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Human Brain Derived Neurotrophic
Facor(BDNF) ELISA Kit
Catalog No. CSB-E04501h
(96 tests)
This immunoassay kit allows for the in vitro quantitative determination of human
BDNF concentrations in serum, plasma and other biological fluids.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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INTRODUCTION
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
PRINCIPLE OF THE ASSAY
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 Horseradish Peroxidase (HRP)
-conjugated antibody preparation specific for BDNF and incubated. Then
substrate solutions are added to each well. 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.
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DETECTION RANGE
0.62 ng/ml-20 ng/ml. The standard curve concentrations used for the
ELISA’s were 20 ng/ml, 10 ng/ml, 4.37 ng/ml,1.87 ng/ml, 0.62ng/ml.
SPECIFICITY
This assay recognizes human BDNF. No significant cross-reactivity or
interference was observed.
SENSITIVITY
The minimum detectable dose of human BDNF is typically less than 0.31
ng/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined
as the lowest protein concentration that could be differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 5 x 0.5 ml
HRP-conjugate 1 x 6 ml
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard S1 S2 S3 S4 S5
Concentration
0.62 1.87 4.37 10 20
(ng/ml)
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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.
OTHER SUPPLIES REQUIRED
Microplate reader capable of measuring absorbance at 450 nm, with
the correction wavelength set at 540 nm or 570 nm.
Pipettes and pipette tips.
Deionized or distilled water.
Squirt bottle, manifold dispenser, or automated microplate washer.
SAMPLE COLLECTION AND STORAGE
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.
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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.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
1. Set a Blank well without any solution. Add 50μl of Standard or Sample
per well.
2. Add 50μl of HRP-Conjugate to each well (Not to Blank!). Incubate for 1
hour at 37°C.
3. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash by filling each well with ddH O (200μl)
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using a squirt bottle, multi-channel pipette, manifold dispenser or
autowasher. Complete removal of liquid at each step is essential to
good performance. After the last wash, remove any remaining Wash
Buffer by aspirating or decanting. Invert the plate and blot it against
clean paper towels.
4. Add 50μl of Substrate A and 50μl Substrate B to each well. Incubate
for 15 minutes at 37°C. Keeping the plate away from drafts and other
temperature fluctuations in the dark.
5. Add 50μl of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
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6. Determine the optical density of each well within 30 minutes, using a
microplate reader set to 450 nm.
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.
If samples generate values higher than the highest standard, dilute the
