Two binding epitopes modulating specificity of immunoassay for β-agonist detection: Quantitative structure-activity...

Area of Science:

• Food Safety
• Veterinary Pharmacology
• Analytical Chemistry

Background:

• Illegal use of beta-agonists (β-agonists) for animal fat reduction is a growing concern.
• Analytical methods often lag behind the emergence of new illicit β-agonist compounds.
• Class-specific antibody-based immunoassays are crucial for timely supervision of β-agonist use.

Purpose of the Study:

• To develop a class-specific immunoassay for detecting multiple β-agonists.
• To identify critical structural features of β-agonists that influence antibody specificity.
• To provide insights for designing novel β-agonist haptens for improved detection.

Main Methods:

• Development of a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) using a clenbuterol monoclonal antibody.
• Recognition of 23 β-agonists and analogues by the developed ciELISA.
• Application of holographic and three-dimensional quantitative structure-activity relationship (HQSAR and 3D QSAR) to analyze structure-activity relationships.

Main Results:

• The developed ciELISA successfully recognized 23 different β-agonists and analogues.
• HQSAR and 3D QSAR identified two critical binding epitopes on β-agonist haptens affecting antibody specificity.
• Validation using a ractopamine antibody confirmed the importance of the tert-butyl group at the C-2' epitope and substituents on the benzene ring for antibody specificity.

Conclusions:

• A class-specific ciELISA for detecting a wide range of β-agonists was successfully developed.
• Understanding the structure-activity relationships of β-agonists is key to designing specific and broad-spectrum antibodies.
• This study provides a valuable reference for the rational design of future β-agonist haptens and immunoassays for food safety monitoring.