Rat C-Fos ELISA Kit

Rat C-Fos ELISA Kit

principle

This experiment uses the double antibody sandwich ABC-ELISA method. Coated with anti-rat C-Fos monoclonal antibody on the enzyme plate, the C-Fos in the standard and the sample are combined with the monoclonal antibody, and the biotinylated anti-rat C-Fos is added to form an immune complex and connected to the plate On the top, the horseradish peroxidase-labeled Streptavidin is combined with biotin, the substrate working solution is added, and the final solution is blue, and the sulfuric acid is added to the stop solution. The OD value is measured at 450nm. The C-Fos concentration is proportional to the OD value and can be passed Draw a standard curve to find the C-Fos concentration in the specimen.

Kit composition (stored at 2-8 ° C)

Coated Wells

96 wells

Enzyme Conjugate

12ml

10 × Sample Buffer

12ml

20 × concentrated washing solution (Wash Buffer)

50ml

Standards (Standards): 1.6ng / bottle

2 bottles

Substrate working solution (TMB Solution)

12ml

The first antibody working solution (Biotinylated Antibody)

12ml

Stop Solution (Stop Solution)

12ml

Prepare reagents and collect blood samples

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

2. Preparation of standard solution: add 0.4ml of distilled water to mix well before use to make a solution of 4000pg / ml. Set 8 standard tubes, and add 200ul of sample diluent to each tube. Add 200 ul of the standard solution of 4000 pg / ml to the first tube and mix it. Then aspirate the 200 ul with the sampler and move to the second tube. Repeat the double dilution in this way, draw 200ul from the seventh tube and discard. The eighth tube is a blank control.

3. The 10 × specimen dilution is diluted 1:10 with distilled water (example: 1ml concentrated dilution + 9ml distilled water).

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

Specimen activation method

1. Add 450ul of specimen dilution to a 1.5ml imported polypropylene tube and add 10ul of serum or plasma specimen.

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. Ready to use, or store at -20 / -70 ℃ for 3 days. Multiply the dilution factor by 50 when calculating the result. (Note: The C-Fos level of different specimens may be quite different, please flexibly grasp the dilution according to the actual situation)

5. Cell culture supernatant or tissue homogenate is diluted 10-fold (410ul of specimen dilution + 50ul of sample + 20ul of 1N HCI + 20ul of 1N NaOH).

Testing procedures

1. Add sample: add 100ul of standard or sample to be tested (activated) to each well, mix the reaction plate thoroughly and set it at 37 ℃ for 120 minutes.

2. Wash the plate: wash the reaction plate 4-6 times with the washing solution, and print it on the filter paper.

3. Add 100ul of the first antibody working solution to each well. The reaction plate was mixed thoroughly and then placed at 37 ° C for 60 minutes.

4. Wash board: same as above.

5. Add 100ul of enzyme-labeled antibody working solution to each well. Place the reaction plate at 37 ° C for 30 minutes.

6. Wash board: same as above.

7. Add 100ul of substrate working solution to each well, and place in a dark place at 37 ℃ for 15 minutes.

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

9. 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. With the standard products 2000, 1000, 500, 250, 125, 62.5, 31.2, 0 pg / ml as the abscissa, and the OD value as the ordinate, draw a graph on the graph paper and draw a standard curve.

3. Find the corresponding C-Fos content on the graph according to the OD value of the sample, and then multiply it by the dilution factor.

Kit performance

1. Sensitivity: The smallest C-Fos detection concentration is less than 15pg / ml.

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

3. Repeatability: The coefficient of variation within the board and the board is less than 8.9%.

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!