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Product name : Human BDNF ELISA kit elisa

Catalog number : LF-EK50006

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Human BDNF ELISA kit elisa
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Human BDNF ELISA
Catalog # LF-EK50006 ( 4x96T)

 


Sandwich Enzyme-Linked Immunosorbent Assay for Quantitative Detection of human BDNF
For research use only


Not for diagnostic or therapeutic purposes


1. Introduction
Brain-derived neurotrophic factor (BDNF) is a prosurvival factor induced by cortical neurons that is necessary for survival of striatal neurons in the brain. It is a secreted protein with the molecular weight of 27.8kDa, consisting of 247 amino acids. It is known to promote neuronal survival and differentiation. BDNF shares substantial amino acid sequence identity with nerve growth factor (NGF). BDNF and neurotrophin-3 (NT-3) are two recently cloned neurotrophic factors that are homologous to NGF. mRNA products of the BDNF and NT-3 genes are detected in the adult human brain, suggesting that these proteins are involved in the maintenance of the adult nervous system.1 BDNF and other neurotrophins are critically involved in long-term potentiation (LTP). BDNF-mediated LTP is induced postsynaptically.2 BDNF has trophic effects on serotonergic (5-HT) neurons in the central nervous system.3 BDNF has an essential maintenance function in the regulation of anxiety-related behavior and in food intake through central mediators in both the basal and fasted state.4 It plays a role in treating breathing disorders such as respiratory insufficiency after spinal injury.5 The mature form of BDNF is identical in all mammals examined, and the gene encoding human BDNF to chromosome 11, band p13. 6
2. Principles of Method
AbFrontier’s human BDNF ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. Human BDNF specific-specific polyclonal antibodies were precoated onto 96-well plates. The human specific detection monoclonal antibodies were biotinylated. The test samples and biotinylated detection antibodies were added to the wells subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the human BDNF amount of sample captured in plate.
3. Intended Use
The AbFrontier human BDNF ELISA kit is to be used for the in vitro quantitative determination of human BDNF in human serum, human plasma, cell lysate and buffered solution. The assay will recognize both native and recombinant human BDNF.
This kit has been configured for research use only and is not to be used in diagnostic procedures.
4. Storage and Stability
All kit components of this kit are stable at 2 to 8℃. Any unused reconstituted standard should be discarded or frozen at -70℃. Standard can be frozen and thawed one time only without loss of immunoreactivity.
5. Chemical Hazard
- Stop solution: This reagent is an irritant to eyes, skin and mucous membranes. Avoid contact with eyes, skin and clothing. Wear suitable protective clothing, gloves and eye protection. In the event of contact with eyes or skin, wash immediately with plenty of water.
- All reagents containing Sodium Azide also contain Thimerosal as a preservative. Thimerosal contains Hg thus should be handled with great care.
6. Kit Contents
① 96 Well Plate
: Human BDNF microtiter plate, one plate of 96 wells.
A plate using break-apart strips coated with a monoclonal antibody specific to human
BDNF.
② Standard Protein
: Recombinant human BDNF.
③ Secondary Antibody
: Biotin labeled anti human BDNF antibody.
④ AV-HRP
: Avidin-Biotin-Peroxidase Complex (ABC)
⑤ Substrate (Stabilized chromogen)
: Tetramethylbenzidine (TMB) solution
⑥ Stop Solution
: 1 N solution of sulfuric acid (H2SO4)

