The following video tutorial described how to run a sandwich ELISA, also known as enzyme-linked immunosorbent assay, using a general PeproTech protocol.
接下來的實(shí)驗(yàn)指導(dǎo)講述PeproTech夾心法ELISA，即酶聯(lián)免疫吸附試驗(yàn)的通用步驟
The capture, standard, and detection are supplied as stable lyophilized products, and should be stored at -20℃ until ready for use.
捕獲抗體、標(biāo)準(zhǔn)品和檢測(cè)抗體均為凍干粉，使用前應(yīng)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 ℃.
捕獲抗體、標(biāo)準(zhǔn)品和檢測(cè)抗體重懸后，在4℃時(shí)zui長保存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.
如果您已重懸了EDK的各組份，并準(zhǔn)備在2周之后使用，請(qǐng)將重懸的組分分裝并凍存于-20℃，zui長可保存6個(gè)月。
In contrast to the other three EDK components, the Avidin-HRP vial comes ready to use
與EDK的其它三個(gè)組分不同，Avidin-HRP為即用型
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.
目的是避免反復(fù)凍融或長期4℃保存對(duì)該組分的功能損害。收到Avidin-HRP后，立即將其分裝為6ul/管，共10管，凍存于-20℃，zui長可保存2年
Phase 1:
第1階段：
Coating the plate with capture antibody
捕獲抗體包板
Centrifuge the vial briefly to bring the capture antibody to the bottom
將捕獲抗體稍作離心，使抗體集中于管底
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.
用無菌水將捕獲抗體重懸至說明書上要求的濃度，靜置10分鐘以使抗體*溶解
Centrifuge the reconstituted vial for 3 minutes at maximum speed
重懸的抗體以zui高速度離心3分鐘
Dilute the capture antibody in 1×PBS to the concentration specified on the datasheet.
用1xPBS稀釋捕獲抗體至說明書上要求的濃度
Gently mix or vortex the vial, try to ensure the air bubbles don’t mix into the solution,
輕輕顛倒或振蕩混勻，一定不要產(chǎn)生氣泡
Immediay add 100ul of the capture antibody solution into the ELISA plate wells.
立即在每個(gè)ELISA板孔中加入100uL捕獲抗體
Press firmly to seal the plate, take care not to let the reagent splash on the film
將封板膜用力壓蓋在ELISA板上，注意不要使抗體滴濺在封板膜上
Incubated the plate overnight at 25 ℃, alternatively the incubation for this phase can be done at 37℃ for 2-4 hours
25 ℃孵育過夜，或者37℃孵育2-4個(gè)小時(shí)
To wash the plate, discard the liquid and blot on a clean paper towel,
洗板時(shí)需將液體倒掉，并在干凈的吸水紙上拍干
Add 300ul of the wash buffer to every well and then aspirate the plate
每孔加入300ul 洗液，然后再將液體吸除
Repeat the step three additional times, totaling four washes in all.
該步驟再重復(fù)3次，總共洗滌4次
After the last wash, invert the plate to move liquid, and blot on a clean paper towel.
zui后一次洗滌后，將板倒置以去除液體，并在干凈的吸水紙上拍干
There are several other methods to wash an ELISA plate.Whatever you choose, be consistent with your washing technique throughout the whole process.
ELISA板還有其它幾種洗滌方法。無論使用哪種方法，請(qǐng)?jiān)谡麄€(gè)ELISA實(shí)驗(yàn)過程中保持一致
Phase 2: Blocking non-specific binding
第二階段：封閉非特異性結(jié)合
Bovine serum albumin is used as the blocking reagent to block any unbound open sites within the plastic wells
牛血清白蛋白作為封閉試劑，可封閉塑料孔內(nèi)任何未結(jié)合蛋白的位點(diǎn)
Add 300ul of the blocking buffer to each well.
每孔中加入300ul封閉液
Seal the plate and incubate for at least 1 hour at 25 ℃.
封板，并在25 ℃孵育至少1小時(shí)
詳細(xì)請(qǐng)看：ELISA操作指南
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