人氧化低密度脂蛋白（OxLDL）ELISA 檢測(cè)試劑盒

【資料簡(jiǎn)介】

本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷

人氧化低密度脂蛋白（OxLDL）ELISA 檢測(cè)試劑盒

使用說(shuō)明書

檢測(cè)原理

試劑盒采用雙抗體夾心法酶聯(lián)免疫吸附試驗(yàn)（ELISA）。往預(yù)先

包被人氧化低密度脂蛋白（OxLDL）捕獲抗體的包被微孔中，依次加

入標(biāo)本、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測(cè)抗體，經(jīng)過溫育并*洗滌。用底

物TMB顯色，TMB在過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人氧化低密度脂蛋白

（OxLDL）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。

樣品收集、處理及保存方法

1. 血清：使用不含熱原和內(nèi)毒素的試管，操作過程中避免任何細(xì)

胞刺激，收集血液后，3000 轉(zhuǎn)離心10 分鐘將血清和紅細(xì)胞迅速小心

地分離。

2. 血漿：EDTA、檸檬酸鹽或肝素抗凝。3000 轉(zhuǎn)離心30 分鐘取上清。

3. 細(xì)胞上清液：3000 轉(zhuǎn)離心10 分鐘去除顆粒和聚合物。

4. 組織勻漿：將組織加入適量生理鹽水搗碎。3000 轉(zhuǎn)離心10 分鐘

取上清。

5. 保存：如果樣本收集后不及時(shí)檢測(cè)，請(qǐng)按一次用量分裝，凍存

于-20℃，避免反復(fù)凍融，在室溫下解凍并確保樣品均勻地充分解凍。

自備物品

1. 酶標(biāo)儀（450nm）

2. 高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL

3. 37℃恒溫箱

操作注意事項(xiàng)

1. 試劑盒保存在2-8℃，使用前室溫平衡20 分鐘。從冰箱取出的

濃縮洗滌液會(huì)有結(jié)晶，這屬于正?，F(xiàn)象，水浴加熱使結(jié)晶*溶解

后再使用。

2. 實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中，密封（低溫干燥）保

存。

3. 預(yù)處理后的樣本無(wú)需稀釋，直接取10μL 加樣即可。

4. 嚴(yán)格按照說(shuō)明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作。

5. 所有液體組分使用前充分搖勻。

試劑盒組成

名稱96 孔配置48 孔配置備注

微孔酶標(biāo)板12 孔×8 條12 孔×4 條無(wú)

標(biāo)準(zhǔn)品0.3mL 0.3mL 無(wú)

*6mL 3mL 無(wú)

檢測(cè)抗體-HRP 10mL 5mL 無(wú)

20×洗滌緩沖液25mL 15mL 按說(shuō)明書進(jìn)行稀釋

底物A 6mL 3mL 無(wú)

底物B 6mL 3mL 無(wú)

終止液6mL 3mL 無(wú)

封板膜2 張2 張無(wú)

說(shuō)明書1 份1 份無(wú)

自封袋1 個(gè)1 個(gè)無(wú)

注：標(biāo)準(zhǔn)品濃度依次為：800、400、200、100、50、0 ng/mL.

試劑的準(zhǔn)備

20×洗滌緩沖液的稀釋：蒸餾水按1：20 稀釋，即1 份的20×洗滌

緩沖液加19 份的蒸餾水。

洗板方法

1. 手工洗板：甩盡孔內(nèi)液體，每孔加滿洗滌液，靜置1min 后甩盡

孔內(nèi)液體，在吸水紙上拍干，如此洗板5 次。

2. 自動(dòng)洗板機(jī)：每孔注入洗液350μL，浸泡1min，洗板5 次。

操作步驟

1. 從室溫平衡20min 后的鋁箔袋中取出所需板條，剩余板條用自

封袋密封放回4℃。

2. 設(shè)置標(biāo)準(zhǔn)品孔和樣本孔，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50μ

L；

3. 待測(cè)樣本孔先加待測(cè)樣本10μL，再加*40μL；

4. 隨后標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標(biāo)記

的檢測(cè)抗體100μL，用封板膜封住反應(yīng)孔，37℃水浴鍋或恒溫箱溫

育60min。

5. 棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置1min，甩去

洗滌液，吸水紙上拍干，如此重復(fù)洗板5 次（也可用洗板機(jī)洗板）。

6. 每孔加入底物A、B 各50μL，37℃避光孵育15min。

7. 每孔加入終止液50μL，15min 內(nèi)，在450nm 波長(zhǎng)處測(cè)定各孔的

OD 值。

結(jié)果判斷

繪制標(biāo)準(zhǔn)曲線：在Excel 工作表中，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對(duì)應(yīng)

