Manual of human 6-ketoprostaglandin (6-K-PG) Elisa kit
This kit is for research use only.
Detection range: 96T
10ng / L – 320ng / L
purpose of usage:
This kit is used to determine the content of 6-ketoprostaglandin (6-K-PG) in human serum, plasma and related liquid samples.
Experimental principle
Human 6-ketoprostaglandin (6-K-PG) Elisa kit instructions This kit uses the double antibody sandwich method to determine the level of human 6-ketoprostaglandin (6-K-PG) in the specimen. With purified human 6-
The ketone prostaglandin (6-K-PG) antibody is coated on the microwell plate to make a solid phase antibody, and 6-ketone is added to the monoclonal antibody-coated microwells in sequence
Prostaglandin (6-K-PG), then combined with HRP-labeled 6-ketoprostaglandin (6-K-PG) antibody to form antibody-antigen-
The enzyme-labeled antibody complex was washed thoroughly and added with substrate TMB for color development. TMB turns into blue under the catalysis of HRP enzyme,
And converted into the final yellow under the action of acid. The shade of the color is normal to the 6-ketoprostaglandin (6-K-PG) in the sample
turn off. Measure the absorbance (OD value) at 450nm with a microplate reader, and calculate the forefront of human 6-keto in the sample through the standard curve
Adenosine (6-K-PG) concentration.
Kit composition
1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 Enzyme label reagent 6ml × 1 bottle 8 standard (640 ng / L) 0.5ml × 1 bottle
3 Enzyme label coated plate 12 wells × 8 strips 9 standard dilutions 1.5ml × 1 bottle
4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy
5 Developer A solution 6ml × 1 bottle 11 2 sealing film
6 Developer B liquid 6ml × 1 / bottle 12 1 sealed bag
Specimen requirements
1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If not
Perform the test immediately. The specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided
2. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard products: this kit provides one original standard product, the user can dilute in a small test tube according to the following chart
release.
320 ng / L No. 5 standard 150μl original standard added 150μl standard dilution
160ng / L No. 4 standard 150μl No. 5 standard added 150μl standard dilution
80ng / L No. 3 standard 150μl No. 4 standard added 150μl standard dilution
40ng / L No. 2 standard 150μl No. 3 standard added 150μl standard diluent
20ng / L No. 1 standard 150μl No. 2 standard added 150μl standard diluent
2. Add sample: set up blank wells separately (the blank control wells do not add samples and enzyme label reagents, the rest of the steps are the same), standard wells,
Sample hole to be tested. Add 50μl of the standard on the enzyme-coated plate accurately, and add 40μl of sample diluent to the sample well.
Then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microtiter plate
Do not touch the wall of the hole, shake gently to mix.
3. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, so
Repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. Determination should be terminated
Within 15 minutes after the solution.
Summary of operating procedures:
Calculation
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, according to the sample
The corresponding concentration of OD value is found by the standard curve; multiplied by the dilution factor; or the standard concentration and OD value are used to calculate
The linear regression equation of the quasi-curve, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor,
This is the actual concentration of the sample.
Precautions
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment.
After use, the slats should be stored in sealed bags.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. One sample loading time is best
Control within 5 minutes, if the number of specimens is large, it is recommended to use a volley gun to add samples.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the specimen is too high (sample OD value
Is greater than the OD value of the first well of the standard well), please dilute it with a certain multiple (n times) of the sample diluent before measuring.
When calculating, please multiply the total dilution factor (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
Storage conditions and validity period
1. Kit storage :; 2-8 ℃.
2. Validity: 6 months Instructions for human 6-keto prostaglandin (6-K-PG) Elisa kit
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