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上海寶葉生物科技有限公司
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- 蔣先生
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- 上海市嘉定區(qū)新源路155弄
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- www.shjsbio.com
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本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷
植物（Plant）脫鎂螯合酶（MDCase）ELISA檢測(cè)試劑盒
使用說(shuō)明書(shū)
檢測(cè)原理
試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)（ELISA）。往預(yù)
先包被脫鎂螯合酶（MDCase）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(guò)溫育并*洗滌。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脫鎂螯合酶（MDCase）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. 樣本不能含疊氮鈉（NaN3），因?yàn)榀B氮鈉（NaN3）是辣根過(guò)氧化
物酶（HRP）的抑制劑。
2. 標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行。
3. 植物萃取液或其它相關(guān)樣本：請(qǐng)1000 x g離心20分鐘，取上清即
可檢測(cè)。
4. 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. 濃度為0 的S0 號(hào)標(biāo)準(zhǔn)品即可視為陰性對(duì)照或者空白；按照說(shuō)明
書(shū)操作時(shí)樣本已經(jīng)稀釋5 倍，zui終結(jié)果乘以5 才是樣本實(shí)際濃度。
4. 嚴(yán)格按照說(shuō)明書(shū)中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作Materials supplied
Name 96 determinations 48 determinations
Microelisa stripplate 12*8strips 12*4strips
Standard 0.3ml*6tubes 0.3ml*6tubes
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 （S0 → S5） concentration was followed by: 0,3,6,12,24,48 nmol/L
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
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.