Notice for Application of Kit
1. Before using Kit, spin tubes and bring down all components to bottom of tube.
2. Duplicate well assay was recommended for both standard and sample testing.
3. Don’t let 96-well plate dry, dry plate will inactivate active components on plate.
4. In order to avoid marginal effect of plate incubation due to temperature difference (reaction may be stronger in the marginal wells), it is suggested that the diluted ABC and TMB solution will be pre-warmed in 37℃ for 30 min before using.
7. Materials Required But Not Provided
① Microtiter plate reader in standard size.
② Automated plate washer.
③ Distilled or deionized water
④ Calibrated, adjustable precision pipettes, preferably with disposable plastic tips (A manifold multi-channel pipette is desirable for large assays.)
⑤ Data analysis and graphing software
⑥ Vortex mixer
⑦ Polypropylene tubes for diluting and aliquoting standard
⑧ Absorbent paper towels
⑨ Calibrated beakers and graduated cylinders of various sizes
⑩ Washing buffer (neutral PBS or TBS). Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g Nacl; 450μl of purified acetic acid or 700μl of concentrated hydrochloric acid to 1000ml
H2O and adjust pH to 7.2-7.6. Finally, adjust the total volume to 1L. Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na2HPO4 and 0.2g NaH2PO4 to 1000ml distilled water and adjust pH to 7.2-7.6. Finally, adjust the total volume to 1L.
8. Reagent Preparation
1) Plate washing
Discard the solution in the plate without touching the side walls. Blot the plate onto paper towels or other absorbent material. Soak each well with at least 0.3 ml PBS or TBS buffer for 1~2 minutes. Repeat this process two additional times for a total of THREE washes.
Note: For automated washing, aspirate all wells and wash THREE times with PBS or TBS buffer, overfilling wells with PBS or TBS buffer. Blot the plate onto paper towels or other absorbent material.
2) Sample Preparation and Storage
Store samples to be assayed within 24 hours at 2-8°C. For long-term storage, aliquot and freeze samples at -20°C. Avoid repeated freeze-thaw cycles.
o Cell culture supernate, tissue lysate or body fluids: Remove particulates by centrifugation, analyze immediately or aliquot and store at -20°C
o Serum: Allow the serum to clot in a serum separator tube (about 30 min) at room temperature. Centrifuge at approximately 1000 X g for 15 min. Analyze the serum immediately or aliquot and store frozen at -20°C.
o Plasma: Collect plasma using heparin, EDTA, citrate as an anticoagulant. Centrifuge for 15 min at 2-8℃ at 1500 x g within 30 min of collection. For eliminating the platelet effect, suggesting that further centrifugation for 10 min at 2-8°C at 10000 x g. Analyze immediately or aliquot and store samples at -20°C.
3) Sample Dilution Guideline
The user needs to estimate the concentration of the target protein in the sample and select a proper dilution factor so that the diluted target protein concentration falls near the middle of the linear regime in the standard curve. Dilute the sample using the provided diluent buffer. The following is a guideline for sample dilution. Several trials may be
necessary in practice. The sample must be well mixed with the diluents buffer.
o High target protein concentration (20-200ng/ml). The working dilution is 1:100. i.e. Add 1 μl sample into 99 μl sample diluent buffer.
o Medium target protein concentration (2-20ng/ml). The working dilution is 1:10. i.e. Add 10 μl sample into 90 μl sample diluent buffer.
o Low target protein concentration (31.2-2,000pg/ml). The working dilution is 1:2. i.e. Add 50 μl sample to 50 μl sample diluent buffer.
o Very Low target protein concentration (≤31.2pg/ml). No dilution necessary, or the working dilution is 1:2.
4) Reagent Preparation and Storage
A. Reconstitution of the human BDNF standard:BDNF standard solution should be prepared no more than 2 hours prior to the experiment. Two tubes of human BDNF standard (10ng per tube) are included in each kit. Use one tube for each experiment.
a. 10,000pg/ml of human BDNF standard solution: Add 1 ml sample diluent buffer into one tube, keep the tube at room temperature for 10 min and mix thoroughly.
b. 2000pg/ml of human BDNF standard solution: Add 0.2 ml of the above 10ng/ml BDNF standard solution into 0.8 ml sample diluent buffer and mix thoroughly.
c. 1000pg/ml→31.2pg/ml of human BDNF standard solutions: Label 6 Eppendorf tubes with 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, respectively. Aliquot 0.3 ml of the sample diluent buffer into each tube. Add 0.3 ml of the above 2000pg/ml human BDNF standard solution into 1st tube and mix. Transfer 0.3 ml from 1st tube to 2nd tube and mix. Transfer 0.3 ml from 2nd tube to 3rd tube and mix, and so on.
Note: The standard solutions are best used within 2 hours. The 10 ng/ml standard solution may be stored at 4°C for up to 12 hours, or at -20°C for up to 48 hours.
Avoid repeated freeze-thaw cycles.
B. Preparation of biotinylated anti-human BDNF antibody working solution: The solution should be prepared no more than 2 hours prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume)
b. Biotinylated anti-human BDNF antibody should be diluted in 1:99 with the antibody diluent buffer and mixed thoroughly.
C. Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The solution should be prepared no more than 1 hour prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume)
b. Avidin- Biotin-Peroxidase Complex (ABC) should be diluted in 1:99 with the ABC dilution buffer and mixed thoroughly.
9. Assay Procedure
The ABC working solution and TMB color developing agent must be kept warm at 37°C for 30 min before use. When diluting samples and reagents, they must be mixed completely and evenly. Standard BDNF detection curve should be prepared for each experiment. The user will decide sample dilution fold by crude estimation of BDNF amount in samples.
1. Aliquot 0.1ml per well of the 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml human BDNF standard solutions into the precoated 96-well plate. Add 0.1ml of the sample diluent buffer into the control well (Zero well). Add 0.1ml of each properly diluted sample of human sera, plasma, body fluids, tissue lysates or cell culture supernatants to each empty well. See “Sample Dilution Guideline” above for details. We recommend that each human BDNF standard solution and each sample is measured in duplicate.
2. Seal the plate with the cover and incubate at 37°C for 90 min.
3. Remove the cover, discard plate content, and blot the plate onto paper towels or other absorbent material. Do NOT let the wells completely dry at any time.
4. Add 0.1ml of biotinylated anti-human BDNF antibody working solution into each well and incubate the plate at 37°C for 60 min.
5. Wash the plate three times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material.
6. Add 0.1ml of prepared ABC working solution into each well and incubate the plate at
37°C for 30 min.
7. Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1-2 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material.
8. Add 90 μl of prepared TMB color developing agent into each well and incubate plate at 37°C for 15-20 min (shades of blue can be seen in the wells with the four most concentrated human BDNF standard solutions; the other wells show no obvious color).
9. Add 0.1ml of prepared TMB stop solution into each well. The color changes into yellow immediately.
10. Read the O.D. absorbance at 450nm in a microplate reader within 30 min after adding
the stop solution. For calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The human BDNF concentration of the samples can be interpolated from the standard curve.
Note: if the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.


