Multi-effect growth factor (PTN) elisa technical specification kit: kit composition 48-well configuration 96-well configuration storage instructions 1 part 1 part sealing film 2 pieces (48) 2 pieces (96) sealed bag 1 one enzyme label Coated plate 1×48 1×96 2-8°C Preservation standard: 1350pg/mL 0.5ml×1 bottle 0.5ml×1 bottle 2-8°C Preservation standard dilution 1.5ml×1 bottle 1.5ml×1 bottle 2 -8 ° C preservation enzyme standard reagent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation sample dilution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation developer A liquid 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation developer B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation concentrated washing solution (20ml ×20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ° C preservation sample processing and requirements: 1. Serum: room temperature blood solidification for 10-20 minutes, centrifugation for about 20 minutes (2000-3000 rev / min ). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again. 2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. 3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to. 4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the intracellular components were detected, the cell suspension was diluted with PBS (pH 7.2-7.4) to a cell concentration of about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes and 2 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use. 6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the 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 horseradish peroxidase (HRP) activity. Operation steps 1. Dilution and loading of standard products: 10 holes of standard wells are placed on the enzyme labeling plate, 100 μl of standard is added to the first and second holes, and then added in the first and second holes. Mix 50 μl of the standard dilution and mix well; then add 100 μl from each of the first and second wells to the third and fourth wells, and then add 50 μl of the standard dilution to the third and fourth wells. Then, 50 μl of each of the third and fourth wells is discarded, and 50 μl of each is added to the fifth and sixth wells, respectively, and 50 μl of the standard dilution is added to the fifth and sixth wells, respectively. Evenly; after mixing, take 50μl from each of the fifth and sixth holes and add them to the seventh and eighth holes respectively. Then add 50μl of the standard dilution in the seventh and eighth holes, and mix them from the seventh. 50 μl of the eighth well was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50 μl after dilution, and the concentrations were 900 pg/mL, 600 pg/mL, 300 pg/mL, 150 pg/mL, and 75 pg/mL, respectively. 2. Adding samples: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix. 3. Incubation: After sealing with a sealing film, incubate at 37 ° C for 30 minutes. 4. Dosing: Dilute 30 (20 times of 48T) concentrated washing solution with distilled water 30 (20 times of 48T) and set aside. 5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry. 6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells. 7. Incubation: operation is the same as 3. 8. Washing: operation is the same as 5. 9. Color development: add 50 μl of color developer A, add 50 μl of color developer B, gently shake and mix, and avoid light at 37 °C. 15 min.10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow). 11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
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