| Clenbuterol is a ?2-adrenergic receptor agonist.It can cause skeletal muscle tremor,arrhythmia,dizziness,weakness and other symptoms of poisoning when human take residual clenbuterol from the animal-derived food.Clenbuterol gold test kit has been widely used in the rapid detection of clenbuterol,the method is simple,fast,intuitionistic.The study optimized the preparation conditions of the clenbuterol colloidal gold test kit and the pre-treatment conditions of the sample,and studied the preparation of antigen and antibody required for clenbuterol colloidal gold test kit preliminary.The colloidal gold were prepared by the method of trisodium citrate reduction.The colloidal gold particles with different particle sizes were prepared by changing the amount of trisodium citrate and scanning in the visible spectrum.The results showed that the size of colloidal gold particles decreased with the addition of trisodium citrate when the amount of trisodium citrate was less than 2.5 mL.When the addition amount of trisodium citrate was more than 2.5 mL,the size of colloidal gold particle increased with the amount of trisodium citrate.The colloidal gold with different particle size was used to prepare the clenbuterol colloidal gold test kit.Through the performance test,it showed that colloidal gold particles with the size around 20 nm were most suitable for the preparation of clenbuterol colloidal gold test kit.From the results of electron microscopy,it could be observed that the colloidal gold size was uniform,its shape was regular and its dispersion was high,and the gold particles were not agglomerated.Through optimizing the preparation process of the clenbuterol colloidal gold test kit.The optimum preparation process was as follows:3 ?L 0.2 mol/L K2CO3 was added in per milliliter colloidal gold solution;the optimal dosage of antibody was 2?g/mL;during preparing the gold-labelled antibody,the centrifugal way was centrifuging at 14000 r/min for 15 min;the volume of gold-labelled antibody was 50?L,the spray-gold amount was 4 ?L/cm;the coating concentration was 0.5 mg/mL on the T line,the coating concentration was 0.5 mg/mL on the C line.Preparing the clenbuterol colloidal gold test kit with this preparation conditions.It can be detected the clenbuterol with the content more than 3 ppb.Through the comparison to three kinds of pork pretreatment methods,the pork pretreatment method was as follows:weighed 1 g homogeneous pork samples,added 1 mL of 1 mol/L hydrochloric acid,the mixture was shaken for 5 min.Added 1 mL of 1 mol/L sodium hydroxide,shaked for 5 min,then added 5 mL of ethyl acetate,shake for 5 min,centrifugated at 4 ?,10000 r/min for 5 min,take the supernatant in another tube and dried with nitrogen at 50 ?.Added 1 mL of sample buffer and add 120 ?L of sample droplets to the test kit.Through pretreating the pork by this method,the detection limit can reach to 12 ng/g.The complete antigen CLB-BSA was prepared by diazo method.Balb/c mice were immunized for four times with this antigens,obtained the mice with immunization titer of 1:320000.The antiserum of the mice was used to prepare the polyclonal antibody.The antiserum was purified by ammonium sulfate fractionation method.The protein concentration of the antiserum was 12.62 mg/mL before purifying,and the protein concentration of the polyclonal antibody was 1.15 mg/mL after purifying.The IC50 of the purified polyclonal antibody was 14.13 ?g/mL.It has been verified that homemade CLB-BSA has been applied to the preparation of clenbuterol colloidal gold test kit. |
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