Quantitative detection of β2-adrenergic agonists using fluorescence quenching by immunochromatographic assay

Abstract

β₂-Adrenergic agonists are banned in China and other areas in the world. In this study, a novel method was developed to quantitatively detect β₂-adrenergic agonists. The clorprenaline (CLP)–bovine serum albumin (BSA) conjugate is mixed with a BSA–fluorescent microsphere (FM) complex and sprayed on a nitrocellulose membrane as the test line; a goat-anti-mouse antibody is mixed with a BSA–fluorescent microsphere (FM) complex and sprayed on a nitrocellulose membrane as the control line. If the target molecule is absent in the sample, the colloidal gold-monoclonal antibody will bind to the CLP–BSA conjugate coated on the test line, and the colloidal gold quenches the fluorescent microspheres, so that no fluorescent signal develops in the test line, indicating a negative result. The target molecule present in the sample at a cutoff level or higher binds to the colloidal gold-monoclonal antibody in the ELISA well. The colloidal gold-monoclonal antibody (Au-mAb) does not bind to the CLP–BSA conjugate coated on the test line. The fluorescent signal developed in the test line indicates a positive result. The limit of detection (LOD) of the immunochromatographic assay test strip was 0.12 ng mL⁻¹ when the antibody amount was 0.8 μg mL⁻¹ with a detection time of 15 min. The immunochromatographic assay test strip could simultaneously detect five β₂-adrenergic agonists, including clorprenaline, bambuterol, terbutaline, clenbuterol, and salbutamol. When spiked swine urine samples (5.0 ng mL⁻¹ and 10.0 ng mL⁻¹) were tested by the novel immunoassay, the recovery was 39.00 ± 3.0 and 32.00 ± 2.0, respectively.