| Clenbuterol Hydrochloride is used to relieve cough and asthma, in recent years, many studies have found that it can promote adipose tissue conversing into protein, as a result increasing the percentage of lean meat. Many farmers illegally put clenbuterol hydrochloride into feed for pigs, sheep and other livestock. After people intake a certain amount of clenbuterol hydrochloride, it will cause metabolic disorders, increase the burden of viscera working, even seriously threaten people’s life.Immune colloidal gold technique is a new kind of immune detection technology, it is widely applied in many fields and developed rapidly owe to its unique characteristics: less time consuming, good sensitivity and specificity, it plays an important role in food testing.This paper will introduce how to develop CLB colloidal gold immunchromatographic strip and use it to test CLB in animal tissues and other processed food which composites meat. First of all, trisodium citrate was used as reducing agent to prepare colloidal gold, UV spectrophotometer was used to evaluate diameter and uniformity of gold particles, then contrasting the absorbance values at wavelength of 400~700 nm, the results showed that when the reducing agent was quantitation, different concentration of trisodium citrate had little effect for colloidal gold, so selected 1% of trisodium citrate and added 1.8 m L, the colloidal gold had the maximum absorption peak at 520 nm, particle size was 18.41 nm. Strip preparation processes were as follows:(1) The optimal labeled antibody proportion:It was 4.5 μg/m L and 0.2 mol/L K2CO3 was added into per milliliter colloidal gold solution 3 μL as the suitable volume for the optimum p H,the final concentration of BSA was 1% which selected as protectant and temperture optima was4℃.(2) The optimal formula for dissolving colloidal gold precipitation:It was that 0.01 mol/L p H 7.4 PB and at the final concentrations of BSA 、sucrose and PEG20000 were 1%、10%、0.5%.(3) The coating concentration of antigen:It was 0.2 mg/m L and anti-mouse antibody was 1.0mg/m L, and optimizing envelope antigen diluent was 0.01 mol/L p H 7.4 PBS.(4) The dilution of gold-labeled antibody was 3×.(5) Selection and pretreatment of cellulose membrane:The suitable cellulose membrane was glass fiber 0194, which was used as conjugate pad. There were some different between 0194 and sample pad GL-b04, the latter had better effect in solution diffusion;The optimal formula for mat sample processing solution were 0.01 mol/L p H 7.4 PB、1% ofsucrose and 1% of Tween-20.(6) Selection of membrane:Comparing different models of nitrocellulose membrane(AE99、Millipore 180、SS24), in order to choose the best performance as the NC membrane, it was Millipore 180.(7) Strip performance:The sensitivity of test strips was 8ng/m L, having no significant cross-reaction with Ractopamine、Salbutamol、Terbutaline and Mabuterol. Colloidal gold test trips could be stability in 60 days at room temperature.There were no difference between same batch, and the high qualifies trips during multi-batch was over 97%.When testing CLB residues in animal meat samples could be extracted as follows: fresh animal tissue(including lean meat, livers) or animal processed foods, 2 g samples with 5 m L 0.2mol/L HCl-4% Na Cl to precipitate protein, reserving supernatant fluid, added 1 m L 1 mol/L Na OH, 4~5 g Na Cl, 10 m L of acetic ether to extract the CLB supernatant fluid, 65℃ nitrogen concentration, with 1 m L 0.01 mol/L ammonium acetate or p H 7.4 PBS solution to redissolve.When tested simulation samples with CLB standard substance, the CLB in pork、dried meat floss or animal liver was no less than 15 ng/g, and it was 20 ng/g in beef jerky could be checked out. |
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