Journal of immunological methods最新文献

{"title":"Natural cross-reactive anti-SARS-CoV-2 antibodies in avian egg yolk","authors":"Gholamreza Nikbakht Brujeni,&nbsp;Pouya Houshmand,&nbsp;Shervin Sadafian,&nbsp;Reza Rezaei","doi":"10.1016/j.jim.2024.113798","DOIUrl":"10.1016/j.jim.2024.113798","url":null,"abstract":"<div><div>Since the beginning of the 21st century, the <em>Coronaviridiae</em> family has caused several life-threatening outbreaks in the world. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the cause of the latest <em>Coronaviridiae</em>-related outbreak, is still a major health issue worldwide. Prevention, diagnosis, and therapeutic actions are the most important strategies to mitigate the spread of the COVID-19 pandemic. Among therapeutics, specific antibodies play a crucial role in controlling the symptoms of patients and preventing others from becoming infected. Here, we have introduced the avian egg yolk as a natural source of cross-reactive anti-SARS-CoV-2 immunoglobulin Y. ELISA, dot blot and western blot were used to identify natural anti-SARS-CoV-2 IgY in the egg yolk of different species of birds. Also, bioinformatics analysis was performed to investigate the possible causes of the presence of these natural antibodies in the egg yolks. The results of blotting and ELISA assays demonstrated that the egg yolk-derived antibodies could identify and bind to the different subunits of SARS-CoV-2. Substantial concentrations of neutralizing antibodies against SARS-CoV-2 were also detected in the egg yolk. In addition, bioinformatics analysis showed structural similarities between the components of infectious bronchitis virus, SARS-CoV-2, and other members of the <em>Coronaviridiae</em> family. It seems that egg yolk can be used as a natural, inexpensive, and accessible source of anti-SARS-CoV-2 antibodies. Diverse diagnostic and therapeutic potentials for avian egg yolk-derived anti-SARS-CoV-2 antibodies are imagined.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"536 ","pages":"Article 113798"},"PeriodicalIF":1.6,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142846882","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":"Identification of the linear Fc-binding epitope on the bovine IgG1 Fc receptor of (boFcγRI) using synthetic peptides","authors":"Qingmei Li ,&nbsp;Ge Li ,&nbsp;Xun Wang ,&nbsp;Ruining Wang ,&nbsp;Jifei Yang ,&nbsp;Junqing Guo ,&nbsp;Gaiping Zhang","doi":"10.1016/j.jim.2024.113799","DOIUrl":"10.1016/j.jim.2024.113799","url":null,"abstract":"<div><h3>Background</h3><div>Bovine IgG1 Fc receptor (boFcγRI) is a homologue to human FcγRI (CD64) that has three extracellular Ig-like domains and can bind bovine IgG1 with high affinity. Identification of the linear epitope for Fc-binding on boFcγRI provides new insights for the IgG-Fcγ interaction and FcγR-targeting drugs development.</div></div><div><h3>Methods</h3><div>The boFcγRI molecules were expressed on cell surface of the boFcγRI -transfected COS-7 cells. The extracellular domain of boFcγRI was expressed in <em>Escherichia coli</em> (<em>E. coli</em>) BL21, and the soluble boFcγRI was purified by Ni-chelation chromatography followed by refolding. To identify the Fc-binding epitope on the boFcγRI, peptides derived from the membrane-distal extracellular domain (EC2) of boFcγRI were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin (BSA). Binding of bovine IgG1 to the different peptides was tested by Dot-blot assay, and the peptide showing maximal IgG-binding was further modified by truncation and mutation. The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay, respectively.</div></div><div><h3>Results</h3><div>The minimal effective peptide TNLSHNGI corresponding to the sequence 142–149 of boFcγRI was found to bind bovine IgG1 specifically in Dot-blot suggesting it represents a linear ligand-binding epitope located in the putative E-F loop of the EC2 domain on the receptor. Mutation analysis of the peptide showed that the residues of Thr¹⁴², Asn¹⁴³, Leu¹⁴⁴, Gly¹⁴⁸ and Ile¹⁴⁹ within the linear epitope are critical for IgG1-binding. The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRII with IC₅₀ of 20.27 μmol/L, and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRI transfected cells with IC₅₀ of 86.75 μmol/L.</div></div><div><h3>Conclusions</h3><div>The linear epitope for Fc-binding as well as its crucial residues of EC2 domain on boFcγRI was identified using synthetic peptides. The Fc-binding peptide showed well capability of regulating boFcγRI-IgG1 interaction on cell surface.