ELISA操作指南

为让中国用户能够更好的熟悉和掌握PeproTech的ELISA试剂盒及其技术，PeproTech专门制作了"ELISA操作指南视频 "，供各位老师和同学观摩！

视频网址为 ：http://v.youku.com/v_show/id_XMzQ3Mzk3NTg4.html  或 http://www.tudou.com/programs/view/tzU8m93o0tI/ ，欢迎大家的批评和指正！

该视频为英文讲解，为便于大家理解，请看下面的英文原文及翻译 ：

The following video tutorial described how to run a sandwich ELISA, also known as enzyme-linked immunosorbent assay, using a general PeproTech protocol.
       接下来的实验指导讲述PeproTech夹心法ELISA，即酶联免疫吸附试验的通用步骤

The capture, standard, and detection are supplied as stable lyophilized products, and should be stored at -20℃ until ready for use.
       捕获抗体、标准品和检测抗体均为冻干粉，使用前应保存于-20℃

Reconstituted Capture, Standard, and Detection components are only guaranteed to be stable for up to 2 weeks when stored at 4 ℃.
       捕获抗体、标准品和检测抗体重悬后，在4℃时最长保存2周。

If you have reconstituted the EDK, and plan on using it for a duration greater than two weeks, aliquot and store at -20℃ for up to 6 months.
       如果您已重悬了EDK的各组份，并准备在2周之后使用，请将重悬的组分分装并冻存于-20℃，最长可保存6个月。

In contrast to the other three EDK components, the Avidin-HRP vial comes ready to use
       与EDK的其它三个组分不同，Avidin-HRP为即用型

In order to avoid harmful repeated freeze/thaw cycles, or long-term storage at 4 ℃ w

hich mean advisory functionality. Aliquot the Avidin-HRP into ten 6uL vials upon receipt and stored at -20 ℃ are stable for up 2 years from the date of receipt.
       目的是避免反复冻融或长期4℃保存对该组分的功能损害。收到Avidin-HRP后，立即将其分装为6ul/管，共10管，冻存于-20℃，最长可保存2年

Phase 1:
       第1阶段：

Coating the plate with capture antibody
       捕获抗体包板

Centrifuge the vial briefly to bring the capture antibody to the bottom
       将捕获抗体稍作离心，使抗体集中于管底

Reconstitute the capture body in sterile water to the concentration specified on the datasheet. And allow the vial to be reconstituted for minimum 10 minutes.
       用无菌水将捕获抗体重悬至说明书上要求的浓度，静置10分钟以使抗体完全溶解

Centrifuge the reconstituted vial for 3 minutes at maximum speed
       重悬的抗体以最高速度离心3分钟

Dilute the capture antibody in 1×PBS to the concentration specified on the datasheet.
       用1xPBS稀释捕获抗体至说明书上要求的浓度

Gently mix or vortex the vial, try to ensure the air bubbles don’t mix into the solution,
       轻轻颠倒或振荡混匀，一定不要产生气泡

Immediately add 100ul of the capture antibody solution into the ELISA plate wells.
       立即在每个ELISA板孔中加入100uL捕获抗体

Press firmly to seal the plate, take care not to let the reagent splash on the film
       将封板膜用力压盖在ELISA板上，注意不要使抗体滴溅在封板膜上

Incubated the plate overnight at 25 ℃, alternatively the incubation for this phase can be done at 37℃ for 2-4 hours
       25 ℃孵育过夜，或者37℃孵育2-4个小时

To wash the plate, discard the liquid and blot on a clean paper towel,
       洗板时需将液体倒掉，并在干净的吸水纸上拍干

Add 300ul of the wash buffer to every well and then aspirate the plate
       每孔加入300ul 洗液，然后再将液体吸除

Repeat the step three additional times, totaling four washes in all.
       该步骤再重复3次，总共洗涤4次

After the last wash, invert the plate to move liquid, and blot on a clean paper towel.
       最后一次洗涤后，将板倒置以去除液体，并在干净的吸水纸上拍干

There are several other methods to wash an ELISA plate.Whatever you choose, be consistent with your washing technique throughout the whole process.
       ELISA板还有其它几种洗涤方法。无论使用哪种方法，请在整个ELISA实验过程中保持一致

Phase 2: Blocking non-specific binding
       第二阶段：封闭非特异性结合

Bovine serum albumin is used as the blocking reagent to block any unbound open sites within the plastic wells
       牛血清白蛋白作为封闭试剂，可封闭塑料孔内任何未结合蛋白的位点

Add 300ul of the blocking buffer to each well.
       每孔中加入300ul封闭液

Seal the plate and incubate for at least 1 hour at 25 ℃.
       封板，并在25 ℃孵育至少1小时

Phase 3:
       第3阶段：

Specific binding of antigen
       抗原的特异性结合

Priority to the end of the previous incubation period
       在上一孵育过程结束前

Centrifuge the standard vial briefly
       将标准品稍作离心

And reconstitute to the specified concentration in sterile water.
       并用无菌水重悬至要求的浓度

