Enzyme-linked immunoassay for simultaneous detection of methyl parathion and sibutramine in apple cider vinegar

Abstract

Methyl parathion, a highly toxic, efficient, and persistent organophosphorus pesticide, is widely used in China. Sibutramine, a non-amphetamine central nervous system depressant, helps lose weight by disrupting hormone regulation, stimulating sympathetic nerves, and suppressing appetite. However, some unethical businesses fail to properly handle raw materials in foods like apple cider vinegar, leading to residual methyl parathion in apples or illegal excessive addition of sibutramine. Therefore, it is imperative to develop an immunoassay for the rapid detection of methyl parathion and sibutramine. The corresponding two haptens were prepared and coupled with the carrier proteins according to methyl parathion–sulfur–bovine serum protein (BSA)/chicken ovalbumin (OVA)–sibutramine (20 : 1 : excess, 15 : 1 : excess, 10 : 1 : excess, and 5 : 1 : excess), and sibutramine–BSA/OVA–methyl parathion (20 : 1 : excess, 10 : 1 : excess: 5 : 1 : excess, and 0 : 1 : excess). The result shows that the inhibition rate of the antibody obtained by methyl parathion–BSA/OVA–sibutramine (20 : 1 : excess) was higher than that of sibutramine–BSA/OVA–methyl parathion, which was 67.93%, and the concentration of methyl parathion was 8.65 ng mL⁻¹ at this inhibition rate. Thus, methyl parathion–BSA/OVA–sibutramine (8.65 : 1 : excess) and the corresponding antibodies were selected for subsequent method establishment. By changing the concentration of the coating and antibody, the inhibition rate was found when the coating was 0.125 ng mL⁻¹ and the antibody was diluted 4000 times. The antibody was used to develop a standard curve for the detection of sibutramine at the half-maximum inhibitory concentration (IC₅₀) is 4.59 ng mL⁻¹, the limit of detection (IC₁₀) is 2.21 ng mL⁻¹, the detection range is 2.89 to 7.28 ng mL⁻¹, methyl p–phosphorus at the half-maximum inhibitory concentration (IC₅₀) is 15.34 ng mL⁻¹, the limit of detection (IC₁₀) is 0.42 ng mL⁻¹, the detection range is ng mL⁻¹. Under these conditions, the recovery rate was between 88% and 102%, within reasonable limits, indicating the successful establishment of a rapid enzyme-linked ELISA assay.