1 principle and use The kit uses indirect competitive ELISA to detect Clenbuterol ( CLE ) in urine samples, tissues, feeds, etc. The kit consists of an enzyme-labeled plate and a horseradish enzyme label pre-coated with antigen. Composition of antibodies, standards and other supporting reagents. At the time of testing, a standard or sample solution is added, and the Clenbuterol hydrochloride and the enzyme-labeled plate in the sample are pre-coated with the antigen to compete against the Clenbuterol antibody, and after adding the enzyme label, the color is developed with the TMB substrate. The sample absorbance value is negatively correlated with the Clenbuterol content contained in the sample, and the residual amount of clenbuterol hydrochloride in the sample can be obtained by comparison with the standard curve. 2 technical indicators 2.1 Kit sensitivity: 0.05ppb (ng / ml) 2.2 Reaction mode: 25 °C, 30min ~ 1 5min 2.3 Detection limit: Urine.................................0.05ppb Organization (Processing Method 1)...............0.2ppb Organization (Processing Method 2)...............0.05ppb Feed....................................0.5ppb 2.4 Cross-reaction rate: Clenbuter..............................100% Teptalin..............................<1% Mabtro...........................<1% Bromobutro..............................<1% Salbutamol...........................<1% Lake Dopamine........................<1% 2.5 Sample recovery rate: Urine sample..............................95%±10% Organization, feed........................85%±15% 3 kit composition Enzyme plate.................................96 hole Standard solution: 1ml each 0ppb, 0.05ppb, 0.15ppb, 0.45ppb, 1.35ppb, 4.05ppb High standard solution (red cover): 100ppb.........1ml Enzyme label (red cover)........................5.5ml Antibody working solution (blue cap)..................5.5ml Substrate A (white cover)........................6ml Substrate B (black cover)........................6ml Stop solution (yellow cover)..............................6ml 20X concentrated washing solution (white cover)..................40ml 10X complex solution (yellow cover)........................50ml Instruction manual..............................1 copy 4 equipment and reagents needed 4.1 Instrument: CSY-E96SR lean meat fast detector , homogenizer, nitrogen blow dryer, oscillator, centrifuge, graduated pipette, balance (sensitivity 0.01g) 4.2 Micropipette: single channel 20μl-200μl, 100μl-1000μl, multi-channel 300μl 4.3 Reagents: sodium hydroxide, ethyl acetate, concentrated HCl, acetonitrile, methanol, n-hexane, anhydrous sodium sulfate 5 sample pretreatment 5.1 Notes before sample processing: Laboratory equipment must be clean and use disposable tips to avoid contamination and interference with experimental results. 5.2 Dosing: Solution 1: 0.1M HCl solution Take 0.86 ml of concentrated HCl and deionized water to 100 ml. Solution 2: 0.1M NaOH solution Weigh 0.4g NaOH plus deionized water to 100ml. Solution 3: acetonitrile-0.1M HCl solution V acetonitrile: V0.1M HCl = 84:16. Solution 4: complex solution The 10× complex solution was diluted 10-fold with deionized water for reconstitution of the sample, and the complex solution was stored at 4 ° C for one month. 5.3 Sample pre-processing steps: 5.3.1 Urine sample processing method: 50 μl of clear urine sample was taken directly for measurement (the turbid urine sample needs to be filtered or centrifuged at 4000 r/min for 5 min to obtain a clear urine sample), and the samples that are not used should be stored frozen. Urine sample dilution factor: 1 detection limit: 0.05ppb 5.3.2 Organizational sample processing method 1: Weigh 2±0.05g of homogenized tissue sample, add 6ml of complex solution, shake well for 2min, and centrifuge at room temperature 4000r/min for 10min. (If the oil content in the tissue sample is high, it can be placed in 85°C water bath for 10min after shaking. Centrifuge again). 50 μl of the supernatant was taken for analysis. Sample dilution factor: 4 lower limit of detection: 0.2ppb 5.3.3 Organizational Sample Processing Method 2: 1) Weigh 2±0.05g of homogenized tissue sample, add 6ml acetonitrile-0.1M HCl, shake well for 2min, and centrifuge at room temperature 4000r/min for 10min. 2) Take 3ml of the supernatant, add 2ml of 0.1M NaOH, add 6ml of ethyl acetate, shake well for 2min, centrifuge at room temperature 4000r/min for 10min, and take all the supernatant at 50-60 °C under nitrogen or air flow to complete dry; 3) Add 1 ml of the complex solution and mix and shake for 30 s, and take 50 μl for analysis. Sample dilution factor: 1 lower limit of detection: 0.05ppb 5.3.4 Method of processing feed samples: 1) Weigh the homogenized feed sample 1.0±0.05g, add 10ml