Rat soluble CD14 molecule (sCD14) ELISA kit

Rat soluble CD14 molecule (sCD14) ELISA kit

(Used in serum, plasma, cell culture supernatant and biological fluid)

principle

This experiment uses the double antibody sandwich ABC-ELISA method. The anti-rat sCD14 monoclonal antibody was coated on the enzyme plate. The sCD14 in the standard and the sample was combined with the monoclonal antibody. Biotinylated anti-rat sCD14 was added to form an immune complex and attached to the plate. Horseradish peroxidation The enzyme-labeled Streptavidin is combined with biotin, the substrate working solution is added, and the final solution is added with sulfuric acid. The OD value is measured at 450nm. The sCD14 concentration is proportional to the OD value. The sCD14 in the specimen can be obtained by drawing a standard curve concentration.

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): 200ng / 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, 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. Before the determination of all specimens, use the specimen dilution to make a 1:10 dilution (take 20ul, add specimen dilution 180ul, and dilute 10 times).

2. Standard solution preparation: add 0.5ml of distilled water to mix well before use to make a 400ng / ml solution. Set 8 standard tubes, and add 300ul of sample dilution to each tube. Add 300 ng of standard solution of 400 ng / ml to the first tube, mix well, then aspirate 300 ul with a pipette and move to the second tube. Repeat the double dilution in this way, draw 300ul 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)

Testing procedures

1. Add sample: add 100ul of standard or sample to be tested to each well, mix the reaction plate thoroughly and place 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. Take the standard products 200, 100, 50, 25, 12.5, 6.25, 3.12, 0 ng / ml as the abscissa, and the OD value as the ordinate.

3. According to the OD value of the sample, find the corresponding sCD14 content on the graph, and then multiply the dilution factor by 10.

Kit performance

1. Sensitivity: The minimum detection concentration of sCD14 is less than 1.5ng / ml.

2. Specificity: simultaneous detection of recombinant or natural rat sCD14. Does not cross-react with other cytokines in rats.

3. Repeatability: The coefficient of variation within the board and the board is less than 10.6%.

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!