The Research Of Colloidal Gold-based Immunochromatography Assay For Rapid And Quantitative Detection Of β2-agonist Residues In Swine Urine

The traditional colloidal gold-based lateral flow immunoassay is only used to qualitatively or semi-quantitatively detection. The result is obtained according to the color of the test line, but it would be influenced by the environment and the operators.Colloidal gold-based lateral flow immunoassay system, that involves the use of a portable strip reader, can rapidly and quantitatively detect targets in samples according to the T/C ratio. It is rapid, convenient, sensitive, cheap, quantitative and suitable for on-site testing. In the research, it aimed to develop the colloidal gold-based lateral flow immunoassay for rapid quantitative detection system, and initially explore the stability of the system. The research will provide the theoretical basis and basic data for extending the application of the rapid quantitative detection system.Recently, clorprenaline has been found to be used as a new kind of “lean meat powder”. It metabolizes slowly and accumulates in animal bodies, the animal product will threat to the health of humankind by food chain. A competitive colloidal gold-based lateral flow immunoassay with a portable strip reader has been successfully developed for rapid quantitative detection of clorprenaline residue in swine urine. The detection system was optimized so that the detection can be completed within 9 min with the limit of detection of 0.15 μg/L. The assay exhibited good linear range from 3.0 μg/L to 20.0 μg/L with reliable correlation of 0.997 and with no obvious cross-reaction with five other β2-agonist compounds. Twenty spiked swine urine samples were tested by lateral flow immunoassay and LC–MS/MS to confirm the accuracy of the system. Results show good correlation between the two methods. This method is convenient, rapid, sensitive, and specific. It can be applied in the field for on-site detection of clorprenaline in urine samples.In the farmed animals industries, clenbuterol, ractopamine and salbutamol have been used to promote animal growth in muscular mass and decrease fat accumulation.In the research, the accelerated aging test that involved storage at 60℃ has beencarried out to explore the stability of the test strips. The results showed that the stability of the control line was the key factor. The ractopamine test strip showed better stability than clenbuterol and salbutamol test strips. The control lines significantly decreased leading to an increase of T/C ratio, the main factor affecting the strip stability was the control line. To improve the stability of the test strip, the goat anti-mouse antibody spotted on the control line was mixed with Wellchampion(5%, v/v). Furthermore, the newly prepared strips showed satisfactory stability by drying the nitrocellulose membrane at 60℃ for one day.Besides, colloidal gold-based lateral flow immunoassay for rapid quantitative detection system has been successfully developed. The limit of detection of clenbuterol, ractopamine and salbutamol residues in swine urine samples were 0.19μg/L, 0.39 μg/L, and 0.83 μg/L, respectively. And the recoveries of the detection system ranged from 86.6% to 109.7% with the coefficient of variations between 4.7%and 14.7%. The limit of detection in pork samples was 0.43 μg/kg, 0.52 μg/kg, and1.01 μg/kg, respectively. The results showed that the recoveries were in the range of71.2% to 101.6% with the coefficient of variations ranged from 7.2% to 17.9%, when the detection system was evaluated by spiked pork samples. In the study, the quantitative detection system has been applied to detect 135 swine urine samples and710 pork samples. The result that all samples were negative showed that clenbuterol,ractopamine and salbutamol have been effectively monitored in our country.