Tetracycline enzyme-linked immunosorbent assay (ELISA) instructions

This kit is for research use only. 1 Purpose of use: This kit is used for quantitative detection of tetracycline residues in feed, fish, shrimp and meat tissues (such as chicken, beef and pork), eggs, honey, milk, serum, urine samples, soil and other samples. 2 Experimental principle The kit uses a competitive ELISA method, coated with tetracycline-conjugated antigen in a microplate, added tetracycline standard or sample, free tetracycline and pre-coated tetracycline-conjugated antigen on microporous strip compete with each other for anti-tetracycline antibody The enzyme label is developed with TMB substrate. After adding the stop solution, the color changes from blue to yellow. The sample is detected by a microplate reader at a wavelength of 450 nm. The absorbance is inversely proportional to the tetracycline content in the sample, and the sample is calculated by a standard curve. The content of tetracycline. 3 kit composition 3.1 pre-coated tetracycline-conjugated antigen detachable plate: 1 (12 holes × 8). 3.2 Tetracycline standard: 6 bottles (1 ml / bottle), the contents are: 0ppb, 3 ppb, 9ppb, 27 ppb, 81 ppb, 243 ppb3.3 anti-tetracycline antibody enzyme conjugate: 1 bottle (6ml). 3.4 Coloring solution A: 1 bottle (6 ml). 3.5 color developing solution B: 1 bottle (6 ml). 3.6 Stop solution: 1 bottle (6 ml), 2 M sulfuric acid. 3.7 Sample Diluent: 1 bottle (10 ×, 6 ml) for sample dilution. 3.8 Concentrated washing solution: 1 bottle (20 x, 20 ml) for washing the plate. 3.9 a copy of the manual. 4 Materials not required but not provided 4.1 Equipment 4.1.1 Wavelength 450nm microplate reader. 4.1.2 Crusher. 4.1.3 Measuring cylinder. 4.1.4 Oscillator. 4.1.5 funnel. 4.1.6 Whatman No 1 or equivalent filter paper. 4.1.7 Micropipettes. 4.2 Reagents 4.2.1 Deionized water or distilled water. 4.2.2 Methanol. 5 Storage 5.1 The kit should be stored at 2~8 °C, do not freeze. 5.2 Unused microplate should be sealed and stored dry. 6 Caution 6.1 Please read the instructions carefully before using the kit. 6.2 Do not use expired kits. 6.3 Before using the kit, return the reagent to room temperature (25±2°C). It is recommended to return to the temperature for at least 2 hours. 6.4 Tetracycline is included in the standard. Special care should be taken when using it. Gloves should be worn during operation. 6.5 Stop solution contains sulfuric acid, which prevents burns on the skin and corrosive clothing. 6.6 The tips used in different standards and samples should not be mixed, otherwise the test results will be affected. 6.7 Reagents in different batches of reagent kits must not be mixed; the tips used in different standards and samples must not be mixed, otherwise the results will be affected. 6.8 Sample dilutions in this kit must be used when diluting the sample, otherwise the results will be affected. 6.9 Avoid foaming when mixing reagents. 7 Working solution preparation 7.1 Tetracycline standard solution: 0ppb, 3 ppb, 9ppb, 27 ppb, 81 ppb, 243 ppb7.2 Concentrated washing solution: diluted with distilled water 1:20 (1+19). 7.3 Sample Diluent: Distilled water is diluted 1:10 (1+9). 7.3 Developer: Already reserved, avoid direct light 7.4 Reaction stop solution: Already 8 Sample processing: (In the extraction process, strictly follow the instructions, the extraction process The sample should be accurately diluted, otherwise the result will be inaccurate, the sample should be stored in a cool and dark place and stored in cold storage. General sample treatment 8.1 Take 10g of crushed sample, add 20ml 70% methanol solution 8.2 strong oscillation for 3 minutes 8.3 with Whatman No 1 filter paper filter 8.4 take 100μl of the treated sample, add 400μl sample dilution 8.5 take 100μl dilution for analysis of animal tissue pretreatment 8.6 accurately weigh 1 ± 0.05 g homogenized tissue sample to 50 ml polystyrene centrifuge tube Add 3ml acetonitrile-acetone extraction solution, violently shake for 20s at 2000rpm, centrifuge for 5min at 4000r/min or more. 8. Take supernatant 0.8ml and blow dry at 50°C under nitrogen flow. Add 3.2mL sample dilution, vortex at 750rpm for 20s8. 9 take 100?l for points Feed pretreatment method 8.10 Accurately weigh 1±0.05 g of crushed feed sample into 50 ml polystyrene centrifuge tube; add 4ml acetonitrile, 1ml acetone, violently shake for 20s at 2000rpm, centrifuge at 4000r/min for 3min, 8.11, take the supernatant. 0.7 ml was blown dry under nitrogen flow at 50 ° C. 8.12 was added to 2.8 mL of sample dilution, vortexed for 20 s, mixed and taken for 100 μl for analysis of milk pretreatment method 8.13 1 ml of milk sample was taken into 5 ml polystyrene centrifuge tube Add 4ml sample dilution, mix and take 100?l for analysis of milk powder pretreatment method 8.14 accurately weigh 0.3g milk powder sample into 7 ml polystyrene centrifuge tube; add 2ml PBS solution, 2 ml n-hexane, Shake and mix for 8.154000r/min or more for 5min, remove the organic layer and intermediate layer, take the lower layer solution 100ml to 400ml sample dilution 8.16 and mix and take 100ml for analysis. 