow Cytometer with the newly introduced integrated system (BD FACSDuet™/BD FACSLyric™). They were validated for accuracy, precision, and external quality assessment. Process mapping and Lean assessment helped identify steps leading to waste. An economic model was developed to characterize workflow and economic impact associated with total daily hands-on time, processing time, and reworks.</div></div><div><h3>Results</h3><div>Strong linear correlation was present between both systems. Precision and accuracy studies revealed that all coefficient of variation (CV)% values were below 20% of allowable limits. External proficiency assessments were within limits. The fully automated workflow of BD FACSDuet™/BD FACSLyric™ permitted better consistency with significantly shorter processing time and batch processing and reduced operator interventions. Lean assessment identified defects with motion, over-processing, waiting, and non-utilized talent. Significant reductions in hands-on and total daily processing time that could increase daily specimen testing efficiency and fewer reworks were associated with the BD FACSDuet™/BD FACSLyric™. Lean improvements translated to significant economic savings associated with operator costs and unnecessary reagent consumption.</div></div><div><h3>Conclusion</h3><div>BD FACSDuet™/BD FACSLyric™ is an accurate, reliable, and cost-effective fully automated system for high-volume flow cytometry labs that perform T-cell enumeration using a single-platform and single-tube approach.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113767"},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142467508","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":"Epitope profiling of cow's milk allergen-specific antibodies with determining IgE content in epitopes-ALL, a 14-epitopes mixture.","authors":"Yoshihiro Watanabe, Ikuo Okafuji, Satoko Tamai, Natsuko Hosokawa, Takako Ohbayashi, Shigeki Kato, Kiyoaki Ito, Mitsuhiro Kawano, Yusei Ohshima","doi":"10.1016/j.jim.2024.113773","DOIUrl":"https://doi.org/10.1016/j.jim.2024.113773","url":null,"abstract":"<p><p>Allergen-specific antibodies (Abs), IgE, and IgG4 increase during the early phase of oral immunotherapy (OIT) of allergen food in patients; subsequently, IgE levels decrease and specific IgG4 levels increase after successful OIT treatment. The detailed profile of these Abs during OIT remains largely unclear. We developed a diagnostic tool to assess the OIT efficacy and extent of responsiveness based on a profiling method by identifying epitopes recognized by the Ab classes of IgE or IgG4. A peptide microarray followed by microplate analysis using synthetic peptides was used to identify 14 epitopes widely recognized by IgE and/or IgG4 in the serum samples of patients with OIT among the amino acid sequences of five major cow's milk allergens. The set of defined 14 epitopes clarified different epitope profiles of allergen-specific IgE and IgG4 in each patient's serum samples. Moreover, the total signal of Abs recognizing all 14 epitopes was equal to the sum of all individual epitope-specific Abs. It was further observed that the quantitative value of IgE concentrations of 14 epitopes-ALL correlated with the ImmunoCAP IgE value. These findings strongly imply that the quantity of IgE and IgG4 recognizing epitopes-ALL may easily be used to measure allergy severity. To investigate this potential, we developed an immunochromatographic method that can detect IgE and IgG4 levels in patient samples. This study clearly demonstrated the usefulness of the defined 14 epitopes and their mixture, \"epitopes-ALL,\" and that the simple and reliable methods of immunochromatography and microplate analyses demonstrating the epitope profile of allergen-specific Abs are applicable for diagnostic use at multiple disease stages and the OIT-treatment course in patients with cow's milk allergy.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":" ","pages":"113773"},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568170","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 for an effective potency determination of recombinant rabies human monoclonal antibody","authors":"Ambika Divase , Sambhaji Pisal , Manjusha Dake , Rajeev Dhere , Pravin Kumar Dakshinamurthy , Peddireddy Srinivas Reddy , Chandrashekhar Kamat , Digamber Singh Chahar , Jayanta Pal , Neelu Nawani","doi":"10.1016/j.jim.2024.113769","DOIUrl":"10.1016/j.jim.2024.113769","url":null,"abstract":"<div><div>Rapid Fluorescence Focus Inhibition Test (RFFIT) is the most widely used cell-based assay to measure the potency of recombinant human rabies monoclonal antibodies. Nonetheless, RFFIT assay is time-consuming and it requires well-equipped biosafety level 2 facility, virulent live rabies virus cultures, permissive cell lines, and well-trained manpower. Therefore, the development of alternative methods to the RFFIT has been encouraged by the World Health Organization (WHO) expert working groups to overcome these barriers.</div><div>An <em>In-vitro</em> ELISA test has been developed as an alternative to the RFFIT assay, for quantifying the rabies monoclonal antibody (mAb) potency using inactivated rabies virus vaccine (Rabivax-S). It is based on the specific interaction between the antigen and the antibody, that induces neutralizing antibody response to rabies virus.</div><div>The ELISA was validated involving accuracy and precision within 20 % coefficient of variance. The validation has been done by 4PL standard curve with linearity r<sup>2</sup> ˃ 0.98 and LLOQ of 0.3 μg/mL indicating high assay sensitivity. The specificity of the assay was ascertained by challenging with another homologous non-rabies humanized mAb, which does not show binding with the rabies virus.</div><div>The indirect ELISA developed here, is precise, robust, and accurate to quantitate the potency of rabies monoclonal antibody. It is highly sensitive and has a broad range of detection. It is easy to perform, and it has a short turnaround time (results available in few hours). Furthermore, it is cost effective and can be performed with low-cost resource setting, as there is no requirement of handling the live cells and live virus and also BSL-2 Facility.