Human Brain derived neurotrophic facor（BDNF）ELISA Kit
Catalogue Number: HG00774
（96 Tests）
Store all reagents at 2-8°C
Collect sample: serum or blood plasma
Assay range ： 50pg/mL -8500pg/mL
FOR LABORATORY RESEARCH USE ONLY.
NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!
PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
INTENDED USE
This BDNF ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of BDNF in the sample, this BDNF ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus BDNF concentration. The concentration of BDNF in the samples is then determined by comparing the O.D. of the samples to the standard curve.
PRINCIPLE OF THE ASSAY
The coated well immunoenzymatic assay for the quantitative measurement of serum BDNF utilizes a monoclonal anti-BDNF as capture antibody and a BDNF-biotin conjugate as detection antibody. The assay sample and standard are incubated together with anti-BDNF antibody coated plate for one hour and aspirated. The diluted BDNF-biotin conjugate is then added to each well and incubated. After the incubation period, the wells are decanted and washed three times. The Streptavidin-HRP is then added to each well and incubated. After the incubation period, the wells are decanted and washed three times. The wells
are then incubated with a substrate for the enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stopping solution is added to stop the reaction, which will then turn the solution yellow.The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is proportional to the BDNF concentration since BDNF from samples and BDNF-biotin conjugate compete for the anti-BDNF antibody binding site. Since the number of sites is limited, as more sites are occupied by BDNF from the sample, fewer sites are left to bind BDNF-biotin conjugate. Standards of known BDNF concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color （Optical Density） to the concentration of BDNF. The unknown BDNF concentration in each sample is interpolated from this curve.
REAGENTS PROVIDED
All reagents provided are stored at 2-8° C. Refer to the expiration date on the label.
1. MICROTITER PLATE 96 wells
2. BIOTIN CONJUGATE 100 uL 1 tube, Dilution Buffer 10ml 1 vial
3. STANDARD 8000pg/ml 1 tube, Dilution Buffer 15ml 1 vial
4. STREPTAVIDIN-HRP 100 uL 1 tube, Dilution Buffer 10ml 1 vial
5. SUBSTRATE A 6.0 mL 1 vial
6. SUBSTRATE B 6.0 mL 1 vial
7. STOP SOLUTION 6.0 mL 1 vial
8. WASH SOLUTION x30 10 mL 1 vial
9. Instruction 1
SAMPLE COLLECTION AND STORAGE
Serum- Use a serum separator tube（SST） and allow samples to clot for 30minutes before centrifugation for 15minutes at approximay 1000 x g.Remove serum and assay immediay or aliquot and store samples at -20 °C or -80°C.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant.Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within 30minutes of collection.Store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.
Cell culture fluid and other biological fluids - Remove particulates by centrifugation and assay immediay or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.
NOTE: Serum, plasma, and cell culture fluid samples to be used within 7 days may be stored at 2-8°C, otherwise samples must stored at -20°C（≤2months） or -80°C（≤6months） to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles .When performing the assay slowly bring samples to room temperature.
DO NOT USE HEAT-TREATED SPECIMENS.
MATERIALS REQUIRED BUT NOT SUPPLIED
1. Microplate reader capable of measuring absorbance at 450 nm.
2. Precision pipettes to deliver 2 ml to 1 ml volumes. .
3. Adjustable 10ml -100ml pipettes for reagent preparation.
4. 100 ml and 1 liter graduated cylinders.
5. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. （A manifold multi-channel pipette is desirable for large assays.）
6. Absorbent paper.
7. 37°C incubator.
8. Distilled or deionized water.
9. Data analysis and graphing software..
10. Tubes to prepare standard or sample dilutions.
PRECAUTIONS
1. Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.
2. Allow kit reagents and materials to reach room temperature （20-25°C） before use. Do not use water baths to thaw samples or reagents.
3. Do not use kit components beyond their expiration date.
4. Use only deionized or distilled water to dilute reagents.
5. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
6. Use fresh disposable pipette tips for each transfer to avoid contamination.
7. Do not mix acid and sodium hypochlorite solutions.
8. Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.
9. All samples should be disposed of in a manner that will inactivate viruses.
10. Solid Waste: Autoclave 60 min. at 121°C.
11. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.
12. Substrate Solution is easily contaminated. If bluish prior to use, do not use.
13. Substrate B contains 20% acetone, keep this reagent away from sources of heat or flame.
14. Remove all kit reagents from refrigerator and allow them to reach room temperature （ 20-25°C）.
SAMPLE PREPARATION
Serum and plasma samples require a dilution into Standard Dilution Buffer. A
suggested 2-fold dilution is 75 μL sample + 75 μL Standard Dilution Buffer. Mix well.
REAGENT PREPARATION
Standard -The Kit provides a stock solution of 8000 pg/mL standard. Allow the standard to sit for a minimum of 5 minutes with gentle mixing prior to making dilutions.Pipette 250μL of Standard Dilution into each tube.
（total 6 tubes） Use the 250μL of stock solution to produce a 2-fold dilution series （including 8000 pg/mL,4000 pg/mL,2000 pg/mL,1000 pg/mL,500 pg/mL,250 pg/mL,0 pg/mL）. Mix each tube thoroughly before the next transfer. The undiluted Human BDNF Standard serves as the high standard （8000 pg/mL）. Standard Dilution serves as the zero standard （0 pg/mL）.
Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have compley dissolved. To prepare enough Wash Buffer for one plate, add 20mL Wash Buffer Concentrate into deionized or distilled water to prepare 600mL of Wash Buffer.
ASSAY PROCEDURE
Prepare all Standards before starting assay procedure （see Preparation Reagents）. It is recommended that all Standards and Samples be added in duplicate to the Microtiter Plate.
1. secure the desired number of coated wells in the holder, then add 100 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate. Cover and incubate for 1 hours at 37°C. Wash the Microtiter Plate using one of the specified methods indicated below： Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well compley with distilled or de-ionized water, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of THREE washes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly hen washing the plate to assure that all strips remain securely in frame. Automated Washing: Aspirate all wells, then wash plate THREE times using distilled or de-ionized water. Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 μL/well/wash （range: 350-400 μL）. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10 seconds or shaking time of 5 seconds between washes
2. Diluted biotin Conjugate to 10ml by Dilution Buffer, Add 100 μL of Diluted Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hours at 37°C. Wash the Microtiter Plate using one of the specified methods indicated in 1.
3. Diluted Streptavidin-HRP to 10ml by Dilution Buffer, Add 100 μL of Working Dilution to each well. Cover the plate and incubate for 20 minutes at room temperature.Avoid placing the plate in direct light. Wash the Microtiter Plate using one of the specified methods indicated in 1.
4. Prepare Substrate Solution no more than 15 minutes before end of incubation （see Preparation of Reagents）. Add 50 μL Substrate A&B to each well. Cover and incubate for 15 minutes at 20-25°C.
5. Add 50 μL of Stop Solution to each well. Mix well.
6. Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.
TYPICAL DATA
CALCULATION OF RESULTS
Average the duplicate readings for each standard,control, and sample and subtract the average zero standard optical density. 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. 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.
