Human Corticosterone ELISA Kit
principle
This experiment used double antibody sandwich ABC-ELISA. The anti-human Corticosterone mAb was coated on the microtiter plate, Corticosterone in the standard and sample was combined with the mAb, and biotinylated anti-human Corticosterone was added to form an immune complex attached to the plate, horseradish peroxidase The 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 Corticosterone concentration is directly proportional to the OD value, and the concentration of Corticosterone in the sample can be obtained by drawing a standard curve.
Kit composition ( 2-8 ° C preservation)
Coated Wells | 96 holes | Enzyme Conjugate | 12ml |
10× specimen dilution (Sample Buffer) | 12ml | 20×Wash Buffer | 50ml |
Standards: 12ng/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. 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) Save to avoid repeated freezing and thawing.
2. Standard solution preparation: Add 0.3ml of distilled water before use and mix well to form a solution of 40,000pg/ml. Set 8 tubes of standard tubes, and add 300 ul of standard dilution solution to each tube. Add 400 ul of the standard solution of 40000 pg/ml to the first tube, mix it, and then suck 300 ul with the sampler and transfer to the second tube. Repeat the dilution in this way, and remove 300 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)
Specimen activation method
1. Add 450 ul of the sample dilution to a 1.5 ml imported polypropylene tube and add 10 ul of serum or plasma samples.
2. Add 20ul 1 N HCI, cover tightly and mix up and down. Leave at 2-8 ° C for 60 ± 2 minutes.
3. Add 20ul 1 N NaOH, cover tightly, and mix up and down.
4. Use it immediately, or store at -20/-70 °C for 3 days. Multiply the dilution factor by 50 when calculating the result. ( Note: The level of different specimens of Corticosterone may vary greatly, please flexibly grasp the dilution according to the actual situation)
    5. Cell culture supernatant or tissue homogenate 10-fold dilution (410 ul of sample dilution + 50 ul sample + 20 ul of 1 N HCI + 20 ul of 1 N NaOH).
Test procedure
1. Loading: Add 100 ul of standard or test sample (activated) to 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 per well, set 37 The reaction was carried out in the dark at °C for 15 minutes.
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. Take the standard products 20000, 10000, 5000, 2500, 1250, 625, 312, 0 pg/ml as the abscissa and OD as the ordinate. Draw on the coordinate paper and draw the standard curve.
3. Find the corresponding Corticosterone content on the graph based on the OD value of the sample, and multiply by the dilution factor.
Kit performance
1. Sensitivity: The minimum Corticosterone detection concentration is less than 150pg/ml.
2. Specificity: Recombinant or natural human Corticosterone can be detected simultaneously. Does not cross-react with other human cytokines.
3. Repeatability: The coefficient of variation in the plate and plate is less than 9.6%.
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 4 o C refrigerator.
5. This kit is for scientific research only and cannot be used for clinical diagnosis!
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