Summary
1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash.
2. Add biotinylated antibodies and incubate the plate at 37°C for 60 min. Wash plate 3 times with 0.01M TBS.
3. Add ABC working solution and incubate the plate at 37°C for 30 min. Wash plate 5 times with 0.01M TBS.
4. Add TMB color developing agent and incubate the plate at 37°C for 10-15 min.
5. Add TMB stop solution and read.
10. Characteristics
1) Typical result
Typical Data Obtained from Human BDNF (TMB reaction incubate at 37℃ for 16 min)
Typical Human BDNF ELISA kit Standard Curve
This standard curve was generated at AbFrontier for demonstration purpose only. A standard curve must be run with each assay.
2) Sensitivity: < 2pg/ml
3) Specificity: No detectable cross-reactivity with any other cytokine.
11. Troubleshooting
12. Reference
1) Jones, K. R.; Reichardt, L. F. Molecular cloning of a human gene that is a member of the nerve growth factor family. Proc. Nat. Acad. Sci. 87: 8060-8064, 1990.
2) Kovalchuk, Y.; Hanse, E.; Kafitz, K. W.; Konnerth, A. Postsynaptic induction of BDNFmediated long-term potentiation. Science 295: 1729-1734, 2002.
Gentaur Molecular Products
Voortstraat 49
1910 Kampenhout, Belgium
3) Lyons, W. E.; Mamounas, L. A.; Ricaurte, G. A; Coppola, V.; Reid, S. W.; Bora, S. H.; Wihler, C.; Koliatsos, V. E.; Tessarollo, L. Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities. Proc. Nat. Acad. Sci. 96: 15239-15244, 1999.
4) Rios, M.; Fan, G.; Fekete, C.; Kelly, J.; Bates, B.; Kuehn, R.; Lechan, R. M.; Jaenisch, R.
Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity. Molec. Endocr. 15: 1748-1757, 2001.
5) Baker-Herman, T. L.; Fuller, D. D.; Bavis, R. W.; Zabka, A. G.; Golder, F. J.; Doperalski, N. J.; Johnson, R. A.; Watters, J. J.; Mitchell, G. S. BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia. Nature Neurosci. 7: 48-55, 2004.
6) Maisonpierre, P. C.; Le Beau, M. M.; Espinosa, R., III; Ip, N. Y.; Belluscio, L.; de la Monte, S. M.; Squinto, S.; Furth, M. E.; Yancopoulos, G. D. Human and rat brain-derived neurotrophic factor and neurotrophin-3: gene structures, distributions and chromosomal localizations. Genomics 10: 558-568, 1991.

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Pathways :
WP1114: MAPK signaling pathway
WP1337: MAPK signaling pathway
WP2107: pathways in human cancer
WP2137: Prenatal Stress & BDNF
WP2145: serotonin and anxiety
WP2148: Brain derived neurotrophic factor
WP2152: BDNF
WP2256: Integrated Pancreatic Cancer Pathway
WP2377: Integrated Pancreatic Cancer Pathway
WP2380: BDNF signaling pathway
WP2431: Spinal Cord Injury
WP2432: Spinal Cord Injury
WP2433: Spinal Cord Injury
WP358: MAPK signaling pathway
WP382: MAPK signaling pathway
WP493: MAPK signaling pathway
WP706: SIDS Susceptibility Pathways
WP769: MAPK signaling pathway
WP880: MAPK signaling pathway
WP998: MAPK signaling pathway

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[KITLG MGF SCF] Kit ligand (Mast cell growth factor) (MGF) (Stem cell factor) (SCF) (c-Kit ligand) [Cleaved into: Soluble KIT ligand (sKITLG)]
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Bibliography :
[30416353] The roles of valerenic acid on BDNF expression in the SH-SY5Y cell.
[30300266] RELATIONSHIP BETWEEN STAGES OF DIABETIC RETINOPATHY AND LEVELS OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN AQUEOUS HUMOR AND SERUM.
[29700574] Serum brain-derived neurotrophic factor level and exercise tolerance complement each other in predicting the prognosis of patients with heart failure.
[28951710] Brain-derived neurotrophic factor is increased in serum levels of patients with symptomatic dermographism.
[28803094] Brief psychoeducation for bipolar disorder: Evaluation of trophic factors serum levels in young adults.
[28341039] Cognitive impairment and BDNF serum levels.
[27314567] Cortisol and Brain-Derived Neurotrophic Factor Levels Prior to Treatment in Children With Obsessive-Compulsive Disorder.
[26200230] Decreased brain-derived neurotrophic factor plasma levels in psoriasis patients.
[26078232] Brain-derived neurotrophic factor concentrations in tetraplegic athletes.
[25587547] Relationship between levels of brain-derived neurotrophic factor and metabolic parameters in patients with type 2 diabetes mellitus.