| Salbutamol is a kind of the artificially synthesized beta-agonists and the drug of bronchial asthma clinically.It may be used illegally in animal feed to increased accretion of skeletal muscle mass.However,when people eat animal tissues that contain high content of salbutamol,there will be potential symptoms including headache,nervousness and heart palpitations.It will cause serious liver damage,threating the health of people.Currently,the detection standards of Salbutamol is chromatography.The confirmatory method has disadvantage of complex sample pretreatment,large instrumentation,professional operation and time-consuming.To control the illegal use of salbutamol,there should be established a rapid,convenient and effective analysis method.Colloidal gold-based immunochromatographic assay is a kind of immunological analysis technology,which is used in food,biology,agriculture,medicine and other fields.It offers the advantage of rapidity,sensitive,simplicity,low costs and easy to realize industrial manufacture.This study aimed at the development of colloidal gold immunochromatographic strip for the rapid detection of salbutamol.The colloidal gold was obtained by reducing the gold chloride with sodium citrate which had two kinds of different particle sizesand labeled SAL-Pc Ab.The gold conjugate pad was oven-dried after soaked specific immunogold to glass fiber of GL0194.The Millipore 135 were used to prepare the test strip,in which the rabbit anti-mouse Ig G coated at 1.0 mg/mL as control line,the SAL-BSA coated at 1.0 mg/mL.Gold conjugating pad,sample pad,nitrocellulose membrance and absording pad were assembled to immunochromatographic test strip.And the test strip was applied to the animal tissue and feed sample pretreatment method for single factor optimization to choose the appropriate program.By comparing the optimal labeled antibody proportion,the optimal pH and the centrifugal condition,the best colloidal gold solution for salbutamol is 1.8 mL in the 0.01% colloidal gold solution 100 mL.The amount of SAL Pc Ab is 6 ?g/mL and the optimum pH is 8 ?L/mL by adding the amount of 0.2 mol/L potash according to the exosyndrome of color and UV-scanning.The formulation of colloidal gold precipitation is 0.01 mol/L pH7.4 PBS which added the final concentrations of 10% sugar,0.5% PEG-20000,1% BSA and 0.1% Na Cl.The formulation of sample pad is 0.01 mol/L pH7.4 PBS which added the final concentrations of 1% BSA and 1%Tween-20.The pretreatment of animal tissues is the method of using trichloroacetic acid by ultrasonic extraction.The pretreatment of feed is the method of using 0.2 mol/L phosphoric acid-method(V:V,4:1)by ultrasonic extraction.The optimized sample dosage(m)is 2 g,the volume extraction liquid is 4 mL,the ultrasonic time is 8 min and the centrifugal time is 5 min by single factor optimization.The sensitivity of test strips was 3 ?g/L using salbutamol standard.The limit of detection in animal tissues and feed were 10 ?g/kg and 12 ?g/kg.The positive samples were detected with 100% positive result within sensitivity range with no false-positive and false-negative phenomenon.On the preparation of salbutamol strip test showed that the strip stability is good and the strip to SAL has strong specificity.It can be well applied in the actual detection of animal tissues and feed samples. |
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