| Salbutamol (Sal), also called tetrahydro-isowuinolin, is a kind ofβ2-receptor agonist which is used to treat BA clinically. It can promote protein. synthesis and increase the lean of body, redistribute the nutrients, improve the feed conversion ratio. It is often illegally used as a feed additive in livestock production, but the residue of SAL is the potential hazard to human health. Such as European Union, USA and China, so many states and regions have made laws to forbid using stimulant ofβ2 as feed additives one by another. SAL has already been the substitute of Clenbuterol for the technology of detection of SAL is relative hysteretic, even it has the same effects with Clenbuterol. The lawbreakers are using SAL as the new means to make economic benefits. So it is very important for ensuing food safety to establish a quick, simple, convenient and effective technology to detect SAL.Many physical and chemical methods to detect residue of SAL have been established at home and abroad. Although the methods of HPLC and GC-MS are sensitive and accurate, they are not suitable for large batch samples'screening because of their expensive equipments, big cost and high technical request. Enzyme-linked immunosorbent assay (ELISA) is gradually being used to detect the residues of medicines, as a method which is sensitive, fast, powerful specificity and can screen a large number of samples at one time.To establish the ciELISA method for detecting SAL, this research synthesized complete antigen through chemical method, developed high-grade antibodies against salbutamol using the technology of monoclonal antibody. The addition and reclamation test of samples proved that the established ciELISA method for detecting the residue of SAL was stable and reliable.1. Synthesis and identification of SAL complete antigens. SAL succinate was prepared through the alcoholysis reaction between SAL free base and succinic anhydride. Then SAL succinate was conjugated with BSA and OVA via mixed anhydride methods and carbodiimide (CDI). And the complete antigen SAL-BSA and SAL-OVA were prepared. The conjugates were demonstrated to be successful by UV-scanning.2. The preparation of the Monoclonal Antibodies against SAL.SAL-BSA synthesized via mixed anhydride methods was used to immunize BALB/c mouse while SAL-OVA was used for detection The titer of blood serum collected after the fifth immunization reached 1:10,000. Then a hybridoma cell line named 3G11 which could excrete anti-salbutamol monoclonal antibody steadily was gained by hybridoma's technique. Hybridoma 3G11 producing anti-salbutamol monoclonal antibody (McAb) was used to inject into BALB/c mice abdominal cavity. The results showed that the protein concentration of McAb was 20.8mg/mL, the titer of the McAb was 1:32,000.3. The foundation of ELISA method. SAL standard curve was prepared by 3G11 ascites, with the linear equation:y=0.2618x+0.0493(R2=0.9862), IC50 53ng/mL, LOD 2 ng/mL, LOQ 16.7 ng/mL. The cross reaction experiment of other (β2-receptor agonists showed that the cross-reactivity with Clenbuterol Hydrochloride was 38%, with less than 0.1 percent inhibition rate anti ractopamine.4. The addition and reclamation test of SAL. This method was initially applied in the addition and reclamation test of the liver tissue sample of swine. The intervention disappeared when the extraction of liver sample was diluted 4 times. In the range from 0 to 1600 ng/mL, the linear equation was y=0.295x-0.0633(R2=0.9829) and LOD was 3.68ng/g when adding 5.20.50.100.200 and 400ng/mL SAL to the liver tissue of swine, the recovery ratio was between 75% and 100%. The intraassay coefficient of variation was between 2.98% and 10.34%, and the interassay coefficient of variation was between 2.61% and 12.47%.
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