| Salbutamol(SAL), also called tetrahydro-isowuinolin, is a kind of selective β2-receptoragonist which is used to treat BA clinically. It can promote muscle(lean) growth, reduce fataccumulation, improve the feed conversion ratio and so on. It is often illegally used as a feedadditive in livestock production. Salbutamol residues in food can cause serious impact onpeople’s health, such as palpitations, headache, dizziness breathing-difficulties. Countries,including China, have made laws to forbid using stimulant of β2as feed additives one byanother. The technology of detection of SAL is relative hysteretic, and SAL has the sameeffect as Clenbuterol. To make economic benefits, some lawbreakers use salbutamol in thefarm instead of clenbuterol as a growth promoter. So it is very important for ensuing foodsafety to establish a quick, simple, convenient and effective technology to detect SAL.There are many methods for salbutamol detection at present, such as GC-MS, HPLC,LC-MS, ELISA and so on. In these methods, GC-MS, HPLC and LC-MS are accurate, stableand reliable, but they are not suitable for production, grass-roots units and on-site inspectionwith the defects of cumbersome pre-treatment and high technical requirements. The ELISAtechnique as long been known as a rapid, specific, and cost-effective analytical method andhas broad application prospects. The aim of this research is to establish the ELISA techniquefor salbutamol.Salbutamol derivatives were prepared by four kinds of reactions, then they were coupledto BSA and OVA, synthesizing complete antigen. The conjugates were demonstrated to besuccessful by UV scanning. The coupling ratios of four immunogens were9:1,6:1,6:1,12:1.Four immunogens were synthesized in this research to develop anti-salbutamol ant-ibodies. All antibodies raised in rabbits and coating antigenes synthesized were screened andcharacterized to select the best combination. The antibody prepared with Hapten D-OVAshowed the best result in the assay using Hapten A-BSA as coating antigens. The antibodyshowed high cross-reactivity with clenbuterol(144%) and terbutaline(169%) and waspromising for the simultaneous determination of terbutalille, salbutamol and clenbuterolresidues in food and food products. The most suitable working condition of ELISA was obtained by several grid tests whichwere used to calculate the most optimal dilution of the coating antigen, antiserum, and HRPsecondary antibody and the results were1:2000,1:4000and l:6000. The standard curve ofELISA was also established and the curve indicated that the linear determination was0.5~20ng/mL with50%inhibitive concentration (IC50) of2.33ng/mL. Methods of sampletreatment were established. The determination limits to swine urine, liver and forage were0.715ng/mL,0.884ng/mL,1.289ng/mL, respectively. The quantitation limits to swine urine,liver and forage were1.358ng/mL,1.522ng/mL,2.613ng/mL, respectively. Recovery ratesfrom the salbutamol fortified feed samples were in the range of80-120%mainly, while theintra-assay and inter-assay coeffieients of variation were below15%. The validity of SAL-Kitat2~8℃was above12months. |
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