Image created by Dr. Michael J. Miller
A new study published in the Journal of Food Composition and Analysis outlines the development of a receptor-binding domain (RBD)-based sandwich ELISA for detecting botulinum neurotoxin type A (BoNT/A) in food products. Conducted by scientists at the Defence Research and Development Establishment (DRDE) in India, this research represents a step forward in food safety monitoring and biodefense preparedness.
Botulinum neurotoxins, produced by Clostridium botulinum, are among the most potent biological toxins known and are classified as Category A bioterrorism agents by the U.S. Centers for Disease Control and Prevention (CDC).
Foodborne botulism, while rare, can be fatal even at nanogram levels of exposure. Early detection is critical for both public health response and national biodefense strategies.
A Novel Approach Targeting the Receptor-Binding Domain
Unlike many current immunoassays that target the enzymatically active light chain of BoNT, the DRDE team focused on the receptor-binding domain (RBD) of the toxin’s heavy chain. This strategic shift addresses limitations in antigen accessibility and enhances diagnostic specificity.
- Target: RBD of BoNT/A heavy chain
- Method: Sandwich ELISA using rabbit and mouse anti-RBD IgG
- Advantage: Avoids requirement for proteolytic activation; more consistent results across food types
Sensitivity Across Diverse Food Matrices
The developed ELISA demonstrated strong performance across a range of food types:
- Recombinant BoNT/A protein: Limit of detection (LOD) = 0.97 ng/mL
- Food matrices:
- Honey and sweet corn: 1.95 ng/mL
- Chicken, pork, mushroom, fish: 3.9 ng/mL
- Beverages (cola, fruit juices): 7.81 ng/mL
This versatility highlights the assay’s potential utility in routine screening, especially in resource-limited or field-deployed settings.
Why This Matters for Public Health and National Security
The extreme potency and lethality of botulinum toxins—even in minute quantities—make them both a public health hazard and a high-priority biothreat. Outbreaks of foodborne botulism have historically been associated with improperly preserved or contaminated foods and remain a threat to food systems globally. The development of a rapid, sensitive, and ethically superior diagnostic method, such as this RBD-based ELISA, strengthens both early outbreak detection and food supply chain surveillance.
Implications for Food Safety Monitoring and Biothreat Preparedness
Botulinum neurotoxins are classified as potential biowarfare agents due to their high lethality and ease of dissemination. Inhalational or foodborne exposure scenarios are particularly concerning in civilian and military contexts.
The DRDE-developed ELISA provides a promising tool for:
- Public Health Agencies: Rapid screening during suspected outbreaks
- Food Safety Inspectors: Routine surveillance of at-risk food products
- Biodefense Programs: Field-deployable diagnostics for emergency response
- Global Health Security: Monitoring toxin circulation across regions with known serotype prevalence
Further validation using real-world samples is essential before broad deployment, but the proof-of-concept shows promise.
Global Relevance and Serotype Trends
BoNT/A, the target of the new assay, is especially common in regions such as the western U.S., China, and South America. Surveillance data from the CDC show that BoNT/A accounted for 34% of foodborne botulism outbreaks in the U.S. from 1920 to 2014. Between 2001 and 2017, the U.S. alone recorded over 320 confirmed cases.
In this context, improvements in diagnostic capacity—particularly tools that can be used outside of highly specialized labs—are essential for global surveillance and response.
Reference:
Dhubkarya, J., Yadav, P.K., & Ponmariappan, S. Development of Heavy Chain Binding Domain Based Sandwich-ELISA for Detection of BoNT/A in Different Food Matrices. Journal of Food Composition and Analysis. July 2025.
Abstract
Botulism is an infrequent yet potentially devastating neuroparalytic illness induced by botulinum toxins, potent neurotoxins synthesized by Clostridium botulinum, an obligate anaerobic, spore-forming bacterium. These toxins disrupt the signalling between nerves and muscles, leading to progressive, flaccid paralysis. Prompt administration of antitoxin, along with supportive care measures, is imperative, as botulism can prove fatal if left untreated. Rapid and sensitive detection methods are crucial for early diagnosis, outbreak control, and biodefense preparedness. In this study, we utilized the receptor-binding domain (RBD) of heavy chains to develop a sandwich ELISA for the detection of BoNT/A in various food matrices. The assay was established using RBD antigen, rabbit anti-RBD IgG as the immobilized antibody, and mice-derived anti-RBD IgG as the detection antibody. The developed assay exhibits high sensitivity, with a minimum detectable concentration of 0.97 ng/mL for purified antigen. When evaluated in diverse food matrices spiked with RBD, the assay showed LODs of 1.95 ng/mL in honey and sweet corn, 3.9 ng/mL in complex solid foods such as chicken, fish, pork, and mushroom, and 7.81 ng/mL in beverages like cola and fruit juices. This immunoassay demonstrates proof-of-concept for RBD as an alternative target for sensitive BoNT detection.