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Human Endorphin-b Quantitative ELISA Kit Instruction Manual
Instructions for use of human Endorphin- b quantitative ELISA kit
principle
This experiment used a double antibody sandwich ELISA method. The anti-human Endorphin-b monoclonal antibody was coated on the microtiter plate, the Endorphin-b in the standard and the sample was combined with the monoclonal antibody, and the enzyme-labeled antibody was added to form an immune complex, which was attached to the plate, and the enzyme substrate TMB was added. Blue appears, the stop solution sulfuric acid is added, the color turns yellow, and the OD value is measured at 450 nm. The Endorphin-b concentration is directly proportional to the OD value, and the Endorphin-b concentration in the sample can be obtained by drawing a standard curve.
Kit composition ( 2-8 ° C preservation)
Coated Wells
96 holes
Enzyme Conjugate
6ml
10× specimen dilution (Sample Buffer)
12ml
20×Wash Buffer
50ml
Standards: 6 bottles
set
Substrate working fluid (TMB Solution)
12ml
Cloth paper
One
Stop Solution
12ml
Prepare reagents and collect blood samples
1. Collection of specimens: serum, plasma (EDTA, citrate, heparin anticoagulation), cell culture supernatant, tissue homogenate, etc., as early as possible, stored 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. Dissolve the standard product in 0.5ml of distilled water for the first time, mix it for half an hour, use it, and store it at -20 o C after use. The concentrations after dissolution were 35, 20, 8, 4, 2, and 0 ng/ml, respectively.
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. Establish a standard curve: set 6 holes of standard holes, and add 50ul of standard to each hole.
2. Adding sample: 50 ul of the sample to be tested is added to each hole of the sample to be tested.
3. Add 50 ul of enzyme-labeled antibody working solution per well. The reaction plate was reacted at 37 ° C for 120 minutes.
4. Wash the plate: Wash the plate thoroughly with the washing solution 4-6 times, and dry it on the filter paper.
5. Add 100 ul of substrate working solution per well, set 37 The reaction was carried out in the dark at °C for 15 minutes.
6. Add 100 ul of stop solution to each well and mix.
7. 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 35, 20, 8, 4, 2, 0ng/ml as the abscissa and OD as the ordinate. Draw on the coordinate paper and draw the standard curve.
3. Find the corresponding Endorphin-b content on the graph based on the OD value of the sample.
Kit performance
1. Sensitivity: The minimum Endorphin-b detection concentration is less than 1 ng/ml.
2. Specificity: Recombinant or natural human Endorphin-b can be detected simultaneously. Does not cross-react with other human cytokines.
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 4 o C refrigerator.
5. This kit is for scientific research only and cannot be used for clinical diagnosis!