E-EL-H0043

scw.产品编号：E-EL-H0043

（本试剂盒仅供体外研究使用、不用于临床诊断!）

声明：尊敬的客户，感谢您选用本公司的产品。本产品选用世界著名生产厂家的原料，采用

专业ELISA kit生产技术制造。适用于体外定量检测人血清、血浆或细胞培养上清液中天然和重组CRP浓度。使用前请仔细阅读说明书并检查试剂组分！如有疑问，请及时联系伊莱瑞特生物科技有限公司。

试剂盒组成： 名称-中文 ELISA酶标板 冻干标准品 标准品&样品稀释液 浓缩生物素化抗体 生物素化抗体稀释液 名称-英文 Micro ELISA Plate Reference Standard Reference Standard & Sample Diluent Concentrated Biotinylated Detection Ab Diluent for Biotinylated Detection Ab 规格 8×12 / 8×6* 2/ 1支* 1瓶 20mL/12mL* 1支 120μL/70μL* 1瓶 10mL/6mL* 1支 120μL/70μL* 1瓶 10mL/6mL* 1瓶 30mL/16mL* 1瓶 10mL/6mL* 1瓶 10mL/6mL* 5/3张* 1份 保存条件 4℃ 4℃ 4℃ 4℃ 4℃ 4℃(避光) 4℃ 4℃ 4℃(避光) 4℃ 浓缩HRP酶结合物 Concentrated HRP Conjugate 酶结合物稀释液 Diluent for HRP Conjugate 浓缩洗涤液（25×） Concentrated Wash Buffer （25×） 底物溶液（TMB） 反应终止液 封板覆膜 产品说明书 Substrate Reagent Stop Solution Plate Sealer Product Description *: [96T/48T]（打开包装后请及时检查所有物品是否齐全完整）

检测原理:

本试剂盒采用双抗体夹心ELISA法。用抗人CRP抗体包被于酶标板上，实验时标本或标准品中的

CRP会与包被抗体结合，游离的成分被洗去。依次加入生物素化的抗人CRP抗体和辣根过氧化物酶标记的亲和素。抗人CRP抗体与结合在包被抗体上的人CRP结合、生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去。加入显色底物，TMB在辣根过氧化物酶的催化下现蓝色，加终止液后变黄。用酶标仪在450nm波长处测OD值，CRP浓度与OD450值之间呈正比，通过绘制标准曲线求出标本中CRP的浓度。

wwienc使用说明书

e.com1. 血清：全血标本于室温放置2小时或4℃过夜后于1000×g离心20分钟，取上清即可检测，收集

血液的试管应为一次性的无热原，无内毒素试管。

2. 血浆：抗凝剂推荐使用EDTA，标本采集后30分钟内于1000×g离心15分钟，取上清即可检测。

避免使用溶血，高血脂标本。

3. 其它生物标本：1000×g离心20分钟，取上清即可检测 4. 标本应清澈透明，悬浮物应离心去除。

5. 标本收集后若不及时检测，请按一次使用量分装，冻存于-20℃/-80℃电冰箱内，避免反复冻

融，1-6月内检测,4℃保存的应在1周内进行检测。

6. 如果您的样本中检测物浓度高于标准品最高值，请根据实际情况，做适当倍数稀释(建议先做

预实验，以确定稀释倍数)。

试验所需自备物品:

1. 酶标仪(450nm波长滤光片)

2. 高精度移液器，EP管及一次性吸头：0.5-10μL, 2-20μL, 20-200μL, 200-1000μL 3. 37℃恒温箱, 双蒸水或去离子水 4. 吸水纸

检测前准备工作:

