Bovine epidermal growth factor (EGF) ELISA kit

Bovine epidermal growth factor (EGF) ELISA kit

Storage conditions and expiration date

1. Storage of reagent kit: 2-8 ℃.

2. Validity period: 6 months

Experimental Principle

This kit uses the double antibody sandwich method to determine the level of bovine epidermal growth factor (EGF) in specimens. Coat a microplate with purified bovine epidermal growth factor (EGF) antibody to prepare a solid-phase antibody. Add EGF to the micropores coated with the monoclonal antibody in sequence, and then bind with HRP labeled EGF antibody to form an antibody antigen enzyme labeled antibody complex. After thorough washing, add substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and to the final yellow under the action of acid. The depth of color is positively correlated with epidermal growth factor (EGF) in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the concentration of bovine epidermal growth factor (EGF) in the sample using a standard curve.

Kit components

130x concentrated washing solution20ml x 1 bottle7Stop Solution6ml x 1 bottle

2Enzyme-labeled reagent6ml x 1 bottle8Standard product (1200pg/ml)0.5ml x 1 bottle

3Enzyme labeled coated plate12 holes x 8 strips9Standard diluent1.5ml x 1 bottle

4Sample diluent6ml x 1 bottle10instruction manual1 copy

5Color reagent A liquid6ml x 1 bottle11Sealing film2 sheets

6Color developer B solution6ml x 1/bottle12sealed bag1 piece

operating steps

1.Dilution of standard samples: This kit provides one original standard sample, and users can dilute it in a small test tube according to the following chart.

600pg/mlStandard 5Add 150 μ l of original standard to 150 μ l of standard dilution solution

300pg/mlStandard No. 4Add 150 μ l of standard 5 to a dilution of 150 μ l of standard solution

150pg/mlStandard product No. 3Add 150 μ l of standard substance No. 4 to a dilution of 150 μ l of standard substance

75pg/mlStandard product No.2Add 150 μ l of standard substance No. 3 to a dilution of 150 μ l of standard substance

37.5pg/mlStandard No.1Add 150 μ l of standard substance No.2 to a dilution of 150 μ l of standard substance

2.Sample addition: Set up blank wells (blank control wells without sample or enzyme-linked immunosorbent assay, all other steps are the same), standard wells, and test sample wells. Accurately add 50 μ l of the standard sample on the enzyme-linked immunosorbent assay (ELISA) coated plate, first add 40 μ l of sample diluent to the well of the test sample, and then add 10 μ l of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay plate well, avoiding touching the well wall as much as possible, and gently shake and mix well.

3.Incubation: Cover the plate with a sealing film and incubate at 37 ℃ for 30 minutes.

4.Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use

5.Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each hole with detergent, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.

6.Enzyme addition: Add 50 μ l of enzyme labeled reagent to each well, except for blank wells.

7.Incubation: Follow the same procedure as in 3.

8.Washing: The operation is the same as 5.

9.Color development: Add 50 μ l of color developing agent A50 to each well, then add 50 μ l of color developing agent B50, gently shake and mix, and develop color at 37 ℃ in the dark for 10 minutes

10.Termination: Add 50 μ l of termination solution to each well to terminate the reaction (at this point, the blue color turns yellow).

11.Measurement: Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the termination solution.

calculate

Draw a standard curve on a coordinate paper with the concentration of the standard substance as the horizontal axis and the OD value as the vertical axis, and determine the corresponding concentration based on the OD value of the sample from the standard curve; Multiply by the dilution factor again; Alternatively, the linear regression equation of the standard curve can be calculated using the concentration and OD value of the standard substance. By substituting the OD value of the sample into the equation, the sample concentration can be calculated, and then multiplied by the dilution factor to obtain the actual concentration of the sample.

Precautions

1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag.

2. There may be crystal precipitation in concentrated washing solution. When diluting, it can be dissolved by heating in a water bath, and washing does not affect the results.

3. Sample dispensers should be used for each step of sample addition, and their accuracy should be regularly checked to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a sampling gun for sample addition.

4. Please make a standard curve at the same time as each measurement, preferably with a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute the sample diluent by a certain multiple (n times) before measuring. When calculating, please multiply by the total dilution multiple (x n x 5) at the end.

5. The sealing film is only for one-time use to avoid cross contamination.

6. Please store the substrate away from light.

7. Strictly follow the instructions for operation, and the judgment of the test results must be based on the reading of the enzyme-linked immunosorbent assay reader

8. All samples, detergents, and various waste materials should be treated as infectious agents.

9．Bovine epidermal growth factor (EGF) ELISA kitDifferent batch numbers of components must not be mixed.