好消息--PeproTech ELISA操作指南視頻已上傳!
美國PeproTech (派普泰克) 公司是世界上領(lǐng)先的重組細胞因子和蛋白生產(chǎn)商，同時也生產(chǎn)多種細胞因子和蛋白的ELISA法檢測試劑盒，為讓中國用戶能夠更好的熟悉和掌握PeproTech的ELISA試劑盒及其技術(shù)，PeproTech專門制作了"ELISA操作指南視頻"，供各位老師和同學觀摩！
視頻網(wǎng)址為：http://v.youku.com/v_show/id_XMzQ3Mzk3NTg4.html 或 http://www.tudou.com/programs/view/tzU8m93o0tI/，歡迎大家的批評和指正！
該視頻為英文講解，為便于大家理解，請看下面的英文原文及翻譯：
The following video tutorial described how to run a sandwich ELISA, also known as enzyme-linked immunosorbent assay, using a general PeproTech protocol.
接下來的實驗指導講述PeproTech夾心法ELISA，即酶聯(lián)免疫吸附試驗的通用步驟
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、標準品和檢測抗體均為凍干粉，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 at4 ℃ .
捕獲抗體、標準品和檢測抗體重懸后，在4℃ 時最長保存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，并準備在2周之后使用，請將重懸的組分分裝并凍存于-20℃ ，最長可保存6個月。
In contrast to the other three EDK components, the Avidin-HRP vial comes ready to use
與EDK的其它三個組分不同，Avidin-HRP為即用型
In order to avoid harmful repeated freeze/thaw cycles, or long-term storage at4 ℃ 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.
目的是避免反復凍融或長期4℃ 保存對該組分的功能損害。收到Avidin-HRP后，立即將其分裝為6ul/管，共10管，凍存于-20℃ ，最長可保存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分鐘以使抗體完全溶解
Centrifuge the reconstituted vial for 3 minutes at maximum speed
重懸的抗體以最高速度離心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
Immediately add 100ul of the capture antibody solution into the ELISA plate wells.
立即在每個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 at25 ℃ , 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個小時
To wash the plate, discard the liquid and blot on a clean paper towel,
洗板時需將液體倒掉，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.
最后一次洗滌后，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.
ELISA板還有其它幾種洗滌方法。
Whatever you choose, be consistent with your washing technique throughout the whole process.
無論使用哪種方法，請在整個ELISA實驗過程中保持一致
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
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 at25 ℃ .
封板，并在25 ℃ 孵育至少1小時
Phase 3:
第3階段：
Specific binding of antigen
抗原的特異性結(jié)合
Priority to the end of the previous incubation period
在上一孵育過程結(jié)束前
Centrifuge the standard vial briefly
將標準品稍作離心
And reconstitute to the specified concentration in sterile water.
并用無菌水重懸至要求的濃度
Allow the vial to reconstitute for minimum 10 minutes.
靜置至少10分鐘，以使標準品完全溶解
When the incubation period is over, remove the plate and wash four times
重懸結(jié)束后，取出板并洗滌4次
Centrifuge the reconstituted standard vial for 3 minutes at maximum speed
重懸的標準品以最高速度離心3分鐘
Dilute to the specified concentration, and gently mix or vortex the solution.
稀釋到所需濃度，輕輕顛倒或振蕩混勻
The standard curve is the area on the plate dedicate to fixed concentrations of our protein standard
標準曲線在ELISA板上的區(qū)域?qū)Ｓ糜跈z測已知濃度的蛋白標準品
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，除第一列外的所有標準曲線孔中均加入100ul稀釋液
Next, add 200ul of standard solution to the first row of standard curve wells.
然后，在標準曲線孔的第一列中加入200ul標準品溶液
Dilute 1:2 down the plate to a minimum of six concentrations
依次倍比稀釋，標準品至少有6個濃度
Each standard curve well should contain the final volume of 100ul of the solvent.
每個標準曲線孔中液體的終體積應(yīng)為100ul
Using the minimum six concentrations in the triplicate, plus a minimum six wells of blanks containing only diluent.
標準品至少有6個濃度，每個濃度為3個復孔，再設(shè)6個空白孔，其中僅含稀釋液
These blanks are essential and later determining the efficacy and ability of the measurements of the samples.
空白孔是必須的，它們將用來評價試劑盒檢測樣本的效能
Next, add your specific samples of interest in triplicate to the remaining wells
然后，在剩余孔中加入待測樣本，每個樣本設(shè)3個復孔
Depending on the concentration of the analyte, dilution may be necessary for optimal results
樣本中若待檢抗原的濃度過高，要得到最佳結(jié)果可能需對樣本進行稀釋
Seal the plate and incubate for two hours at25 ℃ .
封板，25 ℃ 孵育2小時
Phase 4:
第4階段：
Sandwich formation via the addition of biotinylated detection antibody.