OD 值作縱坐標(biāo)，繪制出標(biāo)準(zhǔn)品線性回歸曲線，按曲線方程計(jì)算各樣

本濃度值。

試劑盒性能

1. 準(zhǔn)確性：標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù)R 值，大于等于

0.9900。

2. 靈敏度：zui低檢測(cè)濃度小于1.0 ng/mL。

3. 特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。

4. 重復(fù)性：板內(nèi)變異系數(shù)小于10%、板間變異系數(shù)小于15%。

5. 貯藏：2-8℃，避光防潮保存。

6. 有效期：6 個(gè)月

免責(zé)聲明

1. 試劑盒僅供研究使用，不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn)，否則所

產(chǎn)生的一切后果，由實(shí)驗(yàn)者承擔(dān)，本公司概不負(fù)責(zé)。

2. 嚴(yán)格按照說(shuō)明書操作，實(shí)驗(yàn)者違反說(shuō)明書操作，后果由實(shí)驗(yàn)者

承擔(dān)。

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Human oxidized lowdensity lipoprotein (OxLDL)

ELISA Kit instruction

Intended use

This OxLDL 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 OxLDL in the sample,

this OxLDL 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 OxLDL concentration. The

concentration of OxLDL in the samples is then determined by comparing the O.D.

of the samples to the standard curve.

Sample collection and storages

Serum - Use a serum separator tube and allow samples to clot for 30 minutes

before centrifugation for 10 minutes at approximay 3000×g. Remove serum and

assay immediay or aliquot and store samples at -20℃ or -80℃.Avoid repeated

freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge

samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store

samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates and other biological fluids - Remove particulates

by centrifugation and assay immediay or aliquot and store samples at -20℃or

-80℃. Avoid repeated freeze-thaw cycles.

Note: The samples shoule be centrifugated dequay and no hemolysis or

granule was allowed.

Materials required but not supplied

1. Standard microplate reader(450nm)

2. Precision pipettes and Disposable pipette tips.

3. 37 ℃ incubator

Precautions

1. Do not substitute reagents from one kit to another. Standard, conjugate and

microplates are matched for optimal performance. Use only the reagents supplied by

manufacturer.

2. Do not remove microplate from the storage bag until needed. Unused strips

should be stored at 2-8°C in their pouch with the desiccant provided.

3. Mix all reagents before using.

Remove all kit reagents from refrigerator and allow them to reach room temperature

( 20-25°C)

Materials supplied

Name 96 determinations 48 determinations

Microelisa stripplate 12*8strips 12*4strips

Standard 0.3ml 0.3ml

Sample diluent 6.0ml 3.0ml

HRP-Conjugate reagent 10.0ml 5.0ml

20X Wash solution 25ml 15ml

Chromogen Solution A 6.0ml 3.0ml

Chromogen Solution B 6.0ml 3.0ml

Stop Solution 6.0ml 3.0ml

Closure plate membrane 2 2

User manual 1 1

Sealed bags 1 1

Note: Standard concentration was followed by:

800、400、200、100、50、0 ng/mL.

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1. Prepare all r e a g e n t s before starting assay procedure. It is recommended that

all Standards and Samples be added in duplicate to the Microelisa Stripplate.

2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to

standard well.

3. Add Sample: Add testing sample 10μl Then add sample diluent 40μl to testing

sample well; Blank well doesn’t add anyting.

4. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip

and incubate for 60 minutes at 37°C.

5. Aspirate each well and wash, repeating the process four times for a total of five

washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle,

manifold dispenser or autowasher. Complete removal of liquid at each step is

essential to good performance. After the last wash, remove any remaining Wash

Solution by aspirating or decanting. Invert the plate and blot it against clean paper

towels.

6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well.

Gently mix and incubate for 15 minutes at 37°C. Protect from light.

7. Add 50μl Stop Solution to each well. The color in the wells should change

from blue to yellow. If the color in the wells is green or the color change does not

3. appear uniform, gently tap the plate to ensure thorough mixing.

8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader

within 15 minutes.

Calculation of results

1. This standard curve is used to determine the amount in an unknown sample.

The standard curve is generated by plotting the average O.D. (450 nm)

obtained for each of the six standard concentrations on the vertical (Y) axis

versus the corresponding concentration on the horizontal (X) axis.

2. First, calculate the mean O.D. value for each standard and sample. All O.D.

values, are subtracted by the mean value of the zero standard before result

interpretation. Construct the standard curve using graph paper or statistical

software.

3. To determine the amount in each sample, first locate the O.D. value on the

Y-axis and extend a horizontal line to the standard curve. At the point of

intersection, draw a vertical line to the X-axis and read the corresponding

concentration.

4. Any variation in operator, pipetting and washing technique, incubation time or

temperature, and kit age can cause variation in result. Each user should obtain

their own standard curve.

5. The sensitivity by this assay is 1.0 ng/mL.

6. Standard curve

Storage： 2-8℃.

validity： six months.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR

DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH

ENTIRE PROCEDURE BEFORE BEGINNING!