Abstract:

A direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed for rapidly determining salbutamol. Horseradish peroxidase-labeled salbutamol monoclonal antibodies were synthesized by sodium periodate method. The optimal coating concentration and antibody dilution were determined by chequerboard titration. The effects of surfactants, ion concentration, methanol content and pH on the sensitivity of dcELISA were investigated by single-factor experiments. The results showed that the optimal molar ratio of enzyme-labeled antibody was 2.1. The optimized assay conditions for both the highest sensitivity and the best stability were as follows: coating antigen concentration, 1μg/mL; and dilution fold of antibodyenzyme conjugate in 0.01 mol/L pH 7.4 PBS dilution containing 0.05 % Tween-20, 0.5 mol/mL NaCl, and 5% methanol, 2560. The developed method presented an IC50 of 10.3 ng/mL, a detection limit of 0.049 ng/mL, a linear range of 0.3 to 76.30 ng/mL, with an intra-batch CV of 13.8 % and an inter-batch CV of 22.38%, and 321.18% and 29.09% cross-reactivity rates with clenbuterol and brombuterol, respectively, without notable cross-reactivity with other structural analogues of salbutamol. This study provides valuable experimental data for developing a commercial immunoassay kit for the rapid determination of multiresidues of salbutamol and clenbuterol.

Key words: salbutamol, enzyme-labeled antibody, direct competitive ELISA