加入生物素標記的檢測抗體以形成夾心
Priority to the end of the incubation, centrifuge briefly and reconstitute the detection antibody for ten minutes in sterile water.
孵育結(jié)束前，稍離心檢測抗體，并用無菌水重懸，靜置10分鐘
When the incubation is over, remove the plate and wash four times.
孵育結(jié)束后，取出板，洗滌4次
Centrifuge for 3 minutes at maximum speed
最高速度離心3分鐘
And dilute the detection antibody in diluent to the specified concentration.
用稀釋液將檢測抗體稀釋至要求的濃度
Gently mix the solution and immediately add 100ul per well
輕輕混勻，立即在每孔中加入100ul檢測抗體液
Seal the plate and incubate for 2 hours at25 ℃
封板，25 ℃ 孵育2小時
Phase 5:
第5階段：
Addition of the enzyme-linked Avidin-HRP to the sandwich
夾心上加酶聯(lián)親合素(Avidin-HRP)
While the plate is incubating, remove one 6ul aliquot of Avidin-HRP from the freezer and let it thaw.
板在孵育過時，從冰箱中取出一管Avindin-HRP (6ul)，使其融化
When the incubation is over, wash the plate 4 times
板孵育結(jié)束后，洗滌4次
Dilute the Avidin-HRP 1:2000 in diluent
用稀釋液1:2000稀釋Avidin-HRP
Gently mix the solution and immediately add 100ul per well.
輕輕混勻，立即向每孔中加入100ul
Seal the plate and incubate for 30 minutes at25 ℃ .
封板，25 ℃ 孵育30分鐘
To reduce the background interference, ensure the incubation is for 30 minutes only.
為降低背景干擾，請確保孵育時間正好為30分鐘
Phase 6: Conversion of the colorless substrate into a colored solution.
階段6：底物顯色
Promptly washed the plate after incubation and add 100ul of ABTS substrate solution to each well.
孵育結(jié)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僅可與ABTS配合使用
Using it in conjunction with TMB or other substrate solutions, mainly to a dramatic rise in background development
如與TMB或其它底物溶液配合使用，會顯著提高背景顯色
Incubate at room temperature for color development
室溫孵育顯色
Monitor the plate at 5-minute intervals for up to 60 minutes.
每隔5分鐘讀板一次，最長可監(jiān)測60分鐘
Using a ELISA plate reader set at appropriate wavelength correction for your selected plate
在酶標儀上設(shè)置合適的校正波長
OD readings will vary depending on the specific EDK and its reading time provided on the product datasheet.
EDK不同，以及說明書上所要求的讀板時間不同均可能導致OD值的差異
視頻網(wǎng)址為：http://v.youku.com/v_show/id_XMzQ3Mzk3NTg4.html 或 http://www.tudou.com/programs/view/tzU8m93o0tI/，歡迎大家的批評和指正！
該視頻為英文講解，為便于大家理解，請看下面的英文原文及翻譯：
The following video tutorial described how to run a sandwich ELISA, also known as enzyme-linked immunosorbent assay, using a general PeproTech protocol.
接下來的實驗指導講述PeproTech夾心法ELISA，即酶聯(lián)免疫吸附試驗的通用步驟
The capture, standard, and detection are supplied as stable lyophilized products, and should be stored at
捕獲抗體、標準品和檢測抗體均為凍干粉，使用前應(yīng)保存于
Reconstituted Capture, Standard, and Detection components are only guaranteed to be stable for up to 2 weeks when stored at
捕獲抗體、標準品和檢測抗體重懸后，在
If you have reconstituted the EDK, and plan on using it for a duration greater than two weeks, aliquot and store at
如果您已重懸了EDK的各組份，并準備在2周之后使用，請將重懸的組分分裝并凍存于
In contrast to the other three EDK components, the Avidin-HRP vial comes ready to use
與EDK的其它三個組分不同，Avidin-HRP為即用型
In order to avoid harmful repeated freeze/thaw cycles, or long-term storage at
目的是避免反復凍融或長期
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分鐘以使抗體完全溶解
Centrifuge the reconstituted vial for 3 minutes at maximum speed
重懸的抗體以最高速度離心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
Immediately add 100ul of the capture antibody solution into the ELISA plate wells.
立即在每個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
To wash the plate, discard the liquid and blot on a clean paper towel,
洗板時需將液體倒掉，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.
最后一次洗滌后，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.
ELISA板還有其它幾種洗滌方法。
Whatever you choose, be consistent with your washing technique throughout the whole process.
無論使用哪種方法，請在整個ELISA實驗過程中保持一致
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
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
封板，并在
Phase 3:
第3階段：
Specific binding of antigen
抗原的特異性結(jié)合
Priority to the end of the previous incubation period
在上一孵育過程結(jié)束前
Centrifuge the standard vial briefly
將標準品稍作離心
And reconstitute to the specified concentration in sterile water.