samples and repeat the assay.
Any variation in operator, pipetting technique, washing technique,
incubation time or temperature, and kit age can cause variation in
binding.
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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 Quantikine Immunoassay, the
possibility of interference cannot be excluded.
TECHNICAL HINTS
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.
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人脑源性神经营养因子人脑源性神经营养因子(BDNF)快速检测试剂盒快速检测试剂盒
人脑源性神经营养因子人脑源性神经营养因子 快速检测试剂盒快速检测试剂盒
使用说明书使用说明书
使用说明书使用说明书
本试剂盒仅供研究使用本试剂盒仅供研究使用
本试剂盒仅供研究使用本试剂盒仅供研究使用
产品编号产品编号：CSB-E04501h
产品编号产品编号
检测范围检测范围：：0.62 ng /ml -20 ng /ml
检测范围检测范围：：
最低检测限最低检测限：：0.31 ng/ml
最低检测限最低检测限：：
特异性特异性：：本试剂盒可同时检测天然或重组的BDNF，且与其他相关蛋白基
特异性特异性：：
本无交叉反应。
有效期有效期：6 个月 （2-8℃避光保存）
有效期有效期
预期应用预期应用：：ELISA 法定量测定人血清，血浆及其它相关生物液体中BDNF
预期应用预期应用：：
含量。
说明说明
说明说明
1. 浓洗涤液低温保存会有盐析出，稀释时可在水浴中加温助溶。
2. 刚开启的酶联板孔中可能会含有少许水样物质，此为正常现象，不会对实
验结果造成任何影响。
实验原理实验原理
实验原理实验原理
本试剂盒采用双抗体夹心法检测BDNF 含量。首先用BDNF 抗体包被微
孔板，制备成固相载体，然后加入样品 （标准品与待测标本）同时加入酶标
记的BDNF 抗体，特异性地形成固相抗体-抗原-酶标记抗体复合物，加底物
显色后在酶标仪测定吸光值 （OD 值），根据标准曲线计算出样品的含量。
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试剂盒组成及试剂配制试剂盒组成及试剂配制
试剂盒组成及试剂配制试剂盒组成及试剂配制
1. 酶联板酶联板(Assay plate )： 一块（96 孔）。
酶联板酶联板
2. 标准品标准品（Standard）： 5×0.5ml/瓶。
标准品标准品
Standard 1 Standard 2 Standard 3 Standard 4 Standard 5
0.62ng/ml 1.87ng/ml 4.37ng/ml 10ng/ml 20ng/ml
3. 酶结合物酶结合物（（HRP-conjugate）：） 1×6ml/瓶。
酶结合物酶结合物（（ ））
4. 显色剂显色剂A （（Substrate A）：）： 1×7ml/瓶。
显色剂显色剂 （（ ）：）：
5. 显色剂显色剂B （（Substrate B）：）： 1×7ml/瓶。
显色剂显色剂 （（ ）：）：
6. 终止液终止液（（Stop Solution）：） 1×7ml/瓶。
终止液终止液（（ ））
需要而未提供的试剂和器材需要而未提供的试剂和器材
需要而未提供的试剂和器材需要而未提供的试剂和器材
1. 标准规格酶标仪
2. 高速离心机
3. 电热恒温培养箱
4. 干净的试管和Eppendof 管
5. 系列可调节移液器及吸头，一次检测样品较多时，最好用多通道移液器
6. 蒸馏水，容量瓶等
标本的采集及保存标本的采集及保存
标本的采集及保存标本的采集及保存
1. 血清：全血标本请于室温放置2 小时或4℃过夜后于1000 x g 离心20 分
钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻
融。
2. 血浆：可用EDTA 或肝素作为抗凝剂，标本采集后30 分钟内于2 - 8° C
1000 x g 离心15 分钟，或将标本放于-20℃或-80℃保存，但应避免反复
冻融。
注：注：以上标本置以上标本置4℃℃保存应小于保存应小于1 周，周，-20℃℃或或-80℃℃均应密封保存均应密封保存，，-20℃℃不应超过不应超过1 个个
注注：：以上标本置以上标本置 ℃℃保存应小于保存应小于 周周，， ℃℃或或 ℃℃均应密封保存均应密封保存，， ℃℃不应超过不应超过 个个
月，月，-80℃℃不应超过不应超过2 个月个月；标本溶血会影响最后检测结；标本溶血会影响最后检测结果，果，因此溶血标本不宜进行检因此溶血标本不宜进行检
月月，， ℃℃不应超过不应超过 个月个月；；标本溶血会影响最后检测结标本溶血会影响最后检测结果果，，因此溶血标本不宜进行检因此溶血标本不宜进行检
测。测。
测测。。
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操作步骤操作步骤
操作步骤操作步骤
1. 将各种试剂至室温 〔18-25℃〕平衡半小时。
2. 将酶标板取出，设一个空白对照孔、不加任何液体；每个标准点依次各设
两孔，每孔加入相应标准品50ul；其余每个检测孔直接加待测标本50ul。
3. 每孔加入酶结合物50ul （空白对照孔除外），充分混匀，贴上不干胶封片，
置37℃温育1 小时。
4. 手工洗板，弃去孔内液体。去离子水注满各孔，静置10 秒甩干，重复三
次后拍干；洗板机洗板，选择洗涤三次程序，洗板后拍干。
5. 每孔加显色剂A 液50μl，显色剂B 液50μl，振荡混匀后，37℃避光显色
15 分钟，每孔加终止液50μl。
6. 用酶标仪读数，取波长450nm，先用空白孔调零点，然后测定各孔OD
值。
数据处理数据处理
数据处理数据处理
1. 手工作图：用双对数坐标纸，以标准品浓度为横轴，以对应的0D 值为纵
轴，画出平滑曲线或直线，在曲线上按照待测血清OD 值找到对应的浓度
值。
2. 计算机：使用线性拟合功能，应将标准品S1-S5 的浓度取对数（Log(浓
度)）作为X，将对应的OD 值减去空白对照孔OD 值后取对数（Log(OD
值-NSB)）作为Y，进行线性拟合。再从拟合线上计算出待测血清浓度。
注意事项注意事项
注意事项注意事项
1. 从冷藏环境中取出的试剂盒内全部瓶装试剂及所需预包被板条应置室温
（18-25℃）平衡30 分钟后方可使用，余者应及时封好口，放回2-8℃中避
光保存，以备后用。
2. 使用前试剂应摇匀。
3. 结果判断须在反应终止后10 分钟内完成。
4. 不同批号的试剂不可混用。
5. 加样时应注意避免所用各试剂及样品之间的交又污染。
6. 操作时，试剂盒内每种试剂各使用一个吸头，每一种标准品使用一个吸头，
每一个样品各使用一个吸头，吸头最好一次性使用。
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