Rat blood glucose enzyme-linked immunoassay (ELISA)

Kit instruction manual

This kit is for research use only.

Drug Name:

Generic name: Rat blood glucose (blood glucose) ELISA kit

purpose of usage:

This kit is used to determine the blood glucose content of rat serum, plasma and related liquid samples.

Experimental principle

This kit uses an indirect method to determine the blood glucose level of rats in the specimen. A microplate was coated with purified rat blood glucose antibody to make a solid-phase antibody. A blood glucose standard of known concentration and a blood glucose test sample of unknown concentration were sequentially added to the microwells coated with monoclonal antibody. The biotin-labeled anti-IgG antibody is combined with streptavidin-HRP to form an immune complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the blood glucose in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the blood glucose concentration of rats in the sample was calculated by a standard curve.

Kit composition

1

20 times concentrated washing liquid

50ml × 1 bottle

9

Standard product S1 (480μmol / L)

0.5ml × 1 bottle

2

Streptavidin-HRP

6ml × 1 bottle

Standard product S2 (320μmol / L)

0.5ml × 1 bottle

3

Enzyme coated plate

12 holes × 8

Standard product S3 (160μmol / L)

0.5ml × 1 bottle

4

Biotin-labeled anti-IgG antibody

6ml × 1 bottle

Standard product S4 (80μmol / L)

0.5ml × 1 bottle

5

Developer A liquid

6ml × 1 bottle

Standard product S5 (40μmol / L)

0.5ml × 1 bottle

6

Developer B liquid

6ml × 1 / bottle

10

sealed bag

1

7

Stop solution

6ml × 1 bottle

11

Sealing film

3 sheets

8

Sample diluent

6ml × 1 bottle

12

Instructions

1 serving

Specimen requirements

1. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

2. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided

Steps

1. Determine the number of slats required based on the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used as much as possible.

2. Adding samples: set up blank wells (the blank control wells do not add samples, the rest of the operations are the same), standard wells, and sample wells to be tested. Then add 50μl of the standard to the standard well, add 10μl of the sample to be tested to the sample reaction well and then add 40μl of the sample diluent (the final dilution of the sample is 5 times), cover with the sealing film, shake gently and mix well, 37 ℃ Incubate for 45 minutes.

3. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 times and reserve

4. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds and then discard, repeat this 4 times, pat dry.

5. Add biotin-labeled anti-IgG antibody: Add 50 μl of biotin-labeled anti-IgG antibody to each well. Incubate at 37 ° C for 30 minutes

6. Washing: The operation is the same as 4.

7. Add streptavidin-HRP: add 50μl streptavidin-HRP to each well, gently shake and mix, and incubate at 37 ° C for 30 minutes.

8. Washing: The operation is the same as 4.

9. Color development: Add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.

10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).

11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.

Calculation

Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; Calculate the linear regression equation of the standard curve with the OD value, put the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample.

Precautions

1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.

2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.

3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. 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.

4. 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 by a certain multiple (n times) and then determine it. When calculating, please multiply the dilution factor (× 5 × n).

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

6. The components of different batches of this reagent shall not be mixed. Store developer B in dark place.

7. Strictly follow the instructions, and the test results must be determined by the microplate reader.

8. All samples, washing liquids and various wastes should be treated as infectious agents.

9. If there is any difference with the English manual, the English manual shall prevail.

Linear range:

20μmol / L -500 μmol / L

specification:

96 servings / box

Storage conditions and validity period

1. Kit storage :; 2-8 ℃.

2. Validity: 6 months

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