1. 请提前20分钟从冰箱中取出试剂盒，平衡至室温。

2. 将浓缩洗涤液用双蒸水稀释(1:25)。未用完的放回4℃。从冰箱中取出的浓缩洗涤液可能有结

晶，属于正常现象，可用40℃水浴微加热使结晶完全溶解后再配制洗涤液。（加热温度不要超过50℃）使用时洗涤液应为室温。

3. 标准品: 加入标准品&样品稀释液1.0mL至冻干标准品中，静置10分钟，待其充分溶解后，

轻轻混匀(浓度为50ng/mL)。然后根据需要进行倍比稀释（注：不要直接在板中进行倍比稀释）。建议配制成以下浓度： 50、25、12.5、6.25、3.13、1.56、0.78、0 ng/mL ，样品稀释液直接作为空白孔0ng/mL。如配制25ng/mL标准品：取0.5mL 50ng/mL的上述标准品加入含有0.5mL样品稀释液的EP管中，混匀即可，其余浓度依此类推。

4. 生物素化抗体工作液：实验前计算当次实验所需用量（以100μL/孔计），实际配制时应多配

制100-200μL。使用前15分钟，以生物素化抗体稀释液稀释浓缩生物素化抗体(1:100)成工作浓度。当日使用。

5. 酶结合物工作液：实验前计算当次实验所需用量（以100μL/孔计），实际配制时应多配制

100-200μL。使用前15分钟，以酶结合物稀释液稀释浓缩HRP酶结合物(1:100)成工作浓度。当日使用。

www.elab标本收集:

science.com标准品稀释方法图例：(以500μL/管为例，也可根据实际用量来稀释，如200μL/管)

elabscience.com

www.50

25 12.5 6.25 3.13 1.56 0.78 0 ng/mL

洗涤方法:

1. 自动洗板机：每孔加入洗涤液350μL，注入与吸出间隔60秒。

2. 手工洗板：甩尽孔内液体，在洁净的吸水纸上拍干，每孔加洗涤液350μL，浸泡1-2分钟，吸

去（不可触及板壁）或甩掉酶标板内的液体，在厚的吸水纸上拍干。

操作步骤:

实验开始前，各试剂均应平衡至室温；试剂或样品配制时，均需充分混匀，并尽量避免起泡。

1. 加样：分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液100μL，余孔分别加标

准品或待测样品100μL，注意不要有气泡，加样时将样品加于酶标板底部，尽量不触及孔壁，轻轻晃动混匀。给酶标板覆膜，37℃孵育90分钟。为保证实验结果有效性，每次实验请使用新的标准品溶液。

