Rat Ultra Sensitive Heat Shock
Protein 70,HSP-70
ELISA Kit
（96 T）
This immunoassay kit allows for the in vitro quantitative determination of rat
HSP-70 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.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an antibody specific to HSP-70. Standards or samples are then
added to the appropriate microtiter plate wells with a
biotin-conjugated antibody preparation specific for HSP-70 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 HSP-70, 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 HSP-70 in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
39 pg/ml-2500 pg/ml. The standard curve concentrations used
for the ELISA’s were 2500 pg/ml, 1250 pg/ml, 625 pg/ml, 312
pg/ml, 156 pg/ml, 78 pg/ml, 39 pg/ml.
SPECIFICITY
This assay recognizes recombinant and natural rat HSP-70. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat HSP-70 is typically less
than 9.8 pg/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. The
test kit may be used throughout the expiration date of the kit,
provided it is stored as prescribed above. Refer to the
package label for the expiration date.
2. Opened test plate should be stored at 2-8°C in the aluminum
foil bag with desiccants to minimize exposure to damp air. The
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 Centrifuge the standard vial at 6000-10000rpm
for 30s. Reconstitute the Standard with 1.0 ml of Sample
Diluent. This reconstitution produces a stock solution of 2500
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 （2500 pg/ml）.
The Sample Diluent serves as the zero standard （0 pg/ml）.
Prepare fresh for each assay. Use within 4 hours and discard
after use.
3. Biotin-antibody Centrifuge the vial before opening. Dilute
to the working concentration using Biotin-antibody
Diluent（1:100）, respectively.
4. HRP-avidin Centrifuge the vial before opening. 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.
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.
An incubator which can provide stable incubation conditions
up to 37°C ±0.5°C.
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 g. Remove serum and assay immediay or
aliquot and store samples at -20°C. Centrifuge the sample
again after thawing before the assay. Avoid repeated
freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 g within
30 minutes of collection. Assay immediay or aliquot and
store samples at -20°C. Centrifuge the sample again after
thawing before the assay. 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.
All the reagents should be added directly to the liquid level in the well. The
pipette should avoid contacting the inner wall of the well.
1. Serum and plasma samples require a 20-fold dilution into
Sample Diluent. The suggested 20-fold dilution can be
achieved by adding 15μl sample to 285μl of Sample Diluent.
2. Add 100μl of Standard, Blank, or Sample per well. Cover with
the adhesive strip. Incubate for 2 hours at 37°C.
3. Remove the liquid of each well, don’t wash.
4. 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.
5. Aspirate each well and wash, repeating the process three
times for a total of three washes. Wash: Fill each well with
Wash Buffer （200μl） and let it stand for 2 minutes, then
remove the liquid by flicking the plate over a sink. The
remaining drops are removed by patting the plate on a paper
towel. Complete removal of liquid at each step is essential to
good performance.
6. 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.
7. Repeat the aspiration and wash five times as step 4.
8. Add 90μl of TMB Substrate to each well. Incubate for 10-30
minutes at 37°C. Keeping the plate away from drafts and
other temperature fluctuations in the dark.
9. Add 50μl of Stop Solution to each well when the first four
wells containing the highest concentration of standards
develop obvious blue color. If color change does not appear
uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
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 HSP-70 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 Standard 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 Standard 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
Quantikine Immunoassay, the possibility of interference
cannot be excluded.
TECHNICAL HINTS
Centrifuge vials before opening to collect contents.
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 or light blue until
added to the plate. Keep Substrate Solution protected from
light. Substrate Solution should change from colorless or light
blue 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.
