ELISA Kit for Human Brain-derived Neurotrophic Factor (BDNF)

Instructions for use of human brain-derived neurotrophic factor (BDNF) ELISA kit

The human brain-derived neurotrophic factor (BDNF) enzyme-linked immunoassay kit is based on the principle of double antibody sandwich technology to detect human brain-derived neurotrophic factor (BDNF), which can only be used for research purposes and not for medical diagnosis.

Detection range: 0.5Î¼g / L â†’ 15Î¼g / L

Specification: 96T / box

Uses: For the determination of brain-derived neurotrophic factor (BDNF) in human tissues and related fluid samples.

working principle

This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of human brain-derived neurotrophic factor (BDNF) in the sample. Add brain-derived neurotrophic factor (BDNF) to the enzyme-labeled wells pre-coated with human brain-derived neurotrophic factor (BDNF) monoclonal antibody, incubate; after washing, add HRP-labeled brain-derived neurotrophic Factor (BDNF) antibody. After incubation and washing, the unbound enzyme is removed, and then the substrates A and B are added to produce a blue color, which is converted into a final yellow color under the action of an acid. The color depth is positively correlated with the concentration of human brain-derived neurotrophic factor (BDNF) in the sample.

Composition of human brain-derived neurotrophic factor (BDNF) ELISA kit

1 Standard product (24Î¼g / L) 0.5ml 7 Developer A solution 6ml

2 Standard dilution 3ml 8 Developer B 6ml

3 Enzyme label coated plate 12 well Ã— 8 strips 9 Stop solution 6ml

4 Enzyme label reagent 6ml 10 1 instruction manual

5 30-fold concentrated washing solution 20ml 11 sheets of sealing film

6 Sample diluent 6ml 12 sealed bag 1

Reagents and equipment needed but not provided

1. Standard specification microplate reader

2.7 â„ƒ thermostat

3. Disposable test tubes

4. Absorbent paper

5. Precision pipette and disposable tip

6. Distilled water

Precautions:

The kit taken from 2-8 Â° C should be equilibrated at room temperature for at least 30 minutes before opening the kit. If the enzyme-coated plates are not used up after opening, the slats should be stored in sealed bags. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. It is recommended that all standards and samples be tested in duplicate. If the content of the substance to be tested in the specimen is too high, please dilute it with the sample diluent by a certain multiple (n times) and then perform the operation according to the instructions. When calculating, please multiply the total dilution factor (Ã— n Ã— 5). Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader. To avoid cross-contamination, avoid repeated use of the tip and sealing film in your hand. Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty. Substrate B is sensitive to light and avoid prolonged exposure to light.

Washing method

1. Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.

2. Manual plate washing method: throw away the liquid in the enzyme labeling plate; place a few layers of absorbent paper on the experimental table, and pat the enzyme labeling plate down several times; inject at least 0.35ml of the diluted washing solution into the hole and soak 1-2 minutes. Repeat this process several times as needed.

Specimen requirements

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

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 Â° C, but repeated freezing and thawing should be avoided.

Operating procedures

1. Dilution of standard products: (This kit provides one original standard product, users please follow the instructions to dilute in a small test tube):

2.12Î¼g / L (Standard No. 5) 120Î¼l primary standard added 120Î¼l standard dilution

3.6Î¼g / L (Standard No. 4) 120Î¼l No. 5 Standard plus 120Î¼l Standard Diluent

4.3Î¼g / L (Standard No. 3) 120Î¼l No. 4 Standard plus 120Î¼l Standard Diluent

5.1.5Î¼g / L (Standard No. 2) 120Î¼l No. 3 Standard plus 120Î¼l Standard Diluent

6.0.75Î¼g / L (Standard No. 1) 120Î¼l No. 2 Standard plus 120Î¼l Standard Diluent

7. Separately set up blank wells (the blank control wells do not add samples and enzyme-labeled reagents, the rest of the operations are the same), standard wells, and sample wells to be tested. Add 50Î¼l of the diluted standard to the standard wells of the enzyme-coated plate; add 40Î¼l of the sample diluent to the test sample wells of the enzyme-coated plate, and then add 10Î¼l of the sample to be tested (the final dilution of the sample is 5 Times). Shake gently to mix, and incubate at 37 Â° C for 30 minutes.

8. Discard the liquid, spin dry, fill each well with diluted washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this 5 times, pat dry.

9. Add 50Î¼l of enzyme-labeled reagent to each well, except for blank wells. Shake gently to mix, and incubate at 37 Â° C for 30 minutes.

10. Discard the liquid, spin dry, fill each well with the diluted washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this 5 times, pat dry.

11. Add 50Î¼l of developer A to each well, then add 50Î¼l of developer B, mix gently, and develop at 37 Â° C in the dark for 10 minutes.

12. Remove the enzyme labeling plate and add 50Î¼l of stop solution to each well to stop the reaction (at this time, the blue color turns to yellow).

Determination: Zero the blank holes, and measure the absorbance (OD value) of each well at a wavelength of 450 nm. The measurement should be carried out within 15 minutes after adding the stop solution. The linear regression equation of the standard curve is calculated according to the concentration of the standard product and the corresponding OD value, and then the corresponding sample concentration is calculated on the regression equation according to the OD value of the sample. It can also be calculated using various application software. It should be remembered that since the sample is diluted, its actual concentration should be multiplied by the total dilution factor.

Summary of operating procedures:

1. Prepare reagents, samples and standards

2. Add the prepared samples and standards, and react at 37 â„ƒ for 30 minutes

3. Wash the plate 5 times, add enzyme-labeled reagent, and react at 37 â„ƒ for 30 minutes

4. Wash the plate 5 times, add color developing solutions A and B, and develop at 37 â„ƒ for 10 minutes

5. Add stop solution

Read the OD value within 15 minutes and calculate

Storage: 2-8 â„ƒ

Validity: 6 months.

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