2. 弃去液体，甩干，不用洗涤。每个孔中加入Detection Ab工作液 100μL（在使用前15分

钟内配制），酶标板加上覆膜，37℃温育1小时。

3. 弃去孔内液体，甩干，洗板 3次，每次浸泡1-2分钟，大约350μL/每孔，甩干并在吸水

纸上轻拍将孔内液体拍干。

4. 每孔加HRP Conjugate工作液（临用前15分钟内配制）100μL，加上覆膜，37℃温育30

分钟。

5. 弃去孔内液体，甩干，洗板5次，方法同步骤3。

6. 每孔加底物溶液100μL，酶标板加上覆膜37℃避光孵育15分钟左右（根据实际显色情况

酌情缩短或延长，但不可超过30分钟。当标准孔出现明显梯度时，即可终止）。

7. 每孔加终止液50μL，终止反应，此时蓝色立转黄色。终止液的加入顺序应尽量与底物溶

液的加入顺序相同。

8. 立即用酶标仪在450nm波长测量各孔的光密度（OD值）。应提前打开酶标仪电源，预热

仪器，设置好检测程序。

9. 实验完毕后将未用完的试剂按规定的保存温度放回冰箱保存至有效期结束。

1. 保存：试剂盒中各试剂请按说明书提示合理存放。在储存及温育过程中避免将试剂暴露在强

任何影响。

3. 加样：加样或加试剂时，第一个孔与最后一个孔的加样时间间隔如果太大，将会导致不同的

“预温育”时间，从而明显地影响到测量值的准确性及重复性。每次的加样时间最好控制在10分钟内。推荐设置复孔。

4. 温育：为防止样品蒸发，实验时必须给酶标板覆膜；洗板后应尽快进行下步操作，避免酶标

板处于干燥状态；严格遵守给定的温育时间和温度。

5. 洗涤：洗涤过程中反应孔中残留的洗涤液应在吸水纸上拍干，勿将滤纸直接放入反应孔中吸

水。在读数前要注意清除底部残留的液体和手指印，以免影响酶标仪读数。

6. 试剂配制：Detection Ab及HRP Conjugate体积较小，运输过程会使液体沾到管壁或瓶盖，因

此使用前请手甩几下或1000转/分离心1min，以使附着管壁或瓶盖的液体沉积到管底。取用前，请用移液器小心吹打4-5次使溶液混匀。标准品、Detection Ab工作液、HRP Conjugate工作液请根据所需用量配制，并使用相应的稀释液配制，不能混淆。请精确配制标准品及工作液，尽量不要微量配制（如吸取Detection Ab时，一次不要小于10μL），以避免由于不准确稀释而造成浓度误差；请勿重复使用已稀释过的标准品、Detection Ab工作液、HRP Conjugate工作液。若需要分次使用标准品应按照每一次用量分装，将其放在-20～-80℃贮存。避免反复冻融。

7. 显色时间的控制：加入底物后请定时观察反应孔的颜色变化（比如每隔5分钟），如梯度已很

明显，请提前加入终止液终止反应，避免颜色过深影响酶标仪读数。 8. 底物：底物请避光保存，在储存和温育时避免强光直接照射。

9. 混匀：充分轻微混匀对反应结果尤为重要，最好使用微量振荡器(使用最低频率)，如无微

量振荡器，可在反应前手工轻轻敲击酶标板框混匀。

10. 安全：试验中请穿着实验服并带乳胶手套做好防护工作。特别是检测血液或者其他体液标

本时，请按国家生物试验室安全防护条例执行。

11. 不同批号的试剂盒组份不能混用（洗涤液和反应终止液除外）

12. 试验中所用的EP管和吸头均为一次性使用，严禁混用，否则将影响试验结果！

ww2. 酶标板：刚开启的酶标板孔中可能会有少许水样物质，此为正常现象，不会对实验结果造成

w.光中。所有试剂瓶盖须旋紧以防止蒸发和微生物的污染，否则可能会出现错误的结果。

elab注意事项：

science.com曲线查出相应的浓度，乘以稀释倍数；亦可将样品的OD值代入标准曲线的拟合方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。

3. 若标本OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。

灵敏度、检测范围、特异性和重复性:

● 灵敏度：最小可测0.46ng/mL。 ● 检测范围：0.78 – 50ng/mL。

● 特异性：可同检测重组或天然的人CRP，且与其它相关蛋白无交叉反应。 ● 重复性：板内，板间变异系数均<10%。

问题分析 问题描述 标准曲线梯度差 可能原因 吸液或加液不准 标准品稀释不正确 洗涤不完全 显色很弱或无色 孵育时间太短 实验温度不正确 试剂体积不够或漏加 稀释不正确 读数数值低 酶标仪设置不正确 曲线偏差大 背景值高 加液不正确 检测抗体的工作浓度过高 酶标板洗涤不完全 洗液有污染 灵敏度低 ELISA试剂盒保存不当 读数前未终止 在酶标仪上检查波长及滤光片设置 提前打开酶标仪预热 检查加液情况 使用推荐的稀释倍数 重新阅读操作手册，保证清洗完全；如果用自动洗板机，请检查所有的出口是否有堵塞 配制新鲜的洗液 按说明书要求保存相关试剂 OD读数前应在每孔中加入终止液 相应对策 检查移液器及吸头 溶解标准品时稍微旋转瓶身，轻轻混匀使粉末完全溶解 保证洗涤时间和洗涤次数及每孔的加液量 保证充足的孵育时间 使用推荐的实验温度 检查吸液及加液过程，保证所有试剂按顺序足量添加

www.结果判断:

