Human bladder tumor antigen (BTA) ELISA experimental principle This reagent is for research use only: this kit is used to determine the content of bladder tumor antigen (BTA) in human serum, plasma and related liquid samples. Experimental principle: This kit uses the double antibody sandwich method to determine the level of human bladder tumor antigen (BTA) in the specimen. Microporous plates were coated with purified human bladder tumor antigen (BTA) antibody to prepare solid-phase antibodies. Human bladder tumor antigen (BTA) was added to the microwells coated with mAb in turn, followed by HRP-labeled bladder tumor antigen ( BTA) The antibody binds to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the human bladder tumor antigen (BTA) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human bladder tumor antigen (BTA) in the sample was calculated by a standard curve. Kit composition: kit composition 48 well configuration 96 well configuration storage instructions 1 copy 1 copy sealing film 2 pieces (48) 2 pieces (96) 1 sealed bag 1 enzyme coated plate 1 × 48 1 × 96 2 Store standard at -8 ℃: 135pg / ml 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ℃, store standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle at 2-8 ℃, store enzyme label reagent 3 ml × 1 bottle of 6 ml × 1 bottle of sample diluent at 2-8 ° C 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Developer A 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Developer B solution 3 ml × 1 bottle 6 ml × 1 bottle at 2-8 ° C storage stop solution 3ml × 1 bottle 6ml × 1 bottle at 2-8 ° C Concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 2-8 ° C to store the sample. Processing and requirements: 1. Serum: room temperature blood is naturally coagulated for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage. 2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation. 4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use. 6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP). Operating steps Dilution and sample loading of standards: set up 10 wells of the standard on the enzyme-coated plate, add 100 μl of the standard in the first and second wells, and then add the standard in the first and second wells 50μl of diluent, mix well; then take 100μl from the first well and the second well and add them to the third and fourth wells respectively, and then add 50μl of standard diluent to the third and fourth wells respectively, mix well; Then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eight wells and add them to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentration is 90 pg / ml, 60 pg / ml, 30 pg / ml, 15 pg / ml, 7.5 pg / ml. Sample loading: set blank wells (blank control) No sample and enzyme reagents are added to the wells, the rest of the steps are the same), sample wells to be tested. Add 40μl of sample diluent to the sample wells to be tested on the enzyme coated plate, then add 10μl of the sample to be tested 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes. Solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) times and use it after dilution. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing solution Leave for 30 seconds, discard, repeat 5 times, pat dry. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well. Incubate: operate the same as 3. Wash: operate the same as 5. Color development: add each well first 50μl of developer A, then add 50μl of developer B, gently shake and mix, and develop at 37 ° C in the dark for 15 minutes. Termination: add 50μl of stopper solution to each well, Stop the reaction (blue to yellow at this time). Determination: The absorbance (OD value) of each well is measured in sequence with a blank air conditioner at zero, 450 nm wavelength. The measurement should be performed within 15 minutes after the addition of the stop solution. Note: kit Take out from the refrigerated environment and equilibrate at room temperature for 15-30 minutes before use. If the enzyme label coated plate is not used up after opening, the slats should be stored in a sealed bag. The concentrated washing liquid may crystallize out, when diluted It can be heated and dissolved in a water bath without affecting the results during washing. The sample addition should be used at each step, and the accuracy should be regularly calibrated to avoid test errors. The time of one sample injection is best controlled within 5 minutes. If there are a large number of specimens, it is recommended to use a volley gun to add samples. Please make a standard curve at the same time for each measurement, and it is best to do a double hole. If the content of the substance to be tested in the specimen is too high (the sample OD value is greater than the OD value of the first hole of the standard product hole ), Please dilute a certain multiple (n times) with the sample diluent before measuring, and finally multiply the total dilution factor (× n × 5) when calculating. The sealing film is only for one-time use to avoid cross-contamination. Keep objects away from light. Strictly Follow the instructions, the test results must be determined based on the reading of the microplate reader. All samples, washing liquids and various wastes should be treated as infectious agents. The components of different batches of this reagent should not be mixed. 10. If the instructions are in English If there is any difference, the English instruction manual shall prevail. Calculation: Take the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; Multiply by the dilution factor; or use the concentration and OD value of the standard to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor to get the actual concentration of the sample. ( (This picture is for reference only) Kit performance: 1. The linear regression of the sample and the expected concentration correlation coefficient R value is more than 0.92. 2. The batch and batch see should be less than 9% and 15% respectively. Storage conditions and expiration date: 1. Kit storage :; 2-8 ℃. 2. Validity: 6 months
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