Rat Endorphin-b quantitative EIA kit instructions

Rat Endorphin-b quantitative EIA kit instructions

principle

This experiment uses the double antibody sandwich ELISA method. Coated with anti-rat Endorphin-b monoclonal antibody on the enzyme-labeled plate, Endorphin-b in the standard and the sample is combined with the monoclonal antibody, enzyme-labeled antibody is added to form an immune complex connected to the plate, enzyme substrate TMB is added , Blue appears, add stop solution sulfuric acid, the color turns yellow, the OD value is measured at 450nm, Endorphin-b concentration is proportional to the OD value, the Endorphin-b concentration in the specimen can be obtained by drawing a standard curve.

Kit composition (stored at 2-8 ° C)

Coated Wells

96 wells

Enzyme Conjugate

6ml

Graph paper

One

20 × concentrated washing solution (Wash Buffer)

50ml

Standards (Standards): 6 bottles

set

Substrate working solution (TMB Solution)

12ml

Cover paper

One

Stop Solution (Stop Solution)

12ml

Prepare reagents and collect blood samples

1. Collect specimens: serum, plasma (EDTA, citrate, heparin anticoagulation), cell culture supernatant, tissue homogenate, etc. as soon as possible, stored at 2-8 ℃ for 48 hours; longer time must be frozen (-20 ℃ Or -70 ℃), avoid repeated freezing and thawing.

2. When the standard is used for the first time, dissolve it in 0.5ml of distilled water, let it stand for half an hour and mix well before use. Store it at -20oC after use. The concentration after dissolution was 35, 20, 8, 4, 2, 0ng / ml.

3. Washing solution: 1:20 dilution with redistilled water (example: 1ml concentrated washing solution is added to 19ml of redistilled water)

Testing procedures

1. Establish a standard curve: set 6 standard holes, and add 50ul of standard product to each hole.

2. Add sample: add 50ul of the sample to be tested to each hole of the product to be tested.

3. Add 50ul of enzyme-labeled antibody working solution to each well. The reaction plate was placed at 37 ° C for 120 minutes.

4. Plate washing: Wash the reaction plate 4-6 times with washing solution and print on the filter paper.

5. Add 100ul of substrate working solution to each well, and let it react in the dark at 37 ℃ for 15 minutes.

6. Add 100ul of stop solution to each well and mix well.

7. Measure absorbance at 450nm with a microplate reader within 30 minutes.

Result calculation and judgment

1. All OD values ​​should be calculated after subtracting the blank value.

2. Take the standard products 35, 20, 8, 4, 2, 0ng / ml as the abscissa and the OD value as the ordinate, draw a graph on the graph paper and draw a standard curve.

3. According to the OD value of the sample, find the corresponding Endorphin-b content on the graph.

Kit performance

1. Sensitivity: The minimum detection concentration of Endorphin-b is less than 1ng / ml.

2. Specificity: simultaneous detection of recombinant or natural rat Endorphin-b. Does not cross-react with other cytokines in rats.

3. Repeatability: The coefficient of variation within the board and the board is less than 10%.

Precautions

1. The above standard holes and samples to be tested are recommended to be re-holes, and the standard curve should be made at the same time for each measurement.

2. The washing process is critical. Insufficient washing will lead to an increase in accuracy error and OD value.

3. After opening the slats, 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, not for clinical diagnosis!