1. 每个标准品的OD值减去空白孔的OD值后作图，如设置复孔，则应取其平均值计算。以标准

品的浓度为横坐标，OD值为纵坐标，绘出标准曲线。

2. 推荐使用专业的曲线制作软件，如curve expert 1.3，在软件界面既可根据样品OD值，由标准

elabscience.com说明

1. 限于现有条件及科学技术水平，尚不能对所有原料进行全面的鉴定分析，本产品可能存在

一定的质量技术风险。

2. 最终的实验结果与试剂的有效性、实验者的相关操作以及当时的实验环境密切相关，请

务必准备充足的待测样品。

3. 只有全部使用ElabTM试剂才能保证检测效果，不能混用其他制造商的产品。只有严格遵守

ElabTM试剂的实验说明才会得到最佳的检测结果。 4. 有效期：6个月。

5. 本操作说明同样适用于48T试剂盒。

www.elabscience.comelabHuman CRP (C-Reactive Protein) ELISA Kit

Product Description

Catalog No: E-EL-H0043

（FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGONOSIS !）

Dear customer, Thank you for choosing our products. This product is produced using raw materials from world-renowned manufacturer, and professional manufacturing technology of ELISA kits. Please read the instructions carefully before use and check all the reagent compositions! If in doubt, please contact Elaboratescience Biotechnology Co., Ltd.

Intended use

This immunoassay kit allows for the in vitro quantitative determination of Human CRP concentrations in serum, plasma and other biological fluids.

Kit Components： Item Micro ELISA Plate Reference Standard Reference Standard & Sample Diluent Concentrated Biotinylated Detection Ab Diluent for Biotinylated Detection Ab Concentrated HRP Conjugate Diluent for HRP Conjugate Concentrated Wash Buffer （25×） Substrate Reagent Stop Solution Plate Sealer Product Description *: [96T/48T]

Specifications 8×12 or 8×6 * 2/ 1vial * 1vial 20mL/12mL * 1via l 120μL /70μL * 1vial 10mL/6mL* 1vial 120μL /70μL * 1vial 10mL/6mL * 1vial 30mL/16mL * 1vial 10mL/6mL * 1vial 10mL/6mL * 5/3pieces * 1 copy Storage 4°C 4°C 4°C 4°C 4°C 4°C (shading light) 4°C 4°C 4°C (shading light) 4°C www.science.comThis ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to CRP Standards or samples are then added to the appropriate micro ELISA plate wells and combined to the specific antibody. Then a biotinylated detection antibody specific for CRP and Avidin-Horseradish Peroxidase (HRP) conjugate is added to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain CRP, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color turn yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of CRP. You can calculate the concentration of CRP in the samples by comparing the O.D. of the samples to the standard curve.

Sample collection and storage

Serum - Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000×g. Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.

Other biological fluids –Centrifuge samples for 20 minutes at 1000×g at 2 - 8°C. Collect the supernatant and carry out the assay immediately.

Sample preparation – Samples should be clear and transparent and be centrifuged to remove suspended solids.

Note: Serum and plasma to be used within 7 days when stored at 2-8°C, otherwise samples must be divided and stored at -20°C (≤1 month) or -80°C (≤6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. If the sample concentration is higher than the maximum standard value, please dilute it with appropriate factor according to the actual situation. (A pre-test is recommended to determine the dilute factor)

Other supplies required

Microplate reader with 450nm wavelength filter

High-precision transferpettor, EP tubes and disposable pipette tips 37°C Incubator, Deionized or distilled water. Absorbent paper

www.elabTest principle

science.comBring all reagents to room temperature before use.

or distilled water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely dissolved. The solution should be cooled to room temperature before use. Standard - Reconstitute the Standard with 1.0mL of Sample Diluent, let it stand for 10minutes until it dissolved fully. This reconstitution produces a stock solution of 50ng/mL. Then make serial dilutions as needed (Making serial dilution in the wells directly is not permitted). The recommended concentrations are as follows: 50、25、12.5、6.25、3.13、1.56、0.78、0ng/mL . As if you want to make standard solution at the concentration of 25ng/mL, you can take 0.5mL the standard at 50ng/mL, add it to an EP tube with 0.5mL sample dilution, and mix it. The procedures of making the remaining concentrations are all the same. The undiluted standard serves as the highest standard (50ng/mL). The Sample Diluent serves as the zero (0ng/mL).

