A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). A sensitive and reliable serological diagnostic assay is required, so laboratories can effectively and quickly detect ASFV infection. The p30 protein is abundantly expressed early in cells and has excellent antigenicity. Therefore, this study aimed to produce and characterize p30 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Three monoclonal antibodies against p30 protein that were expressed in E. coli were generated, and their characterizations were investigated. Furthermore, a blocking ELISA based on a monoclonal antibody was developed. To evaluate the performance of the assay, 186 sera samples (88 negative and 98 positive samples) were analyzed and a receiver-operating characteristic (ROC) analysis was applied to determine the cutoff value. Based on the ROC analysis, the area under the curve (AUC) was 0.997 (95% confidence interval: 99.2 to 100%). Besides, a diagnostic sensitivity of 97.96% (95% confidence interval: 92.82 to 99.75%) and a specificity of 98.96% (95% confidence interval: 93.83 to 99.97%) were achieved when the cutoff value was set to 38.38%. Moreover, the coefficients of inter- and intra-batches were <10%, indicating the good repeatability of the method. The maximum dilution of positive standard serum detected by this ELISA method was 1:512. The blocking ELISA was able to detect seroconversion in two out of five pigs at 10 Dpi and the p30 response increasing trend through the time course of the study (0–20 Dpi). In conclusion, the p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity. The assay could be a useful tool for field surveillance and epidemiological studies in swine herd.

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Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera

Journal of Virological Methods ◽

10.1016/j.jviromet.2019.113731 ◽

2019 ◽

Vol 274 ◽

pp. 113731

Author(s):

A. Di Rubbo ◽

L. McNabb ◽

R. Klein ◽

J.R. White ◽

A. Colling ◽

...

Keyword(s):

Monoclonal Antibody ◽

Neutralizing Antibodies ◽

Hendra Virus ◽

Diagnostic Evaluation ◽

Blocking Elisa

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Preparation of Monoclonal Antibody against Ema-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China

Journal of Parasitology ◽

10.1645/19-98 ◽

2020 ◽

Vol 106 (2) ◽

pp. 283

Author(s):

Jingjing Song ◽

Ruiqi Song ◽

Panju Wang ◽

Yang Zhang ◽

Yan Yan ◽

...

Keyword(s):

Monoclonal Antibody ◽

Detection Method ◽

Serological Detection ◽

Theileria Equi

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A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae

Veterinary Microbiology ◽

10.1016/0378-1135(92)90092-8 ◽

1992 ◽

Vol 30 (1) ◽

pp. 35-46 ◽

Cited By ~ 48

Author(s):

Niels C. Feld ◽

Per Qvist ◽

Peter Ahrens ◽

Niels F. Friis ◽

Anders Meyling

Keyword(s):

Mycoplasma Hyopneumoniae ◽

Serum Antibodies ◽

Blocking Elisa

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Structure of P46, an immunodominant surface protein from Mycoplasma hyopneumoniae: interaction with a monoclonal antibody

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320003903 ◽

2020 ◽

Vol 76 (5) ◽

pp. 418-427

Author(s):

Alicia Guasch ◽

Jordi Montané ◽

Alexandra Moros ◽

Jaume Piñol ◽

Marta Sitjà ◽

...

Keyword(s):

Monoclonal Antibody ◽

Structural Information ◽

Surface Protein ◽

Mycoplasma Hyopneumoniae ◽

Economic Losses ◽

Fab Fragment ◽

New Therapeutic Approaches ◽

Complementarity Determining Regions ◽

Extracellular Side

Mycoplasma hyopneumoniae is a prokaryotic pathogen that colonizes the respiratory ciliated epithelial cells in swine. Infected animals suffer respiratory lesions, causing major economic losses in the porcine industry. Characterization of the immunodominant membrane-associated proteins from M. hyopneumoniae may be instrumental in the development of new therapeutic approaches. Here, the crystal structure of P46, one of the main surface-antigen proteins, from M. hyopneumoniae is presented and shows N- and C-terminal α/β domains connected by a hinge. The structures solved in this work include a ligand-free open form of P46 (3.1 Å resolution) and two ligand-bound structures of P46 with maltose (2.5 Å resolution) and xylose (3.5 Å resolution) in open and closed conformations, respectively. The ligand-binding site is buried in the cleft between the domains at the hinge region. The two domains of P46 can rotate with respect to each other, giving open or closed alternative conformations. In agreement with this structural information, sequence analyses show similarities to substrate-binding members of the ABC transporter superfamily, with P46 facing the extracellular side as a functional subunit. In the structure with xylose, P46 was also bound to a high-affinity (K d = 29 nM) Fab fragment from a monoclonal antibody, allowing the characterization of a structural epitope in P46 that exclusively involves residues from the C-terminal domain. The Fab structure in the complex with P46 shows only small conformational rearrangements in the six complementarity-determining regions (CDRs) with respect to the unbound Fab (the structure of which is also determined in this work at 1.95 Å resolution). The structural information that is now available should contribute to a better understanding of sugar nutrient intake by M. hyopneumoniae. This information will also allow the design of protocols and strategies for the generation of new vaccines against this important swine pathogen.

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