Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope

Area of Science:

• Neuroscience and Immunology
• Molecular Biology
• Biotechnology

Background:

• Botulinum neurotoxin type E (BoNT/E) causes flaccid paralysis by cleaving synaptosomal-associated protein (SNAP-25) in motor neurons.
• Accurate detection of the BoNT/E-cleaved SNAP-25 neoepitope is crucial for developing effective anti-BoNT/E antibody preparations and assays.
• Existing methods may lack specificity or require extensive animal use for antitoxin characterization.

Purpose of the Study:

• To isolate highly specific monoclonal antibodies for the in vitro immuno-detection of the BoNT/E-cleaved SNAP-25 neoepitope.
• To develop a novel antibody for sensitive detection of cleaved SNAP-25 in cell-based assays.
• To enable more efficient and potentially reduced animal testing for antitoxin potency determination.

Main Methods:

• Immunization of rabbits and mice with a peptide representing the C-terminus of cleaved SNAP-25.
• Construction of a single-chain variable fragment (scFv) phage-display library from immunized rabbits.
• Isolation and characterization of antibodies, including expression of a chimeric scFv-Fc antibody.

Main Results:

• Rabbits produced a robust polyclonal antibody response, while mice showed a weaker, less specific response.
• Isolated antibodies, particularly a chimeric scFv-Fc construct, exhibited selective, ultra-high affinity (pM) for the SNAP-25 neoepitope.
• The antibody successfully detected cleaved SNAP-25 in BoNT/E-treated cells without cross-reactivity with intact SNAP-25.

Conclusions:

• A novel, specific, high-affinity antibody targeting the BoNT/E-derived SNAP-25 neoepitope has been successfully isolated.
• This antibody is suitable for sensitive in vitro detection of BoNT/E activity.
• The developed antibody can be applied in cell-based assays for antitoxin potency testing, potentially reducing laboratory animal usage.