(500μL/tube，for example. Can also be diluted according to the actual amount，such as 200μL/tube)

www.Wash Buffer - Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized

elabReagent preparation

science.com50

25 12.5 6.25 3.13 1.56 0.78 0 ng/mL

Biotinylated Detection Ab —Calculate the required amount before experiment (100μL /well). In actual preparation you should prepare 100~200μL more. Dilute the concentrated Biotinylated Detection Ab to the working concentration using Diluent for Biotinylated Detection Ab (1:100).

Concentrated HRP Conjugate—Calculate the required amount before experiment (100μL/well). In actual preparation you should prepare 100~200μL more. Dilute the Concentrated HRP Conjugate to the working concentration using Diluent for Concentrated HRP Conjugate (1:100).

1. Automated washer: add 350μL wash buffer into each well, the interval between injection and

suction should be set about 60s.

2. Manual wash: add 350μL wash buffer into each well, soak it for 1~2minutes, suck(no inside wall

touching) or get rid of liquid within the micro ELISA plate and pat it dry on thick clean absorbent paper.

Assay procedure

Allow all reagents to reach room temperature All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming.

1. Add Sample: Add 100μL of Standard, Blank, or Sample per well. The blank well is added with

sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming to the best of your ability. Mix it gently. Cover the plate with sealer we provided. Incubate for 90 minutes at 37°C.

2. Biotinylated Detection Ab: Remove the liquid of each well, don’t wash. Immediately add 100μL

of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C.

3. Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well

with Wash Buffer (approximately 350μL) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. 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 pat it against thick clean absorbent paper.

4. HRP Conjugate : Add 100μL of HRP Conjugate working solution to each well. Cover with the

Plate sealer. Incubate for 30 minutes at 37°C.

5. Wash: Repeat the wash process for five times as conducted in step 3.

6. Substrate: Add 100μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate

for about 15 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.

7. Stop: Add 50μL of Stop Solution to each well. Color turn to yellow immediately. The adding order

of stop solution should be as the same as the substrate solution.

8. OD Measurement: Determine the optical density (OD value) of each well at once, using a

microplate reader set to 450nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.

9. After experiment, put all the unused reagents back into the refrigerator according to the specified

storage temperature respectively until their expiry.

www.elabWashing Procedure:

science.com1. Storage: All the reagents in the kit should be stored following the instructions. Exposure of

reagents should be tightened to prevent evaporation and microbial contamination, or erroneous results may occur.

2. ELISA Plate: Little water-like substance may appear in the ELISA Plate just opened, this is

normal and will not have any impact on the experiment results.

3. Add Sample: The interval of sample adding between the first well and the last well should not be

too long, otherwise will cause different pre-incubation time, which will significantly affect the experiment’s accuracy and repeatability. The interval controlled within 10minutes is good. Parallel measurement is recommended.

4. Incubation: To prevent evaporation, proper adhesion of plate sealers during incubation steps is

necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Do not let the strips dry at any time during the assay. Strict compliance with the given incubation time and temperature.

5. Washing: The wash procedure is critical. Insufficient washing will result in poor precision and

falsely elevated absorbance readings Residual liquid in the reaction wells should be pat dry against absorbent paper in the washing process. But don’t put absorbent paper into reaction wells directly. Note that clear the residual liquid and fingerprint in the bottom before measurement, so as not to affect the microtiter plate reader.

