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Updated: Jul 9, 2026
Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae
Published on: February 24, 2023
Cloning, expression, and immunological characterization of the P30 protein of Mycoplasma pneumoniae
Avanish Kumar Varshney1, Rama Chaudhry, Sushil Kumar Kabra
1Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
Abstract:
Mycoplasma pneumoniae, a self-replicating cell wall-deficient prokaryote, has a differentiated terminal organelle that is essential for cytadherence and gliding motility. P30, an important protein associated with the terminal organelle, is required for the cytadherence and virulence of M. pneumoniae. P30 is a transmembrane protein with an intracytoplasmic N terminus and an exposed C terminus. In the present study, we amplified and sequenced the full-length p30 gene of Mycoplasma pneumoniae directly from 18 Indian asthmatic patients. Sequence diversity was observed in the p30 genes from 16 clinical samples when the sequences were compared with the sequence of strain M-129. We also successfully expressed a fragment of the p30 gene (P30B) that includes the complete C-terminal proline-rich amino acid sequences in different Escherichia coli expression systems. The maltose binding protein (MBP)-P30B fusion protein was recognized by M. pneumoniae-infected patient sera in immunoblots, and the protein was immunogenic in mice. We further analyzed the reactivity of the MBP-P30B fusion protein with patient sera in an enzyme-linked immunosorbent assay (ELISA) and compared it with the reactivity obtained with a commercial kit (the Serion ELISA Classic kit). The sensitivity and the specificity of the in-house ELISA were 78.57% and 89.47%, respectively. This study suggests that the P30 protein can be used as an antigen along with other adhesin proteins for the immunodiagnosis of M. pneumoniae infection.
Insights
The P30 protein from Mycoplasma pneumoniae, crucial for bacterial adherence, shows sequence diversity in Indian asthma patients. This protein fragment is a promising candidate for developing new diagnostic tests for M. pneumoniae infections.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Mycoplasma pneumoniae is a wall-deficient bacterium vital for cytadherence and motility.
- The P30 protein is essential for M. pneumoniae cytadherence and virulence, located in the terminal organelle.
Purpose of the Study:
- To investigate the sequence diversity of the p30 gene in M. pneumoniae from Indian asthmatic patients.
- To express and characterize a P30 protein fragment for potential immunodiagnostic applications.
Main Methods:
- Full-length p30 gene amplification and sequencing from clinical samples.
- Expression of a P30B fragment in E. coli and generation of a fusion protein (MBP-P30B).
- Immunological assays including immunoblot, mouse immunization, and in-house ELISA using patient sera.
Main Results:
- Sequence diversity was found in the p30 genes of M. pneumoniae from 16 out of 18 Indian asthmatic patients.
- The MBP-P30B fusion protein was recognized by patient sera and proved immunogenic in mice.
- The in-house ELISA demonstrated 78.57% sensitivity and 89.47% specificity.
Conclusions:
- The P30 protein exhibits sequence variation in clinical isolates from Indian asthmatic patients.
- The P30 protein fragment is a viable candidate antigen for M. pneumoniae immunodiagnosis.
- P30, alongside other adhesins, can enhance the accuracy of diagnosing M. pneumoniae infections.
