Instruction manual of mouse brain-derived neurotrophic factor (BDNF) ELISA kit

This kit is for research use only

Detection range: 15.6 pg / ml-1000 pg / ml

Minimum detection limit: 3.9 pg / ml

Specificity: This kit can detect natural or recombinant mouse BDNF at the same time, and does not cross-react with other related proteins.

Validity: 6 months

Intended application: ELISA method for quantitative determination of BDNF in mouse serum, plasma, cell culture supernatant or other related biological fluids

content.

Explanation

1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).

2. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted.

3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.

4. The newly opened enzyme-linked plate may contain a little water-like substance in the well. This is a normal phenomenon and will not affect the experimental results.

Into any impact.

Overview

Brain-derived neurotrophic factor (BDNF) is a German neurobiology in 1982

A small molecule protein isolated from pig brain by scientists, because pig, rat, mouse and human BDNF have the same

Amino acid coding sequence, and has an amazing similarity with nerve growth factor (NGF) sequence,

Therefore, it is classified as a member of the nerve growth factor family. BNDF is a basic protein composed of 120 amino acids. It is one of the members of the neurotrophic factor family. It is secreted by the target cells of neurons and reverses the nutrition of neurons. BNDF plays an important role in the growth and development of neurons and the protection and repair , Its biological effects are mainly mediated by two types of transmembrane glycoproteins, one is the high affinity receptor TrKB, and the other is the low affinity neurotrophin receptor (LNGFR), of which TrKB is required for signal transduction of. TrKB's extracellular ligand binding region can be autophosphorylated after binding to BNDF, and then a series of substrate phosphorylation reactions can occur to achieve the transmission of information from the cell membrane to the nucleus and cause a cellular response effect. TrKB is mainly expressed in the brain. BDNF is currently considered to be an anti-apoptotic agent, antioxidant and calcium channel blocker.

Experimental principle

The microtiter plate is coated with purified antibody to make a solid phase carrier, and the standard is added to the microwells coated with anti-BDNF antibody in sequence

This or standard, biotinylated anti-BDNF antibody, HRP-labeled avidin, after thorough washing, use the substrate TMB

Color rendering. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. colour

The depth is positively correlated with BDNF in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.

Kit composition and reagent preparation

1. Assay plate: one piece (96 wells).

2. Standard product (Standard): 2 bottles (lyophilized product).

3. Sample Diluent: 1 × 20ml / bottle.

4. Biotin-antibody Diluent: 1 × 10ml / bottle.

5. HRP-avidin Diluent: 1 × 10ml / bottle.

6. Biotin-antibody: 1 × 120μl / bottle (1: 100)

7. Horseradish peroxidase labeled avidin (HRP-avidin): 1 × 120μl / vial (1: 100)

8. Substrate solution (TMB Substrate): 1 × 10ml / bottle.

9. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.

10. Stop Solution (Stop Solution): 1 × 10ml / bottle (2N H2SO4)

Reagents and equipment needed but not provided

1. Standard Specification Microplate Reader

2. High-speed centrifuge

3. Electric heating thermostat incubator

4. Clean test tubes and Eppendof tubes

5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes

6. Distilled water, volumetric flask, etc.

Collection and preservation of specimens

1. Serum: Whole blood samples should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000 xg for 20 minutes.

It can be detected or stored at -20 ℃ or -80 ℃, but repeated freezing and thawing should be avoided.

2. Plasma: use EDTA or heparin as anticoagulant, centrifuge at 2-8 ° C 1000 xg within 30 minutes after specimen collection

15 minutes, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.

3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for detection, or the specimen

Store at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.

Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.

Dilution principle of specimens:

First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor. Only diluted to standard

Within the scope of the curve, the detection result is accurate. Detailed records should be made during the dilution process. Finally calculate the concentration

When it is diluted by "N" times, the concentration of the specimen should be multiplied by "N".

Standard dilution principle: 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and after being closed, it is allowed to stand for more than 10 minutes.

Then repeatedly invert / scrub to help dissolve, its concentration is 1000 pg / ml, after serial dilutions, respectively dilute 1000

pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.2 pg / ml, 15.6 pg / ml, sample dilution

Directly used as a standard concentration of 0 pg / ml, prepared within 15 minutes before use.

