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LegioTyper: Rapid typing of Legionella pneumophila serogroup 1 by flow-based chemiluminescence sandwich microarray
C Bärwinkel1, A Petzold2, C Lück2
1Chair of Analytical Chemistry and Water Chemistry, School of Natural Sciences, Technical University of Munich, Lichtenbergstraße 4, 85748, Garching, Bavaria, Germany.
Insights
A new LegioTyper panel enables rapid, cost-effective antibody-based subtyping of Legionella pneumophila Sg 1. This method differentiates between Pontiac and non-Pontiac strains and identifies outbreak-associated subtypes.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Legionella pneumophila serogroup 1 (Sg 1) identification is crucial for tracking outbreaks.
- Current methods like indirect ELISA and qPCR are labor-intensive and costly.
- Antibody-based serotyping requires characterization of new antibodies for specific subtypes.
Purpose of the Study:
- To develop and validate the LegioTyper panel for rapid antibody-based subtyping of L. pneumophila Sg 1.
- To optimize the chemiluminescence imaging of a sandwich microarray immunoassay (CL-SMIA) for LegioTyper.
- To characterize monoclonal and polyclonal antibodies for differentiating L. pneumophila Sg 1 subtypes.
Main Methods:
- Manufacturing cost-effective antibody microarrays using Jeffamine-2003-coated polycarbonate sheets.
- Optimizing an automated CL-SMIA procedure for a 34-minute test.
- Covalent immobilization of capture antibodies (cAb) using a specialized microdispensing buffer.
Main Results:
- The LegioTyper successfully differentiated L. pneumophila Sg1 from non-Sg1 strains.
- Distinction between Pontiac and non-Pontiac strains was achieved.
- Most outbreak-associated subtypes, including Philadelphia, were identifiable using the LegioTyper Panel.
Conclusions:
- The LegioTyper principle offers a rapid, automated, and cost-effective alternative for L. pneumophila Sg 1 subtyping.
- It provides a valuable tool for pathogen surveillance and outbreak investigation.
- This method simplifies subtyping compared to traditional ELISA or qPCR techniques.
Abstract:
Chemiluminescence imaging of a sandwich microarray immunoassay (CL-SMIA) is a simple and rapid method for antibody-based serotyping of pathogens like L. pneumophila, which is important to identify even outbreak-associated subtypes like L. pneumophila Sg 1 subtype Philadelphia. The CL image pattern can be interpreted by a decision path similar to the well-known "Dresden Panel", which is typically performed via indirect ELISA. New antibodies needed to be characterized for the so-called LegioTyper panel as capturing antibodies to perform the flow-based CL-SMIA. The following was achieved for the LegioTyper principle: (i) minimizing the costs for antibody microarrays by manufacturing Jeffamine-2003-coated polycarbonate sheets, (ii) optimized automated test procedure which is able to perform the CL-SMIA in 34 min, and (iii) covalent immobilization of cAb using a microdispensing buffer which contains reactions for stabilizing and bioconjugation of cAb. Nine mAb and one polyclonal Ab (pAb) were characterized regarding the selectivity to L. pneumophila Sg 1 and additionally to the virulence-associated subtypes Knoxville, France, Allentown, Benidorm, and Philadelphia (all Pontiac group), OLDA, Oxford, Bellingham, Heysham, and Camperdown (all non-Pontiac group). We could show that by using the LegioTyper it is possible to differentiate between L. pneumophila Sg1 and non-Sg1 and between Pontiac and non-Pontiac strains. Additionally, subtyping with the "LegioTyper Panel" was possible for most of the outbreak-associated subtypes. Overall, our study showed that the LegioTyper principle can be used for rapid antibody-based subtyping of L. pneumophila Sg 1 after cultivation. It is an alternative to indirect ELISA or qPCR, which are more labor- and cost-intensive, and the automation is more complicated.
