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Human Golgi Protein 73 （GP-73）
ELISA Kit
（96T）
This immunoassay kit allows for the in vitro quantitative determination of human
GP-73 concentrations in cell culture supernates, 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.
CUSABIO BIOTECH CO., Ltd.
/ /
: cusabio@ cusabio@
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to GP-73. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for GP-73. 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 GP-73.,
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 GP-73. in the samples is then determined by comparing
the O.D. of the samples to the standard curve.
DETECTION RANGE
0.16 IU/ml-10 IU/ml. The standard curve concentrations used for the
ELISA’s were 10 IU/ml, 5 IU/ml, 2.5 IU/ml, 1.25 IU/ml, 0.62 IU/ml, 0.32 IU/ml,
0.16 IU/ml.
SPECIFICITY
This assay recognizes recombinant and natural human GP-73 No
significant cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of human GP-73 is typically less than 0.04
IU/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 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 with desiccants to
minimize exposure to damp air. The test kit may be used throughout the
expiration date of the kit. Refer to the package label for the expiration
date.
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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.
REAGENT PREPARATION
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 compley
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 10 IU/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 （10 IU/ml）. The Sample Diluent serves as the zero standard
（0 IU/ml）.
3. Biotin-antibody Dilute to the working concentration specified on the
vial label using Biotin-antibody Diluent（1:100）, respectively.
4. HRP-avidin Dilute to the working concentration specified on the vial
label 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.
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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
Cell Culture Supernates Remove particulates by centrifugation and
assay immediay 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 g. Remove
serum and assay immediay 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 immediay 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.
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1. Add 100μl of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37° C.
2. Remove the liquid of each well, don’t wash.
3. Add 100μl of Biotin-antibody working solution to each well. Incubate
for 1 hour at 37°C. Biotin-antibody working solution may appear
