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Rat Glial Cellular Acidic Protein (GFAP) ELISA Kit Instructions for Use
 ( used in serum, plasma, cell culture supernatants and other biological fluids )
principle
This experiment used double antibody sandwich ABC-ELISA. The anti-rat GFAP monoclonal antibody was coated on the plate, the GFAP in the standard and the sample was combined with the monoclonal antibody, and biotinylated anti-rat GFAP 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 GFAP concentration is directly proportional to the OD value, and the GFAP in the sample can be obtained by drawing a standard curve. concentration.
Kit composition ( 2 Store at -8 ° C )
Coated Wells | 96 holes | Enzyme Conjugate | 12ml |
10× specimen dilution (Sample Buffer) | 12ml | 20×Wash Buffer | 50ml |
Standards: 100ng/ml | 1 bottle | Substrate working fluid (TMB Solution) | 12ml |
Primary antibody working solution (Biotinylated Antibody) | 12ml | Stop Solution | 12ml |
Prepare reagents and collect blood samples
1. Collect specimens: serum, plasma (EDTA), cell culture supernatant, tissue homogenate, etc., as soon as possible, store at 2-8 ° C for 48 hours; longer time must be frozen (-20 ° C or -70 ° C) to avoid Repeated freezing and thawing.
2. Standard product preparation: Set 7 tubes of standard tube, add 300ul of sample dilution solution to each tube. Add 100 ng of standard solution of 100 ng/ml to the first tube, mix and aspirate 300 ul with the sampler, and transfer to the second tube. Repeat the dilution in this way, and remove 300 ul from the sixth tube and discard it. The seventh tube is a blank control. Plus a total of eight tubes in the original tube.
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 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 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 GFAP content on the graph based on the OD value of the sample.
Kit performance
1. Sensitivity: The minimum GFAP detection concentration is less than 1 ng/ml.
2. Specificity: Recombinant or natural rat GFAP 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!
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