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"536 ","pages":"Article 113799"},"PeriodicalIF":1.6,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142921958","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":"Characterization of an IL-8 cleavage inhibition assay to determine the functionality of anti-SpyCEP antibodies in human sera","authors":"Luisa Massai ,&nbsp;Martina Carducci ,&nbsp;Luca Rovetini ,&nbsp;Aimee Paterson ,&nbsp;Alana Whitcombe ,&nbsp;Reuben McGregor ,&nbsp;Natalie Lorenz ,&nbsp;Alexander J. Keeley ,&nbsp;Thushan I. de Silva ,&nbsp;Julie Bennett ,&nbsp;Francesco Berlanda Scorza ,&nbsp;Miren Iturriza ,&nbsp;Nicole J. Moreland ,&nbsp;Danilo G. Moriel ,&nbsp;Omar Rossi","doi":"10.1016/j.jim.2024.113786","DOIUrl":"10.1016/j.jim.2024.113786","url":null,"abstract":"<div><div>Exposure to Group A Streptococcus leads to a broad spectrum of disease and sequelae, as the bacterium employs a wide range of virulence factors to facilitate colonization of the host, propagation and onward transmission, disrupting both innate and adaptive immune responses. The protease SpyCEP has a crucial role in contributing to bacterial immune evasion by impairing neutrophil recruitment and killing of bacteria through the cleavage of interleukin-8 (IL-8). Given this critical function, SpyCEP represents a key vaccine antigen and quantifying functional anti-SpyCEP antibodies represents not only an important marker of vaccine efficacy, but also a tool to dissect the natural immune response. Here, we report the development and characterization of an IL-8 cleavage inhibition assay to measure the function of anti-SpyCEP antibodies in human sera. The assay was demonstrated to be sensitive, highly specific, linear and reproducible, and suitable for evaluating the function of anti-SpyCEP antibodies induced in humans in vaccine clinical trials and in observational studies of natural immunity.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"536 ","pages":"Article 113786"},"PeriodicalIF":1.6,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142791979","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":"Plaque reduction neutralization test for smallpox vaccines: Laboratory optimization and validation method for immunogenicity assessment","authors":"Hyun-Jung Kong,&nbsp;You-Jin Kim,&nbsp;Dokeun Kim,&nbsp;Yun-Ho Hwang","doi":"10.1016/j.jim.2024.113787","DOIUrl":"10.1016/j.jim.2024.113787","url":null,"abstract":"<div><div>The eradication of smallpox, a historic triumph in global public health, was accomplished without a complete conception of the mechanisms underlying vaccine-induced protection. Contemporary concerns regarding potential bioterrorism threats and the possibility of smallpox reemergence have spurred research efforts toward developing third-generation vaccines capable of effectively neutralizing the variola virus. Clinical trials for a third-generation smallpox vaccine (KVAC103) are underway to obtain licensure. As a surrogate marker for efficacy, vaccinia virus (VACV) antibody levels can be assessed using the plaque reduction neutralization test (PRNT). In the current study, the PRNT methodology underwent comprehensive development, optimization, and validation in strict adherence to the guidelines for bioanalytical test methods. The VACV PRNT₅₀ was optimized to include the working virus concentration (4 × 10² plaque-forming units/mL), virus-serum neutralization time (60 min), concentration of carboxymethylcellulose sodium salt overlay (1 %), and days of incubation post infection (3 days). Using human serum samples from individuals administered the second-generation smallpox vaccine (CJ-50300), the VACV PRNT₅₀ (cut-off point, 22.58), based on the receiver-operating characteristic curve (area under the curve = 0.9859) and sensitivity and specificity assays, exhibited favorable outcomes, showing 93.75 % specificity (95 % confidence interval [CI], 71.67–99.68 %) and 93.55 % sensitivity (95 % CI, 79.28–98.85 %) against the VACV strain Western Reserve. The validation process encompassed crucial parameters, including intra-assay and inter-assay precision, robustness, dilution linearity, and the lower limit of quantification. The VACV PRNT₅₀ exhibited high accuracy and 100 % intra-assay and inter-assay precision across various ND₅₀ titers (high, middle, and low). Overall, the PRNT was validated as a reliable tool for measuring VACV-neutralizing antibodies and evaluating the effectiveness of new smallpox vaccinations in human serum samples.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"536 ","pages":"Article 113787"},"PeriodicalIF":1.6,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142822098","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 panel of peptides with diagnostic potential for serological identification of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii in human sera","authors":"Antonio C.G. Foddai ,&nbsp;Peter Wilhelmsson ,&nbsp;Per-Eric Lindgren ,&nbsp;Jeremy M. Sternberg ,&nbsp;Alan S. Bowman","doi":"10.1016/j.jim.2025.113802","DOIUrl":"10.1016/j.jim.2025.113802","url":null,"abstract":"<div><div>A novel panel of peptide for serological identification of <em>Borrelia burgdoferi</em> sensu stricto, <em>Borrelia garinii</em> and <em>Borrelia afzelii</em> was developed and assessed in this study. The diagnostic algorithm of the novel test was initially trained testing 10 US human sera including 3 early-stage and 3 late-stage Lyme disease positive sera, 2 sera positive for Babesia and 2 sera positive for Syphilis, all purchased from