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zui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℃，zui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℃，zui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*溶解

Centrifuge the reconstituted vial for 3 minutes at maximum speed
       重悬的抗体以zui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

Immediay 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.
       zui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

详细请看：ELISA操作指南