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Rat Endostatin ELISA Kit Instructions for Use
 ( used in other biological fluids such as serum, plasma, cell culture supernatant, and saliva )
principle
This experiment used double antibody sandwich ABC-ELISA. The anti-rat Endostatin monoclonal antibody was coated on the plate, the endostatin in the standard and the sample was combined with the monoclonal antibody, and biotinylated anti-rat Endostatin was added to form an immune complex attached to the plate, horseradish peroxidation. The enzyme-labeled Streptavidin is combined with biotin, and the substrate working solution is blue. Finally, the stop solution sulfuric acid is added, and the OD value is measured at 450 nm. The concentration of Endostatin is directly proportional to the OD value. The endostatin can be obtained by drawing a standard curve. concentration.
Kit composition ( 2-8 ° C preservation)
Coated Wells
96 holes
Enzyme Conjugate
12ml
10× specimen dilution (Sample Buffer)
12ml
20×Wash Buffer
50ml
Standards: 800ng / bottle
2 bottles
Substrate working fluid (TMB Solution)
12ml
Primary antibody working solution (Biotinylated Antibody)
12ml
Stop Solution
12ml
Prepare reagents and collect blood samples
1. Collection of specimens: serum, plasma (EDTA), cell culture supernatant, tissue homogenate, saliva, etc., as soon as possible, stored at 2-8 ° C for 48 hours; longer time must be frozen (-20 ° C or -70 ° C) to save To avoid repeated freezing and thawing. Before the normal specimen is measured, dilute with the specimen dilution at least 1 : 5 (take 20ul, add the standard dilution 80ul, dilute 5 times). Saliva can be detected directly without dilution.
2. Standard solution preparation: Add 2 ml of distilled water before use and mix well to prepare a 400 ng/ml solution. Set the standard tube 8 tube, the first tube plus the standard dilution 900ul, the second to the eighth tube to add the sample dilution 500ul. Add 100 ul of 400 ng/ml standard solution to the first tube, mix and aspirate 500 ul with a pipette, and transfer to the second tube. Repeat the dilution as described above, and remove 500 ul from the seventh tube and discard it. The eighth tube is a blank control.
3. The 10× specimen dilution was diluted 1:10 with distilled water (example: 1 ml concentrated dilution + 9 ml distilled water).
4. Washing solution: diluted 1:20 with distilled water (example: 1 ml concentrated washing solution added to 19 ml of distilled water)
Test procedure
1. Loading: Add 100 ul of standard or sample to be tested in each well. Mix the reaction plate thoroughly and let it stand at 37 °C for 120 minutes.
2. Wash the plate: Wash the plate thoroughly with washing solution 4-6 times, and dry it on the filter paper.
3. Add 100 ul of the first antibody working solution to each well. The reaction plate was thoroughly mixed and placed at 37 ° C for 60 minutes.
4. Wash the board: the same as before.
5. Add 100 ul of enzyme-labeled antibody working solution per well. The reaction plate was placed at 37 ° C for 30 minutes.
6. Wash the board: same as before.
7. Add 100 ul of substrate working solution to each well and let it react at 37 ° C for 15 minutes in the dark.
8. Add 100 ul of stop solution to each well and mix.
9. Measure the absorbance at 450 nm using a microplate reader within 30 minutes.
Result calculation and judgment
1. All OD values ​​should be subtracted from the blank value before calculation.
2. Using standard products 40, 20, 10, 5, 2.5, 1.25, 0.625, 0 ng/ml as the abscissa and OD as the ordinate, plot on the coordinate paper and draw the standard curve.
3. Find the corresponding Endostatin content on the graph based on the OD value of the sample, and multiply by the dilution factor. .
Kit performance
1. Sensitivity: The minimum Endostatin assay concentration is less than 0.3 ng/ml.
2. Specificity: Recombinant or natural rat Endostatin can be detected simultaneously. Does not cross-react with other cytokines in rats.
3. Repeatability: The coefficient of variation in both the plate and the plate is less than 10%.
Precautions
1. It is recommended to make double holes for the above standard holes and samples to be tested. The standard curve should be made at the same time for each measurement.
2. The washing process is critical. Insufficient washing will result in an accuracy error and an erroneous rise in the OD value.
3. After the slats are opened, the remaining slats should be sealed again to keep the slats dry .
4. This kit should be stored in a 4oC refrigerator.
5. This kit is for scientific research only and cannot be used for clinical diagnosis!