9 Enzyme-free analysis step 9.1 Experimental Notes 9.1.1 Before the experiment Allow all reagents to return to room temperature (25 ± 2 ° C) outside the box for approximately 2 hours. After returning to room temperature (25±2°C), remove the microporous strips. Re-seal the excess microporous strips and dry them at 2~8°C. Note: Ensure that the temperature is sufficient, otherwise the accuracy and accuracy of the detection will be affected. 9.1.2 Put the reagent back to 2~8°C immediately after use. 9.1.3 Please do not change the analysis program 9.1.4 Please use the precise micropipette 9.1.5 Once the operation starts, please do not interrupt any program 9.1.6 The repeatability of the ELISA results depends greatly on the operating procedure. Please operate in strict accordance with the requirements. 9.1.7 To avoid cross-contamination, each standard and sample should be loaded with different tips. 9.1.8 When loading Do not allow the tip to contact the solution or inner surface of the microwell. 9.2 Analytical Procedure 9.2.1 Pre-number, mark the position of B0, standard and sample. It is recommended to perform two-hole detection. 9.2.2 Take the required number of microwells (micro The hole strip is detachable), the excess strip is resealed and immediately returned to 2~8 °C to save. 9.2.3 Sample dilution solution, concentrated washing solution (20×) diluted into working solution (distilled water or deionized water diluted) 9.2.4 Add 50 μl of 0.0 ppb standard solution to B0 well 9.2.5 Add 50 μl of standard solution to each standard well 9.2.6 Add 50 μl of sample solution to each sample well 9.2.7 Add 50 μl of anti-tetracycline to all wells Aproenzyme Conjugate 9.2.8 Gently shake the plate for a few seconds. 9.3 37 °C warm bath for 30min (during the bath during the warm bath, the reaction plate can be tapped from time to time to reduce the double hole error) 9.3.1 Remove the liquid from the well and wash the microplate with the washing solution 5 times. The last time should be tapped on the absorbent paper to completely Remove the liquid from the well. 9.4 Reaction 9.4.1 After the washing procedure is completed, immediately add 50 μl of coloring solution A to each micropore using a micropipette, and add 50 μl of coloring solution B; shake the reaction plate slightly to mix thoroughly. 9.4.2 37 ° C Warm bath 10 min 9.4.3 Add 50 μl of stop solution to each well, mix 9.4.4 to measure absorbance at 450 nm, and read within 5 min. 10 Calculation of results 10.1 Quantitative analysis 10.1.1 The average value of each concentration standard solution and sample absorbance obtained (B) is divided by the absorbance value (B0) of the first standard (0 standard) and multiplied by 100%, ie Percent absorbance value. B—The average absorbance value of the standard solution or sample solution B0—0 ppb The average absorbance value of the standard solution is 10.1.2. The logarithmic value of the tetracycline concentration is the X-axis, and the percent absorbance is the Y-axis, and a standard curve is drawn. According to the sample absorbance value, the abscissa of the corresponding point can be obtained from the curve, which is the logarithm of the concentration of tetracycline. The anti-number is the concentration of tetracycline in the measuring solution C(ppb) 10.1.3 because the sample has been pre-diluted Therefore, the concentration of the sample obtained from the standard curve must be multiplied by its dilution factor. 10.2 Semi-quantitative determination 10.1.1 Visual semi-quantitative determination: First select an appropriate standard solution to run with the sample, and judge whether the sample concentration value is smaller or larger than the standard value based on the comparison between the sample and the standard absorbance value. 10.1.2 Semi-quantitative determination of the instrument: First select an appropriate standard solution to run with the sample, and judge whether the sample concentration value is smaller or larger than the standard value according to the color depth of the sample and the standard. 11 Specific substance cross-reaction tetracycline 100% 12 kit parameters The detection limit of this kit is 3ppbB0 The best value of absorbance should be greater than 1.0 The absorbance of the kit is less than 8%, and the inter-plate error is less than 15%. The recovery rate of the tissue sample extraction method provided by this specification is greater than 80%. 13 Standard Curve Mode (for reference only) The standard curve available from the kit ranges from 3 ppb to 243 ppb. 14 Analytical Limits Samples tested positive for this kit should be confirmed by another method such as HPLC or GC/MS.

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