大鼠超敏熱休克蛋白70（HSP-70）酶聯(lián)免疫分析
試劑盒使用說(shuō)明書
本試劑盒僅供研究使用
檢測(cè)范圍：39 pg/ml - 2500 pg/ml
zui低檢測(cè)限：9.75 pg/ml
特異性：本試劑盒可同時(shí)檢測(cè)天然或重組的大鼠HSP-70，且與其他相關(guān)
蛋白無(wú)交叉反應(yīng)。
有效期：6 個(gè)月
預(yù)期應(yīng)用：ELISA 法定量測(cè)定大鼠血清、血漿或其它相關(guān)生物液體中
HSP-70 含量。
說(shuō)明
11. 試劑盒保存：未開(kāi)封的試劑盒應(yīng)儲(chǔ)存于2-8℃；開(kāi)封后的酶標(biāo)板應(yīng)與干燥
劑一起儲(chǔ)存于鋁箔袋中置于2-8℃保存。僅在此出儲(chǔ)存條件下，產(chǎn)品在有
效期內(nèi)可正常使用。
12. 濃洗滌液低溫保存會(huì)有鹽析出，稀釋時(shí)可在水浴中加溫助溶。
13. 中、英文說(shuō)明書可能會(huì)有不一致之處，請(qǐng)以英文說(shuō)明書為準(zhǔn)。
14. 剛開(kāi)啟的酶聯(lián)板孔中可能會(huì)含有少許水樣物質(zhì)，此為正?，F(xiàn)象，不會(huì)對(duì)實(shí)
驗(yàn)結(jié)果造成任何影響。
實(shí)驗(yàn)原理
用純化的抗體包被微孔板，制成固相載體，往包被抗HSP-70 抗體的微
孔中依次加入標(biāo)本或標(biāo)準(zhǔn)品、生物素化的抗HSP-70 抗體、HRP 標(biāo)記的親和
素，經(jīng)過(guò)*洗滌后用底物TMB 顯色。TMB 在過(guò)氧化物酶的催化下轉(zhuǎn)化成
藍(lán)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HSP-70
呈正相關(guān)。用酶標(biāo)儀在450nm 波長(zhǎng)下測(cè)定吸光度（OD 值），計(jì)算樣品濃度。
試劑盒組成及試劑配制
1. 酶聯(lián)板（Assay plate ）： 一塊（96孔）。
2. 標(biāo)準(zhǔn)品（Standard）： 2 瓶（凍干品）。
3. 樣品稀釋液（Sample Diluent）： 1×20ml/瓶。
4. 生物素標(biāo)記抗體稀釋液（Biotin-antibody Diluent）： 1×10ml/瓶。
5. 辣根過(guò)氧化物酶標(biāo)記親和素稀釋液（HRP-avidin Diluent） 1×10ml/瓶。
6. 生物素標(biāo)記抗體（Biotin-antibody）： 1×120μl/瓶（1：100）。
7. 辣根過(guò)氧化物酶標(biāo)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(shí)每瓶用蒸餾水稀釋25 倍。
10. 終止液（Stop Solution）： 1×10ml/瓶。
需要而未提供的試劑和器材
1. 標(biāo)準(zhǔn)規(guī)格酶標(biāo)儀
2. 高速離心機(jī)
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(cè)樣品較多時(shí)，用多通道移液器
6. 蒸餾水，容量瓶等
標(biāo)本的采集及保存
1. 血清：全血標(biāo)本請(qǐng)于室溫放置2 小時(shí)或4℃過(guò)夜后于1000g 離心20 分鐘，
取上清即可立即檢測(cè)；或進(jìn)行分裝，并將標(biāo)本放于-20℃或-80℃保存，但
應(yīng)避免反復(fù)凍融。解凍后的樣品應(yīng)再次離心，然后檢測(cè)。
2. 血漿：可用EDTA 或肝素作為抗凝劑，標(biāo)本采集后30 分鐘內(nèi)于2 - 8°C
1000 g 離心15 分鐘，取上清即可立即檢測(cè)；或進(jìn)行分裝，并將標(biāo)本放于
-20℃或-80℃保存，但應(yīng)避免反復(fù)凍融。解凍后的樣品應(yīng)再次離心，然后
檢測(cè)。
3. 細(xì)胞培養(yǎng)物上清或其它生物標(biāo)本：1000g 離心20 分鐘，取上清即可立即
檢測(cè)；或進(jìn)行分裝，并將標(biāo)本放于-20℃或-80℃保存，但應(yīng)避免反復(fù)凍融。
解凍后的樣品應(yīng)再次離心，然后檢測(cè)。
注：標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果，因此溶血標(biāo)本不宜進(jìn)行此項(xiàng)檢測(cè)。
標(biāo)本的稀釋原則：
首先通過(guò)文獻(xiàn)檢索的方式了解待測(cè)樣本的大致含量，確定適當(dāng)?shù)南♂尡稊?shù)。
