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Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae
Published on: February 24, 2023
Characterization and application of monoclonal antibodies against Mycoplasma hyorhinis pyruvate dehydrogenase E1
Dongjie Chen1, Yanwu Wei1, Liping Huang1
1Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang District, Harbin, 150001, People's Republic of China.
Abstract:
Mycoplasma hyorhinis is commonly found in the respiratory tract of pigs and is the etiological agent of polyserositis. The metabolic enzymes of M. hyorhinis may play important roles in host-pathogen interactions. We immunized BALB/c mice with sodium deoxycholate-extracted antigens (DOC-Ags) and screened 10 hybridomas that secreted antibodies against various M. hyorhinis proteins. Pyruvate dehydrogenase E1 complex subunit alpha (PDHA) was identified as the protein that reacted with five of the 10 monoclonal antibodies (mAbs). Sequence analysis indicated that PDHA was highly conserved among M. hyorhinis strains, but not among other mycoplasmas. We predicted the three-dimensional structure of PDHA and identified three epitopes ((277)RTEEEEK(283), (299)KDKKYITDE(307), and (350)LKEQKQHAKDY(360)). The mAb 1H12 we generated was used to detect M. hyorhinis PDHA in vitro and in piglets infected with M. hyorhinis. We observed that PDHA was mainly located in the epithelial cells of the lungs. Our results indicate that the mAbs we generated could be used to further investigate the structure and function of M. hyorhinis PDHA. In addition, they could be used in the differential diagnosis of M. hyorhinis and other mycoplasmas.
Insights
Monoclonal antibodies targeting Mycoplasma hyorhinis pyruvate dehydrogenase E1 complex subunit alpha (PDHA) were developed. These antibodies can detect PDHA in infected pigs and aid in diagnosing Mycoplasma infections.
Area of Science:
- Veterinary Microbiology
- Immunology
- Structural Biology
Background:
- Mycoplasma hyorhinis is a significant swine pathogen causing polyserositis.
- Metabolic enzymes of M. hyorhinis are implicated in host-pathogen interactions.
Purpose of the Study:
- To develop monoclonal antibodies (mAbs) against M. hyorhinis antigens.
- To identify specific M. hyorhinis proteins targeted by these mAbs.
- To characterize the diagnostic potential of these mAbs.
Main Methods:
- Immunization of mice with sodium deoxycholate-extracted antigens.
- Screening of hybridomas for antibody production.
- Identification of target antigen using antibody reactivity.
- Sequence analysis and 3D structure prediction of the target protein.
- In vitro and in vivo detection of the target protein using generated mAbs.
Main Results:
- Ten hybridomas secreting antibodies against M. hyorhinis were generated.
- Pyruvate dehydrogenase E1 complex subunit alpha (PDHA) was identified as a key antigen, reacting with five mAbs.
- PDHA showed high conservation among M. hyorhinis strains.
- Three distinct epitopes on PDHA were identified.
- mAb 1H12 successfully detected M. hyorhinis PDHA in vitro and in infected piglets, with PDHA localized in lung epithelial cells.
Conclusions:
- Generated mAbs are valuable tools for studying M. hyorhinis PDHA structure and function.
- These mAbs can be utilized for the differential diagnosis of M. hyorhinis infections in pigs.