Allow the vial to reconstitute for minimum 10 minutes.
       静置至少10分钟，以使标准品完全溶解

When the incubation period is over, remove the plate and wash four times
       重悬结束后，取出板并洗涤4次

Centrifuge the reconstituted standard vial for 3 minutes at maximum speed
       重悬的标准品以最高速度离心3分钟

Dilute to the specified concentration, and gently mix or vortex the solution.
       稀释到所需浓度，轻轻颠倒或振荡混匀

The standard curve is the area on the plate dedicate to fixed concentrations of our protein standard
       标准曲线在ELISA板上的区域专用于检测已知浓度的蛋白标准品

If using a multi-channel pipette, firstly add 100ul of the diluent to all standard curve wells except for the first row
       如果使用多道移液器，除第一列外的所有标准曲线孔中均加入100ul稀释液

Next, add 200ul of standard solution to the first row of standard curve wells.
       然后，在标准曲线孔的第一列中加入200ul标准品溶液

Dilute 1:2 down the plate to a minimum of six concentrations
       依次倍比稀释，标准品至少有6个浓度

Each standard curve well should contain the final volume of 100ul of the solvent.
       每个标准曲线孔中液体的终体积应为100ul

Using the minimum six concentrations in the triplicate, plus a minimum six wells of blanks containing only diluent.
       标准品至少有6个浓度，每个浓度为3个复孔，再设6个空白孔，其中仅含稀释液

These blanks are essential and later determining the efficacy and ability of the measurements of the samples.
       空白孔是必须的，它们将用来评价试剂盒检测样本的效能

Next, add your specific samples of interest in triplicate to the remaining wells
       然后，在剩余孔中加入待测样本，每个样本设3个复孔

Depending on the concentration of the analyte, dilution may be necessary for optimal results
       样本中若待检抗原的浓度过高，要得到最佳结果可能需对样本进行稀释

Seal the plate and incubate for two hours at 25 ℃.
       封板，25 ℃孵育2小时

Phase 4:
       第4阶段：

Sandwich formation via the addition of biotinylated detection antibody.
       加入生物素标记的检测抗体以形成夹心

Priority to the end of the incubation, centrifuge briefly and reconstitute the detection antibody for ten minutes in sterile water.
       孵育结束前，稍离心检测抗体，并用无菌水重悬，静置10分钟

When the incubation is over, remove the plate and wash four times.
       孵育结束后，取出板，洗涤4次

Centrifuge for 3 minutes at maximum speed
       最高速度离心3分钟

And dilute the detection antibody in diluent to the specified concentration.
       用稀释液将检测抗体稀释至要求的浓度

Gently mix the solution and immediately add 100ul per well
       轻轻混匀，立即在每孔中加入100ul检测抗体液

Seal the plate and incubate for 2 hours at 25 ℃
       封板，25 ℃孵育2小时

Phase 5:
       第5阶段：

Addition of the enzyme-linked Avidin-HRP to the sandwich
       夹心上加酶联亲合素(Avidin-HRP)

While the plate is incubating, remove one 6ul aliquot of Avidin-HRP from the freezer and let it thaw.
       板在孵育过时，从冰箱中取出一管Avindin-HRP (6ul)，使其融化

When the incubation is over, wash the plate 4 times
       板孵育结束后，洗涤4次

Dilute the Avidin-HRP 1:2000 in diluent
       用稀释液1:2000稀释Avidin-HRP

Gently mix the solution and immediately add 100ul per well.
       轻轻混匀，立即向每孔中加入100ul

Seal the plate and incubate for 30 minutes at 25 ℃.
       封板，25 ℃孵育30分钟

To reduce the background interference, ensure the incubation is for 30 minutes only.
       为降低背景干扰，请确保孵育时间正好为30分钟

Phase 6: Conversion of the colorless substrate into a colored solution.
       阶段6：底物显色

Promptly washed the plate after incubation and add 100ul of ABTS substrate solution to each well.
       孵育结束后，立即洗板。每孔加入100ul ABTS底物溶液

Use an Avidin-HRP in conjunction with ABTS only.
       Avidin-HRP仅可与ABTS配合使用

Using it in conjunction with TMB or other substrate solutions, mainly to a dramatic rise in background development
       如与TMB或其它底物溶液配合使用，会显著提高背景显色

Incubate at room temperature for color development
       室温孵育显色

Monitor the plate at 5-minute intervals for up to 60 minutes.
       每隔5分钟读板一次，最长可监测60分钟

Using a ELISA plate reader set at appropriate wavelength correction for your selected plate
       在酶标仪上设置合适的校正波长

OD readings will vary depending on the specific EDK and its reading time provided on the product datasheet.
       EDK不同，以及说明书上所要求的读板时间不同均可能导致OD值的差异

美国PeproTech (派普泰克) 公司是世界上领先的重组细胞因子和蛋白生产商，同时也生产多种细胞因子和蛋白的ELISA法检测试剂盒，详细信息请查看www.peprotech.com ，或与PeproTech中国代表处 (电话：0512-6832 5993，Email：china@peprotechasia.com ) 联系。