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Development of a surface plasmon resonance-based immunoassay for Listeria monocytogenes
Paul Leonard1, Stephen Hearty, Gary Wyatt
1School of Biotechnology and National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Insights
Researchers developed a novel surface plasmon resonance assay using a polyclonal antibody to detect Listeria monocytogenes. This method offers a sensitive and reproducible way to identify bacterial cells in solution.
Area of Science:
- Microbiology
- Immunology
- Biosensing Technology
Background:
- Listeria monocytogenes is a significant foodborne pathogen.
- Accurate and rapid detection methods are crucial for food safety.
- Internalin B (InlB) is a key virulence factor of Listeria monocytogenes.
Purpose of the Study:
- To develop a sensitive and reproducible surface plasmon resonance (SPR) based inhibition assay for detecting Listeria monocytogenes.
- To utilize a polyclonal antibody against Internalin B (InlB) for bacterial cell detection.
Main Methods:
- Production of a polyclonal antibody against an InlB-enriched extract.
- Cloning, expression, and purification of recombinant InlB (rInlB).
- Development of an SPR inhibition assay using anti-InlB antibody and rInlB-coated sensor chips.
Main Results:
- The assay demonstrated a decrease in SPR signal with increasing concentrations of Listeria monocytogenes cells.
- The limit of detection was found to be less than 2 x 10^5 cells/ml.
- The assay showed good precision and reproducibility with coefficients of variation between 2.5% and 7.7%.
Conclusions:
- A novel SPR inhibition assay for Listeria monocytogenes detection was successfully developed.
- The assay is sensitive, reproducible, and utilizes an antibody targeting the InlB protein.
- This method holds potential for rapid and accurate bacterial detection in various applications.
Abstract:
A polyclonal antibody was produced against Internalin B (InlB)-enriched extract and used to develop an inhibition assay to detect Listeria monocytogenes cells in solution using surface plasmon resonance. The gene sequence encoding for the InlB protein was cloned into a Qiagen pQE-60 vector, expressed in Escherichia coli, and purified by immobilized metal affinity chromatography. Protein G-purified anti-InlB-enriched extract polyclonal antibody was incubated with various concentrations of L. monocytogenes cells and subsequently injected over a purified-recombinant InlB (rInlB)-immobilized CM5 sensor chip surface. A decrease in antibody binding response was observed with increasing L. monocytogenes cell concentrations. Intraday and interday assay variability studies were carried out to evaluate precision and reproducibility. The assay had a limit of detection of less than 2 x 10(5) cells per ml and could be successfully reproduced with coefficients of variation of between 2.5 and 7.7%.
