Brain derived neurotrophic facor (BDNF) ELISA Kit

Antigen: Brain-Derived Neurotrophic Factor (BDNF)

Synonyms: MGC34632, MGC105254, bdnf-A, BDNF, MGC133995, bdnf, Bdnf

Reactivity: Mouse (Murine)

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN365354

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of mouse BDNF concentrations in cell culture supernates, serum, plasma and other biological fluids.

Alternative name: Brain derived neurotrophic facor (BDNF)

Description: BDNF is a 13 kDa, 119 amino acid (aa) residue non-glycosylated polypeptide whose primary structure is conserved among all mammalian species examined. Initially synthesized as a 247 aa residue prepropeptide, the BDNF molecule is divided into an 18 aa residue signal sequence, a 110 aa residue prosequence, and a 119 aa residue mature segment. Similar to other neurotrophic factors, there is a possibility that the N-terminus is alternatively spliced, giving rise to a longer pre-prosegment (but identical mature segment) with different functional properties. As a mature molecule, BDNF is 52% identical to NGF at the amino acid level, exists as a noncovalently-linked homodimer in solution, and contains six cysteine residues that are believed to form three intrachain disulfide linkages. BDNF in plasma is detected in the pg/mL range, while BDNF in serum is measured in the pg/ml range, the difference apparently attributable to platelet degranulation and BDNF release during clotting. The conservation of BDNF structure potentially allows a BDNF ELISA to be widely applied across species. There are at least two receptors for BDNF, the first being the low affinity 75 kDa Nerve Growth Factor Receptor (LNGFR), and the second being the high affinity 145 kDa TrkB (tropomyosin receptor kinase-B). The LNGFR is a 399 aa residue Type-I (extracellular N-terminus) transmembrane glycoprotein that is currently considered to be a member of the TNF receptor superfamily. Although all neurotrophins bind LNGFR with approximately the same affinity (Kd ~ 1 nM), the significance of such binding is uncertain. What seems clear is that the LNGFR alone can engage certain signal transduction pathways. The biological significance of the activation of these pathways is not well understood. For BDNF specifically, LNGFR may serve as a retrograde transport molecule in neurons, promote Schwann cell migration near injury, and/or modulate TrkB activity in those cells that co-express both LNGFR and TrkB. The second receptor for BDNF is the TrkB high affinity receptor (Kd ~ 10 pM), a receptor that also binds NT-3 and NT-4/5. TrkB is a 792 aa residue type I (extracellular N-terminus) transmembrane glycoprotein that exhibits a number of distinct extracellular domains. These include two N-terminal cysteine-rich regions that flank an intervening leucine-rich domain and two membrane proximal C2 Ig-like domains. Comparing rat and human proteins, these regions exhibit over 90% identity in aa sequence. Alternative splice events have been identified for the TrkB gene in the mouse, the rat, and the human. In each case, nonsignalling, cytoplasmically-truncated variants are produced, leading to speculation that alternative splicing may be one method by which cells down-regulate neurotrophin activity. While full TrkB activity is believed to require TrkB homodimerization, evidence suggests that full length TrkB and TrkC receptors may also form functional heterodimers in select cells where both receptors are co-expressed. These include cerebellar granule neurons and neurons of the hippocampal dentate nucleus. Among the cells known to express TrkB are motoneurons of the spinal cord, pyramidal cells of the hippocampus, almost all neurons in the developing brain, and thymocytes, leading to speculation that BDNF plays a role in lymphopoiesis. The number of functions attributed to BDNF is quite large. During development, BDNF has been implicated in neuronal differentiation, maturation, survival and synapse formation. One of its most promising roles centers on neuroprotection, possibly protecting forebrain neurons from ischemic attack and motor neurons from axotomy-induced death.

Specificity: This assay recognizes recombinant and natural mouse BDNF. No significant cross-reactivity or interference was observed.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to BDNF. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for BDNF and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain BDNF, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of BDNF in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Preparation of Reagents: Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 1000 pg/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (1000 pg/ml). The Sample Diluent serves as the zero standard (0 pg/ml). 3. Biotin-antibody Dilute to the working concentration using Biotin-antibody Diluent(1:100), respectively. 4. HRP-avidin Dilute to the working concentration using HRP-avidin Diluent(1:100), respectively. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. Sample Collection and Storage: Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Assay Procedure: Calculation of Results: Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the BDNF concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Limitations of the Procedure: The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay. Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Application Notes: When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Components: Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20 ml Biotin-antibody Diluent 1 x 10 ml HRP-avidin Diluent 1 x 10 ml Biotin-antibody 1 x 120µl HRP-avidin 1 x 120µl Wash Buffer 1 x 20 ml (25×concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml STORAGE 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Storage: 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. Expiration date: six months from the date of manufacture.

Restrictions: For Research Use only