cloudy. Warm up to room temperature and mix gently until solution
appears uniform.
4. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash by filling each well with Wash Buffer （200μl）
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.
5. Add 100μl of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37° C.
6. Repeat the aspiration and wash three times as step 4.
7. Add 90μl of TMB Substrate to each well. Incubate for 30 minutes at
37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
8. Add 50μl of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well within 30 minutes, using a
microplate reader set to 450 nm.
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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
GP-73. 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.
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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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人高爾基蛋白73（GP-73）酶聯(lián)免疫分析
試劑盒使用說明書
本試劑盒僅供研究使用
檢測范圍：0.16 IU/ml - 10 IU/ml
zui低檢測限：0.04 IU/ml
特異性：本試劑盒可同時檢測天然或重組的人GP-73，且與其他相關(guān)蛋白
無交叉反應(yīng)。
有效期：6 個月
預(yù)期應(yīng)用：ELISA法定量測定人血清、血漿、細胞培養(yǎng)上清或其它相關(guān)生
物液體中GP-73 含量。
說明
1. 試劑盒保存：-20℃（較長時間不用時）；2-8℃（頻繁使用時）。
2. 濃洗滌液低溫保存會有鹽析出，稀釋時可在水浴中加溫助溶。
3. 中、英文說明書可能會有不一致之處，請以英文說明書為準。
4. 剛開啟的酶聯(lián)板孔中可能會含有少許水樣物質(zhì)，此為正常現(xiàn)象，不會對實
驗結(jié)果造成任何影響。
實驗原理
用純化的抗體包被微孔板，制成固相載體，往包被抗GP-73 抗體的微孔
中依次加入標本或標準品、*化的抗GP-73 抗體、HRP 標記的親和素，
經(jīng)過*洗滌后用底物TMB 顯色。TMB 在過氧化物酶的催化下轉(zhuǎn)化成藍色，
并在酸的作用下轉(zhuǎn)化成zui終的黃色。顏色的深淺和樣品中的GP-73.呈正相關(guān)。
用酶標儀在450nm 波長下測定吸光度（OD 值），計算樣品濃度。
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試劑盒組成及試劑配制
1. 酶聯(lián)板（Assay plate ）： 一塊（96孔）。
2. 標準品（Standard）： 2 瓶（凍干品）。
3. 樣品稀釋液（Sample Diluent）： 1×20ml/瓶。
4. *標記抗體稀釋液（Biotin-antibody Diluent）： 1×10ml/瓶。
5. 辣根過氧化物酶標記親和素稀釋液（HRP-avidin Diluent）： 1×10ml/瓶。
6. *標記抗體（Biotin-antibody）： 1×120μl/瓶（1：100）。
7. 辣根過氧化物酶標記親和素（HRP-avidin）： 1×120μl/瓶（1：100）。
8. 底物溶液（TMB Substrate）： 1×10ml/瓶。
9. 濃洗滌液（Wash Buffer）：1×20ml/瓶，使用時每瓶用蒸餾水稀釋25 倍。
10. 終止液（Stop Solution）： 1×10ml/瓶（2N H2SO4）。
需要而未提供的試劑和器材
1. 標準規(guī)格酶標儀
2. 高速離心機
3. 電熱恒溫培養(yǎng)箱
4. 干凈的試管和Eppendof 管
5. 系列可調(diào)節(jié)移液器及吸頭，一次檢測樣品較多時，用多通道移液器
6. 蒸餾水，容量瓶等
標本的采集及保存
1. 血清：全血標本請于室溫放置2 小時或4℃過夜后于1000 x g 離心20 分
鐘，取上清即可檢測，或?qū)吮痉庞?20℃或-80℃保存，但應(yīng)避免反復(fù)凍
融。
2. 血漿：可用EDTA 或肝素作為抗凝劑，標本采集后30 分鐘內(nèi)于2 - 8° C
1000 x g 離心15 分鐘，或?qū)吮痉庞?20℃或-80℃保存，但應(yīng)避免反復(fù)
凍融。
3. 細胞培養(yǎng)物上清或其它生物標本：1000 x g 離心20 分鐘，取上清即可檢
測，或?qū)吮痉庞?20℃或-80℃保存，但應(yīng)避免反復(fù)凍融。
注：標本溶血會影響zui后檢測結(jié)果，因此溶血標本不宜進行此項檢測。
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標本的稀釋原則：
首先通過文獻檢索的方式了解待測樣本的大致含量，確定適當?shù)南♂尡稊?shù)。
