Rat calcitonin gene-related peptide (CGRP) enzyme-linked immunoassay (ELISA)

Rat calcitonin gene-related peptide (CGRP) enzyme-linked immunoassay (ELISA)

Kit instruction manual

This kit is for research use only.

Drug Name:

Generic name: Rat calcitonin gene-related peptide (CGRP) enzyme-linked immunoassay kit

English name: Rat CGRP ELISA Kit

purpose of usage:

This kit is used to determine the content of calcitonin gene-related peptide (CGRP) in rat serum, plasma, or related tissue fluid.

Experimental principle

This kit uses the double antibody sandwich method to determine the level of rat calcitonin gene-related peptide (CGRP) in the specimen. Microporous plates were coated with purified rat calcitonin gene-related peptide (CGRP) antibody to make solid-phase antibodies. Calcitonin gene-related peptide (CGRP) was added to the monoclonal antibody-coated microwells and labeled with HRP The goat anti-mouse antibody binds to form an antibody-antigen-enzyme-labeled antibody 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 rat calcitonin gene-related peptide (CGRP) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the content of rat calcitonin gene-related peptide (CGRP) in the sample was calculated by a standard curve.

Kit composition

1

20 times concentrated washing liquid

30ml × 1 bottle

7

Stop solution

6ml × 1 bottle

2

Enzyme reagent

6ml × 1 bottle

8

Standard product (45ng / L)

0.5ml × 1 bottle

3

Enzyme coated plate

12 holes × 8

9

Standard dilution

1.5ml × 1 bottle

4

Sample diluent

6ml × 1 bottle

10

Instructions

1 serving

5

Developer A liquid

6ml × 1 bottle

11

Sealing film

2 sheets

6

Developer B liquid

6ml × 1 / bottle

12

sealed bag

1

Specimen processing and requirements

1. Serum and plasma samples can be directly measured;

2. Tissue: Take 1g of related tissues in 5ml PBS and homogenize. After incubating for 5 hours at room temperature (vortex mixing from time to time), 2000-3000 rpm / separation heart for 10 minutes, take supernatant for testing.

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

4. Experiment as soon as possible after collection. If the experiment cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided.

Steps

1. Dilution and loading of standard products: 10 standard wells are set in sequence on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add in the first and second well Standard diluent 50μl, mix well; then add 100μl each to the third and fourth wells, and then add standard diluent 50μl to the third and fourth wells, after mixing, in the third well and the first Take 50μl each of the four wells and discard it; then add 50μl each to the fifth and sixth wells; add 50ul of the standard dilution solution to the fifth and sixth wells respectively and mix well; Take 50μl from the sixth well and add it to the seventh and eighth wells respectively; then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Go to the ninth and tenth wells; add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the amount of sample added to each well is 50 μl, and the concentrations are 30 ng / L, 20 ng / L, 10 ng / L, 5 ng / L, 2.5 ng / L).

2. Add samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, shake gently to mix.

3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.

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

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, pat dry.

6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.

7. Incubation: The operation is the same as 3.

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

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 by the dilution factor; Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor to obtain 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. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance 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 it with a certain multiple (n times) of the sample diluent before measuring, and finally multiply the total dilution when calculating Multiple (× n × 5).

5. Strictly follow the instructions, and the test results must be determined by the reading of the microplate reader.

6. The components of different batches of this reagent shall not be mixed.

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

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

9. Please keep the substrate away from light.

examination range:

2 ng / L -40 ng / L

specification:

96 servings / box

Storage conditions and validity period

1. Kit storage :; 2-8 ℃.

2. Validity: 6 months