Human vascular endothelial growth factor (VEGF) quantitative detection kit (ELISA) instruction manual [kit name] human vascular endothelial growth factor (VEGF) quantitative detection kit (ELISA) [kit use] quantitative detection of human serum, plasma and related The content of vascular endothelial growth factor (VEGF) in a liquid sample. [Detection principle] The kit adopts double antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). The standard sample and the sample to be tested are added to a pre-coated human vascular endothelial growth factor (VEGF) monoclonal antibody transparent enzyme-labeled plate, and after culturing for a sufficient time, the unbound component is washed and removed, and the enzyme-labeled working solution is added. After incubation for a sufficient period of time, the unbound components are removed by washing. Substrate A and B were sequentially added, and the substrate (TMB) was converted into a blue product under the catalysis of horseradish peroxidase (HRP), which turned yellow under the action of acid, and the color depth and human vascular endothelial growth in the sample. The factor (VEGF) concentration was positively correlated, and the OD value was measured at a wavelength of 450 nm. The human vascular endothelial growth factor (VEGF) content in the sample was calculated based on the OD value of the standard and the sample. [kit composition] 1 enzyme label coating plate 12 holes × 8 strips 7 color reagent A liquid 6mL2 standard product: 800pg / mL 0.6mL 8 color reagent B liquid 6mL3 20 times concentrated washing liquid 25mL 9 stop liquid 6mL4 standard Diluent 6mL 10 Instructions 1 part 5 Sample dilution 6mL 11 Sealing membrane 2 sheets 6 Enzyme standard reagent 6mL 12 Sealing bag 1 Remarks: Standards are diluted with standard dilutions to: 800, 400, 200, 100, 50 , 25pg/mL [required and not provided reagents and equipment] 1, 37 ° C incubator 2, standard size microplate reader 3, precision pipette and disposable tip 4, distilled water 5, disposable test tube 6, absorbent paper [Operation Procedure] 1. Preparation: Take out the kit from the refrigerator and re-balance at room temperature for 30 minutes. 2. Dosing: Dilute the 20 times concentrated washing solution into the original washing solution with distilled water. 3. Add standard and sample to be tested: Take a sufficient number of enzyme label coated plates, fix them on the frame, set standard hole, sample hole and blank control hole respectively, record the position of each hole, in the standard hole Add 50 μL of the standard; add 10 μL of the sample to be tested first, and add 40 μL of the sample dilution (ie, the sample is diluted 5 times); the blank control well is not added. 4. Incubation: Incubate for 30 min in a 37 °C water bath or incubator. 5, wash the board: discard the liquid, pat dry on the absorbent paper, fill each hole with the washing liquid, let stand for 1min, remove the washing liquid, pat dry on the absorbent paper, and repeat the washing 4 times (can also be used by the washing machine) Instructions for washing the board). 6. Add the enzyme standard working solution: add 50 μL of the enzyme standard working solution to each well, and the blank control well is not added. 7. Incubation: Repeat 4 operations. 8. Wash the plate: Repeat the operation of 5. 9. Color development: 50 μL of the developer A solution was added to each well, and then 50 μL of the developer B solution was added, and the color was developed at 37 ° C for 15 minutes. 10. Termination: The enzyme plate was taken out, and 50 μL of the stop solution was added to each well to terminate the reaction (the color was changed from blue to yellow). 11. Measurement: Zeroing was performed with a blank hole, and the absorbance (OD value) of each well was measured with a wavelength of 450 nm within 15 minutes after termination. 12. Calculation: Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. You can also use various application software to Calculation. The final concentration is the actual measured concentration multiplied by the dilution factor. [Sample Requirements] 1. The sample should not contain sodium azide (NaN3) because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP). 2. The specimens should be extracted as soon as possible after the 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. 3, the sample should be fully centrifuged, no hemolysis and particles. [Notes] 1. The experiment is carried out in strict accordance with the operation of the manual. The results of the experiment must be determined by the reading of the microplate reader. 2. If the enzyme label is not used up after being opened, it should be immediately placed in a sealed bag and desiccant. 3. It is recommended that all standards, samples and blanks be double-tested and averaged to reduce experimental error. 4. Keep in mind that the sample has been diluted 5 times and the calculated result is multiplied by 5 to determine the actual concentration of the sample. 5. The quantitative range of this kit is 25-800pg/mL. If it exceeds this range, it is calculated by the extension of the standard curve. It is not used as an accurate quantitative result. Please dilute with the standard dilution solution to determine the accurate result (25-800pg/mL range) Internal), multiplied by the total dilution factor is the final concentration of the sample. 6. If the color is too light, the substrate incubation time can be extended appropriately. 7. In order to avoid cross-contamination, the standard product, sample and blank control should be replaced once for each additional one; the common components such as enzyme standard working solution, sample diluent and substrate should be cantilevered and should not touch micropores. ; Do not reuse the sealing film. 8. The kit is used during the shelf life. The reagents of different batches must not be mixed. 9. Substrate B is sensitive to light and avoids prolonged exposure to light. [Summary of the operation procedure] Prepare the reagents, samples and standards, add the prepared samples and standards, wash the plate 4 times at 37 ° C for 30 minutes, add the enzyme standard reagent, wash the plate 4 times at 37 ° C for 30 minutes, add the coloring solution A, B, 37 ° C color development 15 minutes to add stop solution within 15 minutes to read OD value calculation [detection range] 25-800pg / mL [Specifications] 96 people / box [storage] 2-8 ° C, protected from light and moisture . [Validity Period] 6 months
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