并用無菌水重懸至要求的濃度
Allow the vial to reconstitute for minimum 10 minutes.
靜置至少10分鐘，以使標準品完全溶解
When the incubation period is over, remove the plate and wash four times
重懸結(jié)束后，取出板并洗滌4次
Centrifuge the reconstituted standard vial for 3 minutes at maximum speed
重懸的標準品以最高速度離心3分鐘
Dilute to the specified concentration, and gently mix or vortex the solution.
稀釋到所需濃度，輕輕顛倒或振蕩混勻
The standard curve is the area on the plate dedicate to fixed concentrations of our protein standard
標準曲線在ELISA板上的區(qū)域?qū)Ｓ糜跈z測已知濃度的蛋白標準品
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，除第一列外的所有標準曲線孔中均加入100ul稀釋液
Next, add 200ul of standard solution to the first row of standard curve wells.
然后，在標準曲線孔的第一列中加入200ul標準品溶液
Dilute 1:2 down the plate to a minimum of six concentrations
依次倍比稀釋，標準品至少有6個濃度
Each standard curve well should contain the final volume of 100ul of the solvent.
每個標準曲線孔中液體的終體積應(yīng)為100ul
Using the minimum six concentrations in the triplicate, plus a minimum six wells of blanks containing only diluent.
標準品至少有6個濃度，每個濃度為3個復孔，再設(shè)6個空白孔，其中僅含稀釋液
These blanks are essential and later determining the efficacy and ability of the measurements of the samples.
空白孔是必須的，它們將用來評價試劑盒檢測樣本的效能
Next, add your specific samples of interest in triplicate to the remaining wells
然后，在剩余孔中加入待測樣本，每個樣本設(shè)3個復孔
Depending on the concentration of the analyte, dilution may be necessary for optimal results
樣本中若待檢抗原的濃度過高，要得到最佳結(jié)果可能需對樣本進行稀釋
Seal the plate and incubate for two hours at
封板，
Phase 4:
第4階段：
加入生物素標記的檢測抗體以形成夾心
Priority to the end of the incubation, centrifuge briefly and reconstitute the detection antibody for ten minutes in sterile water.
孵育結(jié)束前，稍離心檢測抗體，并用無菌水重懸，靜置10分鐘
When the incubation is over, remove the plate and wash four times.
孵育結(jié)束后，取出板，洗滌4次
Centrifuge for 3 minutes at maximum speed
最高速度離心3分鐘
And dilute the detection antibody in diluent to the specified concentration.
用稀釋液將檢測抗體稀釋至要求的濃度
Gently mix the solution and immediately add 100ul per well
輕輕混勻，立即在每孔中加入100ul檢測抗體液
Seal the plate and incubate for 2 hours at
封板，
Phase 5:
第5階段：
Addition of the enzyme-linked Avidin-HRP to the sandwich
夾心上加酶聯(lián)親合素(Avidin-HRP)
While the plate is incubating, remove one 6ul aliquot of Avidin-HRP from the freezer and let it thaw.
板在孵育過時，從冰箱中取出一管Avindin-HRP (6ul)，使其融化
When the incubation is over, wash the plate 4 times
板孵育結(jié)束后，洗滌4次
Dilute the Avidin-HRP 1:
用稀釋液1:2000稀釋Avidin-HRP
Gently mix the solution and immediately add 100ul per well.
輕輕混勻，立即向每孔中加入100ul
Seal the plate and incubate for 30 minutes at
封板，
To reduce the background interference, ensure the incubation is for 30 minutes only.
為降低背景干擾，請確保孵育時間正好為30分鐘
Phase 6: Conversion of the colorless substrate into a colored solution.
階段6：底物顯色
Promptly washed the plate after incubation and add 100ul of ABTS substrate solution to each well.
孵育結(jié)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僅可與ABTS配合使用
Using it in conjunction with TMB or other substrate solutions, mainly to a dramatic rise in background development
如與TMB或其它底物溶液配合使用，會顯著提高背景顯色
Incubate at room temperature for color development
室溫孵育顯色
Monitor the plate at 5-minute intervals for up to 60 minutes.
每隔5分鐘讀板一次，最長可監(jiān)測60分鐘
Using a ELISA plate reader set at appropriate wavelength correction for your selected plate
在酶標儀上設(shè)置合適的校正波長
OD readings will vary depending on the specific EDK and its reading time provided on the product datasheet.
EDK不同，以及說明書上所要求的讀板時間不同均可能導致OD值的差異
PeproTech(派普泰克)中國代表處
江蘇省蘇州市金河國際大廈1918室
郵編：215011
電話：0512-6832 5993/6832 5983
Email：
Website：
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