methanol, add 5g anhydrous sodium sulfate, shake for 2min, centrifuge at room temperature 4000r/min for 10min; 2) Aspirate 1 ml of the supernatant after centrifugation, blow dry at 50-60 ° C under nitrogen or air, dissolve the dried residue with 1 ml of the complex solution, add 1 mL of n-hexane for 30 s, and centrifuge at room temperature 4000 r/min for 5 min. 3) Take 50 μl of the lower layer for analysis. Sample dilution factor: 10 detection limit: 0.5ppb 6 enzyme-linked immunoassay steps Remove the required reagent from the 4 ° C refrigerated environment, and equilibrate at room temperature for more than 30 min. When the washing solution is refrigerated, the crystal may be returned to room temperature to be fully dissolved. Each liquid reagent should be shaken before use. Remove the required number of microplates and frames, place the unused microplates in a ziplock bag and store at 2-8 °C. Before the start of the experiment, the 20X concentrated washing solution was diluted with 20 times of deionized water into a working washing solution. 6.1 No.: The micropores corresponding to the sample and the standard are numbered sequentially, and each sample and the standard are made in parallel with 2 holes, and the position of the standard hole and the sample hole is recorded. 6.2 Addition reaction: Add standard or sample 50μl/well to the respective microwells, then add 50μl/well of enzyme label, then add 50μl/well of antibody working solution, seal the plate with cover membrane, and gently shake 5 Mix in seconds and react at 25 ° C for 30 minutes. 6.3 Washing: Carefully uncover the cover film, dry the liquid in the well, and wash it thoroughly with the working washing liquid 250μl/well 5 times, each time interval 30 seconds, pat dry with absorbent paper (bubbles that have not been removed after pat drying) A clean gun puncture). 6.4 Color: 50 μl of substrate A was added to each well, and 50 μl of substrate B was added. The mixture was gently shaken for 5 seconds, and mixed at 25 ° C for 15 minutes. 6.5 termination: 50 μl of stop solution was added to each well, and the mixture was gently shaken to terminate the reaction. 6.6 Measured absorbance: The absorbance value of each well was measured at 450 nm using a clenbuterol tester (double wavelength 450/630 nm is recommended). The assay should be completed within 10 minutes of terminating the reaction. 7 results analysis 7.1 Calculation of percent absorbance The percent absorbance of the standard solution or sample is equal to the average value of the absorbance value of the standard solution or sample (double well) divided by the absorbance value of the first standard solution (0 ppb), multiplied by 100%, ie Percent absorbance value (%) = A ×100% A0 A—the average absorbance value of the standard solution or sample solution Average absorbance value of A0—0ppb standard solution 7.2 Drawing and calculation of standard curve The semi-logarithmic plot of the standard solution is plotted with the percent absorbance of the standard solution as the ordinate and the logarithm of the corresponding standard solution concentration (ppb) as the abscissa. Substituting the percent absorbance of the sample into the standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the corresponding dilution factor is the actual concentration of the analyte in the sample. If the kit professional analysis software is used for calculation, it is more convenient for accurate and rapid analysis of a large number of samples. (Welcome to call) 8 considerations 8.1 Room temperature below 25 ° C or reagents and samples not returned to room temperature (25 ° C) will result in low OD values ​​for all standards. 8.2 If the plate hole is dry during the washing process, the standard curve will not be linear and the repeatability is not good. Therefore, the next step should be taken immediately after the plate is patted dry. 8.3 The mixing should be uniform, the washing should be thorough, and the reproducibility in the ELISA analysis depends largely on the consistency of the washing. 8.4 Seal the microplate with a cover film during all incubations to avoid light exposure. 8.5 Do not use kits that have expired. Do not exchange reagents from different batches. 8.6 If any color of the coloring solution indicates deterioration, it should be discarded. A 0 standard absorbance value of less than 0.5 units (A450nm < 0.5) indicates that the reagent may deteriorate. 8.7 The reaction stop solution is corrosive and avoids contact with the skin. 9 storage and storage period Storage conditions: The kit is stored at 2-8 ° C to avoid freezing. Shelf life: The product is valid for 1 year, and the production date can be found in the box. 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