Instructions for use of human brain-derived neurotrophic factor (BDNF) ELISA kit
This kit is for research use only
Intended application
Quantitative determination of BDNF in human serum, cell culture supernatant or other related liquids by ELISA.
Overview
Brain-derived neurotrophic factor (BDNF) is a small molecule protein isolated from porcine brain by German neurobiologists in 1982. BDNF in pigs, humans, mice and humans have the exact same amino acid encoding Sequence, and has a striking similarity to nerve growth factor (NGF) sequence, so 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 target cells of neurons and reverses neurotrophic neurons. BNDF plays an important role in the growth and development of neurons and 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), where TrKB is required for signal transduction of. TrKB's extracellular ligand binding region can undergo autophosphorylation 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 an anti-apoptotic agent, antioxidant and calcium channel blocker.
Experimental principle
This kit uses double antibody sandwich enzyme-labeled immunoassay to determine the level of BDNF in the specimen. The microtiter plate was coated with purified antibody to make a solid phase antibody. BDNF antigen, biotinylated anti-human BDNF antibody, and HRP-labeled avidin were added to the monoclonal antibody-coated microwells in turn. Substrate TMB color development. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color 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. ELISA plate: one piece (96 wells)
2. Standard product (lyophilized product): 2 bottles, each of which is diluted with sample diluent to 1ml before use. After being capped, it is allowed to stand for more than 10 minutes, and then repeatedly inverted / rubbed to help dissolve. Its concentration is 10000 pg / mL, after serial dilutions, dilute to 10000 pg / mL, 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156 pg / mL, and its stock solution As the highest standard concentration directly, the sample dilution is directly used as the standard concentration of 0 pg / mL, prepared within 15 minutes before use.
For example, to prepare a 5000 pg / mL standard: Take 0.5 ml of the above-mentioned standard 10000 pg / mL and add it to an Eppendorf tube containing 0.5 ml of sample diluent, mix well. The rest of the concentration can be deduced by analogy.
3. Sample diluent: 1 × 20ml / bottle.
4. Test the diluent A: 1 × 10ml / bottle.
5. Test diluent B: 1 × 10ml / bottle.
6. Detection solution A: 1 × 120ul / bottle (1: 100), diluted with detection diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use.
7. Detection solution B: 1 × 120ul / bottle (1: 100) is diluted with detection diluent B1: 100 before use. The dilution method is the same as that of Test Solution A.
8. Substrate solution: 1 × 10ml / bottle.
9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 × 10ml / bottle (2N H2SO4).
Collection and preservation of specimens
1. Supernatant of cell culture: collect the supernatant after centrifugation, and store the specimen at -20 ℃, and avoid repeated freezing and thawing.
2. Serum: Please leave the specimen at room temperature for 2 hours or overnight at 4 ° C and centrifuge at 1000 xg for 20 minutes. Take the supernatant for testing, or store the specimen at -20 ° C, but avoid repeated freezing and thawing.
3. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection. To further remove platelets from the plasma, centrifuge again at 2-8 ° C 1000 xg for 10 minutes, or Store the specimen at -20 ° C, but avoid repeated freezing and thawing.
Note: BDNF is contained in platelets, and it will be released into the blood with the activation of platelets. Therefore, if you want to determine the amount of circulating BDNF, you must first remove platelets from the specimen.
Steps
Each reagent is equilibrated to room temperature before use.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. In addition to the blank wells, add 100 ul of standard solution or sample to be tested to the remaining wells, taking care not to have air bubbles, mix gently, cover the enzyme plate, and react at 37 ° C for 120 minutes.
2. Discard the liquid and spin dry without washing.
3. Add 100ul of detection solution A working solution to each well at 37 ℃ for 60 minutes. Wash the plate 3 times, 350ul / per well, spin dry.
4. Add 100ul of detection solution B working solution to each well at 37 ° C for 60 minutes, wash the plate 5 times and spin dry.
5. Add 90ul of substrate solution to each well in sequence, and develop the color for 30 minutes at 37 ° C in the dark (at this time, the first 3-4 wells of the standard product have a clear blue gradient, and the latter 3-4 wells have no obvious gradient).
6. Add 50ul of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). The optical density (OD value) of each well was measured sequentially with an enzyme-linked instrument at a wavelength of 450 nm.
Note:
1. One hole is left for each experiment as a blank zero-adjusting hole. No reagents are added to this hole, only the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
2. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test.
Plate washing method Manual plate washing method: suck (do not touch the wall) or shake off the liquid in the microplate; place a few layers of absorbent paper on the experimental table, and force the microplate down several times; pat the recommended wash buffer Inject at least 0.4ml of solution into the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Specificity
The kit can detect recombinant or natural human BDNF at the same time, and has no cross reaction with other related proteins.
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. The washing process is very important, inadequate washing is easy to cause false positives.
2. 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.
3. Please make a standard curve at the same time of each measurement, it is best to make a double hole.
4. 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.
5. When preparing the standard and the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused.
6. Please keep the substrate away from light.
examination range:
156 pg / mL -10000 pg / mL
Explanation
1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. Validity: 6 months
3. The concentrated washing liquid will have salt precipitation, and it can be heated and dissolved in the water bath when diluted.
4. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.
5. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
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