If preparing 500 pg / ml standard: take 0.5ml (not less than 0.5ml) 1000 pg / ml of the above standard and add 0.5ml

In the Eppendorf tube of the sample dilution, mix well, and the rest of the concentration can be deduced by analogy.

Dilution principle of biotinylated antibody:

Dilute with biotinylated antibody diluent before use, and prepare according to the pre-calculated total amount required for each experiment before dilution

(100μl per well), 0.1-0.2ml should be prepared in actual preparation. For example, 10μl biotinylated antibody plus 990μl biotinylated

Record the proportion of antibody dilution, mix gently, and prepare within one hour before use.

Dilution principle of horseradish peroxidase-labeled avidin:

Dilute with horseradish peroxidase-labeled avidin dilution before use, according to the pre-calculated requirements for each experiment before dilution

The total amount of preparation (100μl per well), the actual preparation should be more 0.1-0.2ml. Such as 10μl horseradish peroxidase labeled affinity

Prepared by adding 990μl of horseradish peroxidase-labeled avidin dilution, mix gently, and within one hour before use

Preparation.

Steps

Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they should be mixed well. Try to avoid foaming when mixing.

A standard curve should be made for each test. If the sample concentration is too high, dilute with sample diluent to make the sample comply with the test

The detection range of the kit.

1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100μl of sample diluent to blank wells and add to the remaining wells

100μl of standard or sample to be tested, be careful not to have bubbles, add the sample to the bottom of the well of the microplate, try not to touch

And the well wall, gently shake to mix, add the microplate to the cover or membrane, and react at 37 ℃ for 120 minutes.

To ensure the validity of the experimental results, please use a new standard solution for each experiment.

2. Discard the liquid and spin dry without washing. Add 100μl biotinylated antibody working solution to each well (take 1μl biotinylated

Antibody plus 99μl biotinylated antibody dilution is prepared, mix gently and prepare within one hour before use),

37 ° C, 60 minutes.

3. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350μl / well, shake

dry.

4. Add horseradish peroxidase-labeled avidin working solution (with biotin-labeled antibody working solution) to each well at 37 ℃,

60 minutes.

5. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350μl / well, shake

dry.

6. Add 90 μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, the first 3-4 of the standard product can be seen by the naked eye

The well has a clear gradient blue, and the gradient of the last 3-4 wells is not obvious, it can be terminated).

7. Add 50μl of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). The order of adding stop solution should be as much as possible

The order of adding the substrate liquid is the same. In order to ensure the accuracy of the experimental results, the substrate reaction time should be added as soon as possible

Stop solution.

8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Within 15 minutes after adding stop solution

To be tested.

Note:

1. When using the reagent kit for the first time, the user should centrifuge various reagent tubes for several minutes so that the reagents are concentrated to the bottom of the tube.

2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate solution and 2N H2SO4 are added at the end.

Use this hole to adjust the OD value to zero first during measurement.

3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.

4. Store unused microplates or reagents at 2-8 ° C. Standards, biotinylated antibody working solution, horseradish

Oxidase-labeled avidin working solution should be used according to the required amount. Do not reuse diluted standards

Products, biotin-labeled antibody working solution, or horseradish peroxidase-labeled avidin working solution.

5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.

Washing method

Manual plate washing method: suck (do not touch the wall) or shake off the liquid in the enzyme plate; place a few layers of absorbent paper on the experimental table

Immediately tap the microtiter plate down several times; pour at least 0.3ml of the recommended wash buffer into the well and soak for 1-2 minutes. As needed

Yes, repeat this process several times.

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

Calculation

Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor to obtain the actual concentration of the sample.

Precautions

1. When mixing protein solutions, try to be as gentle as possible to avoid foaming.

2. The washing process is very important. Insufficient washing can easily cause false positives.

3. 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 the standard curve at the same time of each measurement, it is better to make the complex hole.

5. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.

6. When preparing standard products and testing solution working fluid, please prepare with corresponding diluent, not to be confused.

7. Please keep the substrate away from light.

8. Do not replace the reagents in the kit with reagents from other manufacturers.