Instruction Manual of Canine Endothelin-1 (ET-1) ELISA Kit

Instruction Manual of Canine Endothelin-1 (ET-1) ELISA Kit

This kit is for research use only

Intended application

Quantitative determination of endothelin-1 (ET-1) in dog serum, plasma or other related fluids by ELISA.

Experimental principle

This kit uses double antibody sandwich enzyme-labeled immunoassay to determine the level of ET-1 in the specimen. The microtiter plate is coated with purified antibody to make a solid phase antibody. ET-1 antigen, biotinylated anti-canine ET-1 antibody, and HRP-labeled avidin are added to the monoclonal antibody-coated microwell in sequence After thorough washing, color was developed with substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with ET-1 in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.

Kit composition and reagent preparation

1. ELISA plate: one piece (96 wells)

2. Standard product (lyophilized product): 2 bottles, each of which is diluted with sample diluent to 1ml before use. After being capped, it is allowed to stand for more than 10 minutes, and then repeatedly inverted / rubbed to help dissolve. Its concentration is 1000 pg / mL, after serial dilution, dilute to 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 56 pg / mL, 31 pg / mL, 15.6 pg / mL As the highest standard concentration directly, the sample dilution is directly used as the standard concentration of 0 pg / mL, prepared within 15 minutes before use.

For example, to prepare a 500 pg / mL standard: Take 0.5ml 1000 pg / mL of the above standard into an Eppendorf tube containing 0.5ml of sample diluent, mix well, and the rest of the concentration can be deduced by analogy.

3. Sample diluent: 1 × 20ml / bottle.

4. Test the diluent A: 1 × 10ml / bottle.

5. Test diluent B: 1 × 10ml / bottle.

6. Detection solution A: 1 × 120ul / bottle (1: 100), diluted with detection diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use.

7. Detection solution B: 1 × 120ul / bottle (1: 100) is diluted with detection diluent B1: 100 before use. The dilution method is the same as that of Test Solution A.

8. Substrate solution: 1 × 10ml / bottle.

9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.

10. Stop solution: 1 × 10ml / bottle (2N H2SO4).

Collection and preservation of specimens

1. Serum: Please leave the specimen at room temperature for 2 hours or overnight at 4 ° C and centrifuge at 1000 xg for 20 minutes. Take the supernatant for testing, or store the specimen at -20 ° C, but avoid repeated freezing and thawing.

2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000 xg at 2-8 ° C for 15 minutes within 30 minutes after the specimen is collected, or store the specimen at -20 ° C, but avoid repeated freezing and thawing.

Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.

Steps

Each reagent is equilibrated to room temperature before use.

1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. In addition to the blank wells, add 100 ul of standard solution or sample to be tested to the remaining wells, taking care not to have air bubbles, mix gently, cover the enzyme plate, and react at 37 ° C for 120 minutes.

2. Discard the liquid and spin dry without washing.

3. Add 100ul of detection solution A working solution to each well at 37 ℃ for 60 minutes. Wash the plate 3 times, 350ul / per well, spin dry.

4. Add 100ul of detection solution B working solution to each well at 37 ° C for 60 minutes, wash the plate 5 times and spin dry.

5. Add 90ul of substrate solution to each well in sequence, and develop the color for 30 minutes at 37 ° C in the dark (at this time, the first 3-4 wells of the standard product have a clear blue gradient, and the latter 3-4 wells have no obvious gradient).

6. Add 50ul of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). The optical density (OD value) of each well was measured sequentially with an enzyme-linked instrument at a wavelength of 450 nm.

Note:

1. One hole is left for each experiment as a blank zero-adjusting hole. No reagents are added to this hole, only the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.

2. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test.

Plate washing method Manual plate washing method: aspirate (do not touch the wall) or shake off the liquid in the enzyme label plate; place a few layers on the experimental table and suck 0.4ml into the hole, soak for 1-2 minutes, repeat this as needed Process several times. Water-paper, with the microtiter plate down and vigorously pat a few times; wash the recommended washing buffer at least automatically: If there is an automatic washing machine, it should be used after proficient use in the formal experiment process.

Specificity

This kit can detect recombinant or natural canine ET-1 at the same time and has no cross-reactivity with other related proteins.

Calculation

Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor to obtain the actual concentration of the sample.

Precautions

1. The washing process is very important, inadequate washing is easy to cause false positives.

2. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.

3. Please make a standard curve at the same time of each measurement, it is best to make a double hole.

4. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.

5. When preparing the standard and the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused.

6. Please keep the substrate away from light.

examination range:

15.6 pg / mL -1000 pg / mL

Explanation

1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).

2. Validity: 6 months

3. The concentrated washing liquid will have salt precipitation, and it can be heated and dissolved in the water bath when diluted.

4. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.

5. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.