6. Reagent Preparation: As the volume of Detection Ab and HRP Conjugate is very small, liquid

may adhere to the tube wall or tube cap when being transported. You better hand-throw it or centrifugal it for 1 minute at 1000rpm. Please pipette the solution for 4-5 times before pippeting. Please carefully reconstitute Standards, working solutions of Detection Ab and HRP Conjugate according to the instructions. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting. Do not reuse standard solution, working solution of Detection Ab and HRP Conjugate, which have been diluted. If you need to use standard repeatedly, you can divide the standard into small pack according to the amount of each assay, keep them at -20～-80°C and avoid repeated freezing and thawing. 7. Reaction Time Control: Please control reaction time strictly following this product description! 8. Substrate: Substrate Solution is easily contaminated. Please protect it from light.

9. Mixing: You’d better use microoscillator at the lowest frequency, as sufficient and gentle mixing

is particularly important to reaction result. If there is no microsocillator available, you can knock the ELISA plate frame gently with your finger before reaction.

www.reagents to strong light should be avoided in the process of incubation and storage. All the taps of

elabImportant Note:

science.comblood or other body fluid, please perform following the national security columns of biological laboratories. exception)

11. Do not use component from different batches of kit(washing buffer and stop solution can be an 12. To avoid cross-contamination, change pipette tips between adding of each standard level, between

sample adding, and between reagent adding. Also, use separate reservoirs for each reagent. Otherwise, the results will be inaccurate!

Calculation of results

Average the duplicate readings for each standard and samples and subtract the average zero standard optical density. Create a standard curve by plotting the mean OD value 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. It is recommended to use some professional software to do this calculation, such as curve expert 1.3. In the software interface, a best fitting equation of standard curve will be calculated using OD values and concentrations of standard sample. The software will calculate the concentration of samples after entering the OD value of samples. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper limit of the standard curve, you should re-test it after appropriate dilution. The actual concentration is the calculated concentration multiplied dilution factor.

Sensitivity

The minimum detectable dose of Human CRP is 0.46ng/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).

Detection Range

0.78 – 50 ng/mL.

Specificity

This kit recognizes recombinant and natural Human CRP. No significant cross-reactivity or interference was observed.

Repeatability:

Coefficient of variation were<10%

www.elab10. Security: Please wear lab coats and latex gloves for protection. Especially detecting samples of

science.comProblem elabTroubleshooting Poor standard curve Causes sciencSolutions Check pipettes Ensure briefly spin the vial of standard and dissolve the powder thoroughly by a gentle mix. Wells not completely aspirated Low signal Too brief incubation times Incorrect assay temperature Inadequate reagent volumes Improper dilution Deep color but low value Large CV High background Plate reader settings not optimal Inaccurate pipetting Concentration of detector too high Plate is insufficiently washed Contaminated wash buffer Low sensitivity Improper storage of the ELISA kit Stop solution Stop solution not added Verify the wavelength and filter setting in the plate reader. Open the Plate Reader ahead to pre-heat Check pipettes Use recommended dilution factor. Review the manual for proper wash. If using a plate washer, check that all ports are unobstructed. Make fresh wash buffer All the reagents should be stored according to the instructions Stop solution should be added to each well before measurement Completely aspirate wells between steps. Ensure sufficient incubation time; Use recommended incubation temperature. Bring substrate to room temperature before use. Check pipettes and ensure correct preparation Declaration:

1. Limited by the current conditions and scientific technology, we can't completely conduct the

comprehensive identification and analysis on all the raw material provided. So there might be some qualitative and technical risks for users using the kit.

2. The final experimental results will be closely related to validity of the products, operation skills of the operators and the experimental environments. Please make sure that sufficient samples are available.

3. To get the best results, please only use the reagents supplied by the manufacturer and strictly comply with the instructions in the description! 4. Valid period: 6 months.

5. This description is also suitable for 48T kit.

www.Inaccurate pipetting Improper standard dilution e.com