Construction and Application of Indirect Competitive Enzyme-Linked Immunosorbent Assay for Acetamiprid in Traditional...

The contamination of traditional Chinese medicines (TCMs) with neonicotinoid pesticides, notably acetamiprid (ACE), poses a significant challenge to product safety. Conventional detection methods are often hampered by operational complexity, prolonged analysis times, and dependence on sophisticated instrumentation, rendering them impractical for rapid on-site screening. To address these limitations, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the efficient quantification of ACE residue in TCM matrices. A monoclonal antibody-based ic-ELISA was developed through the synthesis of an ACE antigen. Critical assay parameters-including coated antigen concentration, antibody dilution ratio, and blocking buffer composition-were systematically optimized. The validated protocol was subsequently applied to ACE detection in five representative TCMs. The sensitivity (IC₅₀), limit of detection (IC₁₅), and detection range (IC₂₀-IC₈₀) of the developed ic-ELISA for ACE were 13.61 ng/mL, 0.50 ng/mL, and 1.00-150.99 ng/mL, respectively. The ic-ELISA demonstrated good stability and specificity, with cross-reactivity for ACE analogs all below 1.5%. Additionally, the ic-ELISA for ACE achieved recoveries of 86.87-104.80% in spiked TCM samples (Lonicerae Japonicae Flos, Lycii Fructus, Bulbus Lilii, Citri Reticulatae Pericarpium, and Jasminum sambae Flos), with relative standard deviations (RSDs) of 3.33-12.05%. The recovery rate of ic-ELISA was verified to be in good consistency with that of high-performance liquid chromatography (86.09-102.10%), indicating that ic-ELISA has acceptable accuracy and precision. This approach is simple and sensitive, making it suitable for the rapid quantitative detection of ACE residues in TCM products. It also provides technical references for the development of ic-ELISA for other small-molecule contaminants.