只有稀釋至標(biāo)準(zhǔn)曲線的范圍內(nèi)，檢測(cè)的結(jié)果才是準(zhǔn)確的。稀釋的過(guò)程中，應(yīng)
做好詳細(xì)的記錄。zui后計(jì)算濃度時(shí)，稀釋了“N”倍，標(biāo)本的濃度應(yīng)再乘以“N”。
標(biāo)準(zhǔn)品的稀釋原則：2 瓶，使用前于6000-10000rpm 離心30 秒。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(dòng)以助溶解，其濃度為2500 pg/ml，做系列倍比稀釋后，分別稀釋2500
pg/ml, 1250 pg/ml, 625 pg/ml, 312 pg/ml, 156 pg/ml, 78 pg/ml, 39 pg/ml，樣
品稀釋液直接作為標(biāo)準(zhǔn)濃度0 pg/ml，臨用前15 分鐘內(nèi)配制，用完丟棄，下
次檢測(cè)使用新鮮配置的標(biāo)準(zhǔn)品。
如配制1250 pg/ml 標(biāo)準(zhǔn)品：取0.5ml（不要少于0.5ml）2500 pg/ml 的上述
標(biāo)準(zhǔn)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。
生物素標(biāo)記抗體的稀釋原則：
打開(kāi)瓶蓋前請(qǐng)離心，收集瓶壁上的溶液。臨用前以生物素標(biāo)記抗體稀釋液稀
釋，稀釋前根據(jù)預(yù)先計(jì)算好的每次實(shí)驗(yàn)所需的總量配制（每孔100μl），實(shí)際
配制時(shí)應(yīng)多配制0.1-0.2ml。如10μl 生物素標(biāo)記抗體加990μl 生物素標(biāo)記抗體
稀釋液的比例配制，輕輕混勻，在使用前一小時(shí)內(nèi)配制。
辣根過(guò)氧化物酶標(biāo)記親和素的稀釋原則：
打開(kāi)瓶蓋前請(qǐng)離心，收集瓶壁上的溶液。臨用前以辣根過(guò)氧化物酶標(biāo)記親和
素稀釋液稀釋，稀釋前根據(jù)預(yù)先計(jì)算好的每次實(shí)驗(yàn)所需的總量配制（每孔
100μl），實(shí)際配制時(shí)應(yīng)多配制0.1-0.2ml。如10μl 辣根過(guò)氧化物酶標(biāo)記親和素
加990μl 辣根過(guò)氧化物酶標(biāo)記親和素稀釋液的比例配制，輕輕混勻，在使用
前一小時(shí)內(nèi)配制。
操作步驟
實(shí)驗(yàn)開(kāi)始前，請(qǐng)?zhí)崆芭渲煤盟性噭噭┗驑悠废♂寱r(shí)，均需混勻，混勻
時(shí)盡量避免起泡。每次檢測(cè)都應(yīng)該做標(biāo)準(zhǔn)曲線。如樣品濃度過(guò)高時(shí)，用樣品
稀釋液進(jìn)行稀釋，以使樣品符合試劑盒的檢測(cè)范圍。加樣時(shí)，槍頭應(yīng)直接對(duì)
準(zhǔn)液面，切勿沿孔壁加樣。
4. 血清，血漿樣本用樣本稀釋液進(jìn)行1:20 倍稀釋后進(jìn)行檢測(cè)，具體操作如
下：取15μl 樣本加入到285μl 的樣本稀釋液（1:20 稀釋）中混勻。得
到的即為1:20 倍稀釋后的樣本。
5. 加樣：分別設(shè)空白孔、標(biāo)準(zhǔn)孔、待測(cè)樣品孔?？瞻卓准訕悠废♂屢?00μl，
余孔分別加標(biāo)準(zhǔn)品或待測(cè)樣品100μl，注意不要有氣泡，加樣將樣品加于
酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動(dòng)混勻，酶標(biāo)板加上蓋或覆膜，
37℃反應(yīng)120 分鐘。
為保證實(shí)驗(yàn)結(jié)果有效性，每次實(shí)驗(yàn)請(qǐng)使用新的標(biāo)準(zhǔn)品溶液。
6. 棄去液體，甩干，不用洗滌。每孔加生物素標(biāo)記抗體工作液 100μl（取1μl
生物素標(biāo)記抗體加99μl 生物素標(biāo)記抗體稀釋液的比例配制，輕輕混勻，
在使用前一小時(shí)內(nèi)配制），37℃,60 分鐘。
7. 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。
8. 每孔加辣根過(guò)氧化物酶標(biāo)記親和素工作液（同生物素標(biāo)記抗體工作液）
100μl，37℃，60 分鐘。
9. 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。
10. 依序每孔加底物溶液90μl，37℃避光顯色（30 分鐘內(nèi)，此時(shí)肉眼可見(jiàn)標(biāo)
