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Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis
Włodzimierz Przewodowski1, Agnieszka Przewodowska1
1Plant Breeding and Acclimatization Institute - National Research Institute, Department of Potato Protection and Seed Science at Bonin, Poland.
Abstract:
The quarantine bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) causes bacterial ring rot (BRR) in potato but is difficult to detect, hampering the diagnosis of this disease. ELISA immunoassays have not been widely used to detect Cms because commercially available anti-Cms antibodies detect mainly EPS-producing bacteria and can fail to detect strains that do not produce EPS. In the current study, we developed a new type of polyclonal antibody that specifically detects Clavibacter michiganensis subsp. sepedonicus bacteria irrespective of their EPS level. We first found that the presence of bacterial EPS precluded quantitative measurement of bacteria by currently available immunoenzymatic methods, but that washing Cms cells with acidic and basic buffers to remove EPS before analysis successfully standardized ELISA results. We used a mix of three strains of Cms with diverse EPS levels to generate antigen for production of antibodies recognizing Cms cells with and without an EPS layer (IgG-EPS and IgG-N-EPS, respectively). The resulting IgG-N-EPS recognized almost all Cms strains tested in this work regardless of their mucoidal level. The availability of this new antibody renders immunological diagnostics of Cms more sensitive and reliable, as our newly developed antibodies can be used in many type of immunoassays. This work represents an important step forward in efforts to diagnose and prevent the spread of BRR, and the methods and solutions developed in this work are covered by six Polish, one European and one US patents.
Insights
New antibodies improve detection of Clavibacter michiganensis subsp. sepedonicus (Cms), the cause of potato bacterial ring rot (BRR). This advancement enhances diagnostic accuracy for Cms, regardless of its EPS levels.
Area of Science:
- Plant pathology
- Immunology
- Bacteriology
Background:
- Clavibacter michiganensis subsp. sepedonicus (Cms) causes potato bacterial ring rot (BRR), a disease difficult to diagnose due to detection challenges.
- Existing ELISA methods struggle with Cms detection, as antibodies often target EPS-producing strains, failing to identify non-EPS producers.
Purpose of the Study:
- To develop novel polyclonal antibodies for specific and reliable detection of Cms, irrespective of its exopolysaccharide (EPS) levels.
- To overcome limitations in current immunoassays for accurate Cms diagnosis and management.
Main Methods:
- Developed and produced new polyclonal antibodies (IgG-EPS and IgG-N-EPS) using Cms strains with varying EPS production.
- Standardized ELISA by washing Cms cells with acidic and basic buffers to remove EPS prior to analysis.
- Tested antibody efficacy against diverse Cms strains with different mucoidal levels.
Main Results:
- The newly developed IgG-N-EPS antibodies demonstrated high specificity and recognized nearly all tested Cms strains, regardless of EPS production.
- EPS removal through buffer washing successfully standardized ELISA results, enabling quantitative bacterial measurement.
- The new antibodies significantly enhance the sensitivity and reliability of immunological diagnostics for Cms.
Conclusions:
- The developed antibodies represent a significant advancement for sensitive and reliable Cms detection in immunological assays.
- This breakthrough aids in the diagnosis and prevention of potato bacterial ring rot (BRR).
- The patented methods and solutions offer robust tools for disease management.
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