Direct Method Of Surface Plasmon Resonance For Rapid Detection Of Clenbuterol And Salbutamol In Cow Urine

Clenbuterol and Salbutamol areβ₂-adrenoceptor agonists,usually called"nutrient redistribution agents"or"lean meat powder".They have been often used illegally as feed additives in the livestock product breeding industry for profit because of its physiological effects such as nutrient redistribution,changing the metabolic pathways of substances in animals,accelerating protein synthesis,and significantly increasing the growth rate of food animals.Theβ₂-agonist residues in the animal would have certain toxic and side effects on the consumers,even poisoning phenomenon when it enters the human body through the food chain.At present,China,the European Union and many other countries have promulgated a number of laws and regulations to banned the use ofβ₂-agonist as animal feed additives clearly.Therefore,in order to strengthen food safety supervision and protect the health of consumers,it is urgent to establish a more rapid and reliableβ₂-agonist detection method based on the existing methods.The existing methods which are used for confirmatory detection include chromatography,conventional enzyme-linked immunoassay,etc.,however,high-throughput detection of actual samples cannot be achieved due to troublesome pre-processing,complicated operations,and long detection time.Nowadays,surface plasmon sensors have important application prospects in the detection ofβ₂-agonists in animals due to the advantages such as high sensitivity,low interference,and fast detection speed.Monoclonal antibodies with high specificity were prepared for clenbuterol and salbutamol as the detection targets.CLB-BSA and SAL-BSA coupling immunogen were prepared as antigen to immunize BALB/c female mice,using hybridoma technique by fusing spleen cells of BALB/c with mice which had best immune to myeloma cells,and positive cells were subcloned,hybridoma cell lines with specificity,high titer,and stable secretion of monoclonal antibodies could be obtained by indirect ELISA.Then the selected hybridoma cells were injected into the abdominal cavity of mice,and ascites were collected.The ascites titer of the CLB and SAL monoclonal antibody were 1:4.5×10⁶ and 1:3.40×10⁵respectively.According to the results of indirect competitive ELISA,the IC₅₀ of CLB monoclonal antibody to clenbuterol is 2 ng/m L,and the IC₅₀ of ractopamine and salbutamol drugs were greater than 10000,so cross reaction rate is less than 0.2%.The IC₅₀ of SAL monoclonal antibody to salbutamol is 2 ng/m L,and to clenbuterol is 11.3 ng/m L,cross reaction rate was 22%.The cross reaction rate to ractopamine and other drugs was less than0.2%.An SPR immunosensor method was developed for the direct detection of clenbuterol and salbutamol in cow urine.The CM7 chip was modified by the amino coupling method and the antibody was fixed on the chip.and the antibody coupling conditions were optimized.Optimize antibody coupling conditions,such as using 10 m M p H 5.0 acetic acid-sodium acetate buffer as coupling buffer,mixing EDC and NHS at a ratio of 1:1 to activate the chip,setting the activation time is 15 min and the antibody reaction time is 20min,finally,the chip was blocked with ethanolamine for 15 min.Using the direct detection method,the cow urine was passed through the CM7 chip which was immobilized with antibody to construct a standard curve after centrifugating and filtrating cow urine.The linear range of the standard curves was 0.1～6 ng/m L and the limit of detection was 0.05ng/m L.Within the range of 1 ng/m L～5 ng/m L,the average recoveries of clenbuterol in cow urine ranged from 82.46%to 98.60%,the intraassay coefficient of variation was between 1.67%and 8.50%and interassay coefficient of variation was between 2.61%and10.14%.The same method was used for the determination of salbutamol,the limit of detection was 0.01 ng/m L with a good linear relationship in the concentration range of 0.1～6ng/m L.Within the range of 1 ng/m L～5 ng/m L,the average recoveries of clenbuterol in cow urine ranged from 87.82%to 91.67%,the intraassay coefficient of variation was between1.51%and 2.67%and interassay coefficient of variation was between 2.67%and5.53%.UPLC-MS/MS and SPR biosensor were used to compare the contents of CLB and SAL in cow urine samples,the result showed that SPR method could be used for the detection of clenbuterol and salbutamol in animals.