本試劑盒只能用于科學研究，不得用于醫學診斷
人氧化低密度脂蛋白（OxLDL）ELISA 檢測試劑盒
使用說明書
檢測原理
試劑盒采用雙抗體夾心法酶聯免疫吸附試驗（ELISA）。往預先
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
入標本、標準品、HRP標記的檢測抗體，經過溫育并*洗滌。用底
物TMB顯色，TMB在過氧化物酶的催化下轉化成藍色，并在酸的作用
下轉化成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。用酶標儀在450nm 波長下測定吸光度（OD 值），
計算樣品濃度。
樣品收集、處理及保存方法
1. 血清：使用不含熱原和內毒素的試管，操作過程中避免任何細
胞刺激，收集血液后，3000 轉離心10 分鐘將血清和紅細胞迅速小心
地分離。
2. 血漿：EDTA、檸檬酸鹽或肝素抗凝。3000 轉離心30 分鐘取上清。
3. 細胞上清液：3000 轉離心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 轉離心10 分鐘
取上清。
5. 保存：如果樣本收集后不及時檢測，請按一次用量分裝，凍存
于-20℃，避免反復凍融，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
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
濃縮洗滌液會有結晶，這屬于正常現象，水浴加熱使結晶*溶解
后再使用。
2. 實驗中不用的板條應立即放回自封袋中，密封（低溫干燥）保
存。
3. 預處理后的樣本無需稀釋，直接取10μL 加樣即可。
4. 嚴格按照說明書中標明的時間、加液量及順序進行溫育操作。
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
微孔酶標板12 孔×8 條12 孔×4 條無
標準品0.3mL 0.3mL 無
*6mL 3mL 無
檢測抗體-HRP 10mL 5mL 無
20×洗滌緩沖液25mL 15mL 按說明書進行稀釋
底物A 6mL 3mL 無
底物B 6mL 3mL 無
終止液6mL 3mL 無
封板膜2 張2 張無
說明書1 份1 份無
自封袋1 個1 個無
注：標準品濃度依次為：800、400、200、100、50、0 ng/mL.
試劑的準備
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：甩盡孔內液體，每孔加滿洗滌液，靜置1min 后甩盡
孔內液體，在吸水紙上拍干，如此洗板5 次。
2. 自動洗板機：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. 設置標準品孔和樣本孔，標準品孔各加不同濃度的標準品50μ
L；
3. 待測樣本孔先加待測樣本10μL，再加*40μL；
4. 隨后標準品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標記
的檢測抗體100μL，用封板膜封住反應孔，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，如此重復洗板5 次（也可用洗板機洗板）。
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，在450nm 波長處測定各孔的
OD 值。
結果判斷
繪制標準曲線：在Excel 工作表中，以標準品濃度作橫坐標，對應
OD 值作縱坐標，繪制出標準品線性回歸曲線，按曲線方程計算各樣
本濃度值。
試劑盒性能
1. 準確性：標準品線性回歸與預期濃度相關系數R 值，大于等于
0.9900。
2. 靈敏度：zui低檢測濃度小于1.0 ng/mL。
3. 特異性：不與其它可溶性結構類似物交叉反應。
4. 重復性：板內變異系數小于10%、板間變異系數小于15%。
5. 貯藏：2-8℃，避光防潮保存。
6. 有效期：6 個月
免責聲明
1. 試劑盒僅供研究使用，不得用于臨床實驗或人體實驗，否則所
產生的一切后果，由實驗者承擔，本公司概不負責。
2. 嚴格按照說明書操作，實驗者違反說明書操作，后果由實驗者
承擔。
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!