只有稀釋至標準曲線的范圍內(nèi)，檢測的結(jié)果才是準確的。稀釋的過程中，應(yīng)
做好詳細的記錄。zui后計算濃度時，稀釋了“N”倍，標本的濃度應(yīng)再乘以“N”。
標準品的稀釋原則：2 瓶，每瓶臨用前以樣品稀釋液稀釋至1ml，蓋好
后靜置10 分鐘以上，然后反復(fù)顛倒/搓動以助溶解，其濃度為10 IU/ml，做
系列倍比稀釋后，分別稀釋10 IU/ml, 5 IU/ml, 2.5 IU/ml, 1.25 IU/ml, 0.62
IU/ml, 0.32 IU/ml, 0.16 IU/ml，樣品稀釋液直接作為標準濃度0 IU/ml,，臨用
前15 分鐘內(nèi)配制。
如配制5 IU/ml 標準品：取0.5ml（不要少于0.5ml）10 IU/ml 的上述標準品
加入含0.5ml 樣品稀釋液的Eppendorf 管中，混勻即可，其余濃度以此類推。
*標記抗體的稀釋原則：
臨用前以*標記抗體稀釋液稀釋，稀釋前根據(jù)預(yù)先計算好的每次實驗所
需的總量配制（每孔100μl），實際配制時應(yīng)多配制0.1-0.2ml。如10μl 生物
素標記抗體加990μl *標記抗體稀釋液的比例配制，輕輕混勻，在使用
前一小時內(nèi)配制。
辣根過氧化物酶標記親和素的稀釋原則：
臨用前以辣根過氧化物酶標記親和素稀釋液稀釋，稀釋前根據(jù)預(yù)先計算好的
每次實驗所需的總量配制（每孔100μl），實際配制時應(yīng)多配制0.1-0.2ml。如
10μl 辣根過氧化物酶標記親和素加990μl 辣根過氧化物酶標記親和素稀釋液
的比例配制，輕輕混勻，在使用前一小時內(nèi)配制。
操作步驟
實驗開始前，請?zhí)崆芭渲煤盟性噭噭┗驑悠废♂寱r，均需混勻，混勻
時盡量避免起泡。每次檢測都應(yīng)該做標準曲線。如樣品濃度過高時，用樣品
稀釋液進行稀釋，以使樣品符合試劑盒的檢測范圍。
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1. 加樣：分別設(shè)空白孔、標準孔、待測樣品孔。空白孔加樣品稀釋液100μl，
余孔分別加標準品或待測樣品100μl，注意不要有氣泡，加樣將樣品加于
酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻，酶標板加上蓋或覆膜，
37℃反應(yīng)120 分鐘。
為保證實驗結(jié)果有效性，每次實驗請使用新的標準品溶液。
2. 棄去液體，甩干，不用洗滌。每孔加*標記抗體工作液 100μl（取1μl
*標記抗體加99μl *標記抗體稀釋液的比例配制，輕輕混勻，
在使用前一小時內(nèi)配制），37℃,60 分鐘。
3. 溫育60 分鐘后，棄去孔內(nèi)液體，甩干，洗板3 次，每次浸泡1-2 分鐘，
200μl/每孔，甩干。
4. 每孔加辣根過氧化物酶標記親和素工作液（同*標記抗體工作液）
100μl，37℃，60 分鐘。
5. 溫育60 分鐘后，棄去孔內(nèi)液體，甩干，洗板5 次，每次浸泡1-2 分鐘，
200μl/每孔，甩干。
6. 依序每孔加底物溶液90μl，37℃避光顯色（30 分鐘內(nèi)，此時肉眼可見標
準品的前3-4 孔有明顯的梯度藍色，后3-4 孔梯度不明顯，即可終止）。
7. 依序每孔加終止溶液50μl，終止反應(yīng)（此時藍色立轉(zhuǎn)黃色）。終止液的加
入順序應(yīng)盡量與底物液的加入順序相同。為了保證實驗結(jié)果的準確性，底
物反應(yīng)時間到后應(yīng)盡快加入終止液。
8. 用酶聯(lián)儀在450nm 波長依序測量各孔的光密度（OD 值）。在加終止液
后15 分鐘以內(nèi)進行檢測。
實驗備注
1. 用戶在初次使用試劑盒時，應(yīng)將各種試劑管離心數(shù)分鐘，以便試劑集中到
管底。
2. 每次實驗留一孔作為空白調(diào)零孔，該孔不加任何試劑，只是zui后加底物溶
液及2N H2SO4。測量時先用此孔調(diào)OD 值至零。
3. 為防止樣品蒸發(fā)，試驗時將反應(yīng)板放于鋪有濕布的密閉盒內(nèi)，酶標板加上
蓋或覆膜。
4. 未使用完的酶標板或者試劑，請于2-8℃保存。標準品、*標記抗體
工作液、辣根過氧化物酶標記親和素工作液請依據(jù)所需的量配置使用。請
勿重復(fù)使用已稀釋過的標準品、*標記抗體工作液、或辣根過氧化物
酶標記親和素工作液。
5. 建議檢測樣品時均設(shè)雙孔測定，以保證檢測結(jié)果的準確性。
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洗板方法
手工洗板方法：吸去（不可觸及板壁）或甩掉酶標板內(nèi)的液體；在實驗臺上
鋪墊幾層吸水紙，酶標板朝下用力拍幾次；將推薦的洗滌緩沖液至少0.3ml
注入孔內(nèi)，浸泡1-2 分鐘。根據(jù)需要，重復(fù)此過程數(shù)次。
自動洗板：如果有自動洗板機，應(yīng)在熟練使用后再用到正式實驗過程中。
計算
以標準物的濃度為橫坐標（對數(shù)坐標），OD 值為縱坐標（普通坐標），在半對
數(shù)坐標紙上繪出標準曲線，根據(jù)樣品的OD 值由標準曲線查出相應(yīng)的濃度；
再乘以稀釋倍數(shù)；或用標準物的濃度與OD 值計算出標準曲線的直線回歸方
程式，將樣品的OD 值代入方程式，計算出樣品濃度，再乘以稀釋倍數(shù)，即
為樣品的實際濃度。
注意事項
1. 底物請避光保存。
2. 當混合蛋白溶液時應(yīng)盡量輕緩，避免起泡。
3. 請每次測定的同時做標準曲線，做復(fù)孔。
4. 洗滌過程非常重要，不充分的洗滌易造成假陽性。
5. 不要用其它生產(chǎn)廠家的試劑替換試劑盒中的試劑。
6. 一次加樣時間控制在5 分鐘內(nèi)，如標本數(shù)量多，推薦使用排槍加樣。
7. 在配制標準品、檢測溶液工作液時，請以相應(yīng)的稀釋液配制，不能混淆。
8. 如標本中待測物質(zhì)含量過高，請先稀釋后再測定，計算時請zui后乘以稀釋
倍數(shù)。