準(zhǔn)品的前3-4 孔有明顯的梯度藍(lán)色，后3-4 孔顯色不明顯，即可終止）。
11. 依序每孔加終止溶液50μl，終止反應(yīng)（此時(shí)藍(lán)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(shí)驗(yàn)結(jié)果的準(zhǔn)確性，底
物反應(yīng)時(shí)間到后應(yīng)盡快加入終止液。
12. 用酶聯(lián)儀在450nm 波長(zhǎng)依序測(cè)量各孔的光密度（OD 值）。在加終止液
后15 分鐘以內(nèi)進(jìn)行檢測(cè)。
實(shí)驗(yàn)備注
1. 用戶在初次使用試劑盒時(shí)，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(shí)驗(yàn)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
液及終止液。測(cè)量時(shí)先用此孔調(diào)OD 值至零。
3. 為防止樣品蒸發(fā)，試驗(yàn)時(shí)將反應(yīng)板放于鋪有濕布的密閉盒內(nèi)，酶標(biāo)板加上
蓋或覆膜。
4. 未使用完的酶標(biāo)板或者試劑，請(qǐng)于2-8℃保存。標(biāo)準(zhǔn)品、生物素標(biāo)記抗體
工作液、辣根過(guò)氧化物酶標(biāo)記親和素工作液請(qǐng)依據(jù)所需的量配置使用。請(qǐng)
勿重復(fù)使用已稀釋過(guò)的標(biāo)準(zhǔn)品、生物素標(biāo)記抗體工作液、或辣根過(guò)氧化物
酶標(biāo)記親和素工作液。
5. 建議檢測(cè)樣品時(shí)均設(shè)雙孔測(cè)定，以保證檢測(cè)結(jié)果的準(zhǔn)確性。
洗板方法
手工洗板方法：吸去（不可觸及板壁）或甩掉酶標(biāo)板內(nèi)的液體；在實(shí)驗(yàn)臺(tái)上
鋪墊幾層吸水紙，酶標(biāo)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(guò)程數(shù)次。
自動(dòng)洗板：如果有自動(dòng)洗板機(jī)，應(yīng)在熟練使用后再用到正式實(shí)驗(yàn)過(guò)程中。
計(jì)算
請(qǐng)從我們的下載專業(yè)軟件"Curve Exert 1.3"，并根據(jù)提示制作標(biāo)準(zhǔn)曲線。
以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)（對(duì)數(shù)坐標(biāo)），OD 值為縱坐標(biāo)（普通坐標(biāo)），在半對(duì)
數(shù)坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線，根據(jù)樣品的OD 值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；
再乘以稀釋倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與OD 值計(jì)算出標(biāo)準(zhǔn)曲線的直線回歸方
程式，將樣品的OD 值代入方程式，計(jì)算出樣品濃度，再乘以稀釋倍數(shù)，即
為樣品的實(shí)際濃度。
注意事項(xiàng)
1. 本操作說(shuō)明適用于48T試劑盒， 48T試劑盒中酶聯(lián)板、標(biāo)準(zhǔn)品、生物素
標(biāo)記抗體及辣根過(guò)氧化物酶標(biāo)記親和素減半。
2. 當(dāng)混合蛋白溶液時(shí)應(yīng)盡量輕緩，避免起泡。
3. 洗滌過(guò)程非常重要，不充分的洗滌易造成假陽(yáng)性。
4. 一次加樣時(shí)間控制在5 分鐘內(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。
5. 請(qǐng)每次測(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線，做復(fù)孔。
6. 如標(biāo)本中待測(cè)物質(zhì)含量過(guò)高，請(qǐng)先稀釋后再測(cè)定，計(jì)算時(shí)請(qǐng)zui后乘以稀釋
倍數(shù)。
7. 在配制標(biāo)準(zhǔn)品、檢測(cè)溶液工作液時(shí)，請(qǐng)以相應(yīng)的稀釋液配制，不能混淆。
8. 底物請(qǐng)避光保存。
9. 不要用其它生產(chǎn)廠家的試劑替換試劑盒中的試劑。
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