a private biorepository. Findings were then corroborated testing (a) 33 additional EU follow-up positive sera from seroconverted patients bitten by ticks that tested positive for <em>B. burgdorferi</em> sensu stricto (No 2), <em>Borrelia garinii</em> (No 14), <em>Borrelia afzelii</em> (No 15) <em>Borrelia valaisiana</em> (No 2), and (b) 40 negative sera from US healthy donors. Results of preliminary US sera testing showed successful detection of IgM and IgG antibodies and correct identification of <em>Borrelia burgdorferi</em> sensu stricto in all the samples tested. Analysis of EU follow-up sera showed much higher sensitivity and accuracy when IgM and IgG were tested combined together rather than separately. Sensitivity and accuracy in species identification of the anti-IgM + IgG multiplex peptide ELISA was 93.5 % and 96.5 % respectively; lower test performance was observed when IgM (i.e. sensitivity = 58.1 %; correct identification = 88.8 %) and IgG testing (i.e. sensitivity = 74.1 %; correct identification = 96.5 %) were carried out separately. Overall specificity of the anti-IgM, anti-IgG and anti-IgM + IgG multiplex peptide ELISA calculated on a total number of 46 negative sera included in this study was 91.3 %, 95.6 and 93.4 %, respectively.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"536 ","pages":"Article 113802"},"PeriodicalIF":1.6,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142965567","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":"Epitope profiling of cow's milk allergen-specific antibodies with determining IgE content in epitopes-ALL, a 14-epitopes mixture","authors":"Yoshihiro Watanabe ,&nbsp;Ikuo Okafuji ,&nbsp;Satoko Tamai ,&nbsp;Natsuko Hosokawa ,&nbsp;Takako Ohbayashi ,&nbsp;Shigeki Kato ,&nbsp;Kiyoaki Ito ,&nbsp;Mitsuhiro Kawano ,&nbsp;Yusei Ohshima","doi":"10.1016/j.jim.2024.113773","DOIUrl":"10.1016/j.jim.2024.113773","url":null,"abstract":"<div><div>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.</div><div>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.</div><div>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.</div><div>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.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"535 ","pages":"Article 113773"},"PeriodicalIF":1.6,"publicationDate":"2024-12-01","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation of anti-Ancylostoma-secreted protein 5 (ASP5) antibody from a naïve antibody phage library","authors":"Brenda Pei Chui Song ,&nbsp;Jing Yi Lai ,&nbsp;Yee Siew Choong ,&nbsp;Nafiseh Khanbabaei ,&nbsp;Andreas Latz ,&nbsp;Theam Soon Lim","doi":"10.1016/j.jim.2024.113776","DOIUrl":"10.1016/j.jim.2024.113776","url":null,"abstract":"<div><div><em>Ancylostoma</em> species are parasitic nematodes that release a multitude of proteins to manipulate host immune responses to facilitate their survival. Among the released proteins, <em>Ancylostoma</em>-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 <em>Ancylostoma</em> 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 highlights its potential for diagnostic applications for hookworm infections.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"535 ","pages":"Article 113776"},"PeriodicalIF":1.6,"publicationDate":"2024-12-01","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+ T cell proliferation by different antigen presenting cells","authors":"Fabienne Mazerolles ,&nbsp;Frédéric Rieux-Laucat","doi":"10.1016/j.jim.2024.113775","DOIUrl":"10.1016/j.jim.2024.113775","url":null,"abstract":"<div><div>We have shown in previous studies that naive CD4⁺ T cells isolated from human peripheral blood are induced to proliferate by CD4^negCD11c⁺CD14^negCD16^neg 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⁺ B cells, CD11c⁺CD14⁺ and CD11c⁺CD14^neg monocytes, CD11c⁺CD14^negCD16⁺ and CD16^neg 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⁺ B cells, CD14^neg and more specifically CD14^negCD16⁺, are also able to induce T cell proliferation as previously described with CD14^negCD16^neg DCs, but under different conditions. No proliferation was induced with CD14⁺ monocytes. However, these three APCs are less potent than CD16^neg 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⁺ B cells in contrast to CD11c⁺CD14^negCD16⁺ and CD11c⁺CD14^negCD16^neg.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"535 ","pages":"Article 113775"},"PeriodicalIF":1.6,"publicationDate":"2024-12-01","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A note of caution for using calmodulin antibodies","authors":"Mads Munk,&nbsp;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}

{"title":"Generation and characterization of a monoclonal antibody Fab fragment targeting PBP2a in methicillin-resistant Staphylococcus aureus","authors":"Juliana Pascarelli Compan Boechat ,&nbsp;Felipe Rodrigues Semcovici Ramos ,&nbsp;Felipe Betoni Saraiva ,&nbsp;Vinicius de Lima Gonçalves ,&nbsp;Franklin Souza da Silva ,&nbsp;Carlos Roberto Alves ,&nbsp;José Procópio Moreno Senna ,&nbsp;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}