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。
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 ℃ which 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.
ELISA板还有其它几种洗涤方法。
Whatever you choose, be consistent with your washing technique throughout the whole process.
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