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113769"},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502036","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 novel and broadly applicable sandwich ELISA assay based on rabbit single-chain variable fragments and a modified Ig-binding domain of protein L fused to a polystyrene-binding peptide","authors":"Yodai Yamamoto , Haruka Taniguchi , Ngoc Minh Nguyen , Fuki Yokoyama , Kiattawee Choowongkomon , Alessandro Angelini , Jun-Ichi Horiuchi , Yoichi Kumada","doi":"10.1016/j.jim.2024.113771","DOIUrl":"10.1016/j.jim.2024.113771","url":null,"abstract":"<div><div>Most of currently available sandwich-type enzyme-linked immunosorbent assays (ELISA) require the use of full-length animal-derived antibodies which poses welfare criticisms and are often expensive to produce. There is therefore a strong demand for the development of more affordable and animal-free methods to produce antibodies for sandwich ELISA assay. To address these issues, we propose here the development of a new technology based on two complementary rabbit single-chain variable fragments (scFvs) and an Ig-binding domain of protein L (PpL1) fused to a polystyrene-binding peptide (PS-tag) that can be recombinantly produced in bacteria. Toward this goal, we developed a rabbit scFv capable to bind the antigen via its variable regions while engaging protein L through its constant framework domain. To enhance the density of captured scFv and enable a better solvent exposure, we generated multiple PpL1 variants bearing polystyrene-binding peptides (PS) tags fused to its ends. The tandem trimer of PpL1 variant bearing PS-tags located at the N-terminus (PpL1′-T-PSN) revealed increased antigen-binding signal when immobilized on hydrophilic polystyrene (phi-PS) plates. By CDR-grafting different antigen-binding specificities into our engineered protein L-binding scFv we validated our technology against a different antigen. Finally, to further enhance the sensitivity of our assay, we implemented a protein L-based pretreatment to remove potential inhibitory immunoglobulin often present in the blood samples. The ability to rapidly and cost-effectively generate animal-free recombinant antibody fragments that can be adsorbed and specifically oriented on plates while retaining their antigen-binding properties could lead to the development of innovative and widely applicable sandwich ELISA systems for the efficient, versatile and sensitive detection of different types of antigens.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113771"},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568110","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}
Paul M. Hargarten , Cassandra G. Porth , Mark Berrong , Darin Weed , Miranda Carper , Thomas N. Denny , Guido Ferrari , Wes Rountree
{"title":"Decreased variability in the site-specific results during participation in the External Quality Assurance Program Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay","authors":"Paul M. Hargarten , Cassandra G. Porth , Mark Berrong , Darin Weed , Miranda Carper , Thomas N. Denny , Guido Ferrari , Wes Rountree","doi":"10.1016/j.jim.2024.113770","DOIUrl":"10.1016/j.jim.2024.113770","url":null,"abstract":"<div><div>The NIAID DAIDS-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) manages an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) external proficiency program. The ELISpot program evaluates the accuracy and variability of results across laboratories. The variability in the program is quantified via the dispersion, which is the ratio of the variance over the mean of the background-corrected spot-forming cells (SFC) replicates obtained under stimulation with different peptide pools (CMV, CEF). This report includes the longitudinal analysis of the ELISpot program cohort composed of 22 laboratories from 2011 to 2022 to assess whether the within-lab variability has improved over time. Random intercept models of the dispersion over time showed a significant decrease in overall dispersion from an average of approximately 1.8 in 2011 to approximately 1.25 in 2022. Out of the 21 sites, 16 sites (4 being statistically significant) had a negative trend for dispersion over time. Our finding of a reduction of overall within-lab variability demonstrates the need for and benefit of proficiency testing programs.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113770"},"PeriodicalIF":1.6,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502035","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}
Zachary Ende , Margarita Mishina , Robert C. Kauffman , Amrita Kumar , Rashmi Kumari , Paul R. Knight , Suryaprakash Sambhara
{"title":"Human monoclonal antibody cloning and expression with overlap extension PCR and short DNA fragments","authors":"Zachary Ende , Margarita Mishina , Robert C. Kauffman , Amrita Kumar , Rashmi Kumari , Paul R. Knight , Suryaprakash Sambhara","doi":"10.1016/j.jim.2024.113768","DOIUrl":"10.1016/j.jim.2024.113768","url":null,"abstract":"<div><div>Monoclonal antibodies are powerful therapeutic, diagnostic, and research tools. Methods utilized to generate monoclonal antibodies are evolving rapidly. We created a transfectable linear antibody expression cassette from a 2-h high-fidelity overlapping PCR reaction from synthesized DNA fragments. We coupled heavy and light chains into a single linear sequence with a promoter, self-cleaving peptide, and poly(A) signal to increase the flexibility of swapping variable regions from any sequence available <em>in silico</em>. Transfection of the linear cassette tended to generate similar levels to the two-plasmid system and generated an average of 47 μg (14–98 μg) after 5 days in 2 ml cultures with 15 unique antibody sequences. The levels of antibodies produced were sufficient for most downstream applications in less than a week. The method presented here reduces the time, cost, and complexity of cloning steps.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113768"},"PeriodicalIF":1.6,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502037","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}