Dataset Information

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

ABSTRACT: Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope ¹¹⁰DLDGAW¹¹⁵ was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

SUBMITTER: Wang J

PROVIDER: S-EPMC10812811 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Wang Junli J Lei Baishi B Zhang Wuchao W Li Lijie L Ji Jiashuang J Liu Mandi M Zhao Kuan K Yuan Wanzhe W

Animals : an open access journal from MDPI 20240111 2

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel ...[more]

PMID: 38254404

Similar Datasets

Project description:Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

Project description:Pyroptosis is a newly discovered type of pro-inflammatory programmed cell death that plays a vital role in various processes such as inflammations, immune responses, and pathogen infections. As one of the main executioners of pyroptosis, gasdermin D (GSDMD) is a membrane pore-forming protein that typically exists in a self-inhibitory state. Once activated, GSDMD will be cleaved into an N-terminal fragment with pore-forming activity, becoming the key indicator of pyroptosis activation, and a C-terminal fragment. Although commercial antibodies against human and murine GSDMD proteins are currently available, their reactivity with porcine GSDMD (pGSDMD) is poor, which limits research on the biological functions of pGSDMD and pyroptosis in pigs in vivo and in vitro. Here, five monoclonal antibodies (mAbs) were prepared by immunizing BALB/c mice with procaryotically expressed full-length pGSDMD, all of which did not cross react with human and murine GSDMD proteins. Epitope mapping demonstrated that 15H6 recognizes amino acids (aa) at positions 28-34 of pGSDMD (LQTSDRF), 19H3 recognizes 257-260aa (PPQF), 23H10 and 27A10 recognize 78-82aa (GPFYF), and 25E2 recognizes 429-435aa (PPTLLGS). The affinity constant and isotype of 15H6, 19H3, 23H10, 27A10, and 25E2 mAbs were determined to be 1.32 × 10-9, 3.66 × 10-9, 9.04 × 10-9, 1.83 × 10-9, and 8.00 × 10-8 mol/L and IgG1/κ, IgG2a/κ, IgG2a/κ, IgG1/κ, and IgG1/κ, respectively. Heavy- and light-chain variable regions sequencing showed that the heavy-chain complementarity-determining region (CDR) sequences of all five mAbs are completely different, while the light-chain CDR sequences of the four mAbs that recognize the N-terminus of pGSDMD are identical. Our prepared mAbs provide valuable materials for studying pGSDMD function and pyroptosis. KEY POINTS: • A total of five mouse anti-pGSDMD mAbs were prepared, of which four recognize the N-terminus of pGSDMD and one recognize its C-terminus. • The main performance parameters of the five mAbs, including epitope, antibody titer, affinity constant, isotype, and heavy- and light-chain CDR, were characterized. • All five mAbs specifically recognize pGSDMD protein and do not cross react with human and murine GSDMD proteins.

Project description:BackgroundPorcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.ResultsIn this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.ConclusionsWe have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

Dataset Information

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Publications

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets