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Recombinant multiepitope protein for diagnosis of leptospirosis
1Department of Medical Microbiology and Parasitology, School of Medicine, Zhejiang University, Hangzhou, China.
Abstract:
Leptospirosis is an emerging infectious disease and is considered to be the most widespread zoonotic disease in the world. It can be misdiagnosed because manifestations of this febrile disease vary from mild flu-like symptoms to severe illness involving vital organs such as the liver and lungs. Therefore, accurate diagnosis for differentiation of leptospirosis from other pyrogenic infections prevailing in the same locality is imperative for proper treatment. Here, we report a customized recombinant leptospirosis multiepitope protein (r-LMP) that can specifically detect the immunoglobulin class of anti-leptospirosis antibodies in patient sera. Immunodominant epitopes from leptospire outer membrane proteins OmpL1, LipL21, and LipL32 were predicted and confirmed using phage display and immunity reaction. On the basis of the sequences of the identified epitopes, five major immunodominant epitopes were selected to construct a synthetic gene, recombinant lmp. The recombinant lmp gene was doubled and expressed in Escherichia coli. The recombinant protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay for detection of special immunoglobulin M (IgM) or IgG in sera from patients with leptospirosis or other febrile illnesses and healthy subjects. The results showed that the r-LMP protein recognized IgG and IgM in all the sera that were microscope agglutination test positive, and there were no cross-reactions with other patient sera. This approach of creating customized antigens coupled to overexpression and simple purification offers a promising alternative option for leptospirosis diagnosis, with the potential to circumvent the drawbacks of whole-leptospirosis-antigen-based assays.
Insights
A new recombinant leptospirosis multiepitope protein (r-LMP) accurately detects antibodies for leptospirosis. This diagnostic tool shows high specificity, offering a promising alternative for identifying this widespread zoonotic disease.
Area of Science:
- Infectious Diseases
- Immunology
- Biotechnology
Background:
- Leptospirosis is a globally prevalent zoonotic disease with diverse clinical presentations, often leading to misdiagnosis.
- Accurate differentiation from other febrile illnesses is crucial for effective leptospirosis treatment.
Purpose of the Study:
- To develop a customized recombinant leptospirosis multiepitope protein (r-LMP) for specific detection of anti-leptospirosis antibodies.
- To create a novel diagnostic tool for leptospirosis, overcoming limitations of existing assays.
Main Methods:
- Identified and confirmed immunodominant epitopes from leptospire outer membrane proteins (OmpL1, LipL21, LipL32) using phage display.
- Constructed a synthetic gene for r-LMP, expressed and purified it in Escherichia coli.
- Developed an enzyme-linked immunosorbent assay (ELISA) using r-LMP for detecting specific IgM and IgG antibodies.
Main Results:
- The r-LMP protein successfully recognized both IgG and IgM antibodies in microscope agglutination test-positive leptospirosis patient sera.
- The developed ELISA demonstrated no cross-reactivity with sera from patients with other febrile illnesses or healthy individuals.
- The r-LMP assay showed high specificity and sensitivity for leptospirosis diagnosis.
Conclusions:
- Customized antigen development, coupled with efficient expression and purification, provides a robust platform for leptospirosis diagnostics.
- The r-LMP-based ELISA offers a promising, specific, and sensitive alternative to conventional methods for leptospirosis detection.
- This approach has the potential to improve diagnostic accuracy and clinical management of leptospirosis worldwide.
