Human Transferrin (TRF) Elisa Kit

Instructions for use of human transferrin (TRF) Elisa kit

The human transferrin (TRF) Elisa kit is for research use only.

Detection range: 96T 20μmol / L -650μmol / L

Purpose: This kit is used to determine the content of transferrin (TRF) in human serum, plasma and related liquid samples.

Experimental principle: This kit uses the double antibody sandwich method to determine the level of human transferrin (TRF) in the specimen. The microplate was coated with purified human transferrin (TRF) antibody to make a solid phase antibody. Transferrin (TRF) was added to the monoclonal antibody-coated microwells in turn, followed by HRP labeled transferrin (TRF) ) The 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 transferrin (TRF) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human transferrin (TRF) in the sample was calculated by a standard curve.

The human transferrin (TRF) Elisa kit consists of:

1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle

2 Enzyme label reagent 6ml × 1 bottle 8 standard (1200μmol / L) 0.5ml × 1 bottle

3 Enzyme label coated plate 12 wells × 8 strips 9 standard dilutions 1.5ml × 1 bottle

4 Sample diluent 6ml × 1 bottle 10 instructions 1 copy

5 Developer A solution 6ml × 1 bottle 11 2 sealing film

6 Developer B liquid 6ml × 1 / bottle 12 1 sealed bag

Specimen requirements:

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

2. The specimens should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. If the test cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided.

Steps:

1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart.

600μmol / L No. 5 standard 150μl original standard added 150μl standard dilution

300μmol / L No. 4 standard 150μl No. 5 standard added 150μl standard dilution

150μmol / L No. 3 standard 150μl No. 4 standard added 150μl standard diluent

75μmol / L No. 2 standard 150μl No. 3 standard added 150μl standard diluent

37.5μmol / L No. 1 standard 150μl No. 2 standard added 150μl standard diluent

2. Adding samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same), standard wells, sample wells to be tested. Accurately add 50μl of standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, and then add 10μl of 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, try not to touch the wall of the well, shake gently to mix.

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

4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve for use

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 and 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, and then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.

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

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

Calculation: take the concentration of the standard as the abscissa and the OD 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; The linear regression equation of the standard curve is calculated by the concentration and OD value of the sample, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample.

Precautions:

1. The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unsealed after opening, 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 the water bath during dilution, and the results will not be affected during washing.

3. The sample adder should be used in each step of sample addition, and its 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 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 it with a certain multiple of the sample diluent (n times) before measuring, and finally multiply the total dilution when calculating Multiple (× n × 5).

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

6. Please keep the substrate away from light.

7. Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader.

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

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

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

Storage conditions and validity period:

1. Kit storage: 2-8 ℃.

2. Validity: 6 months