利用固體承載物,以及抗體抗原專一性,對於樣品

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Enzyme-linked immunosorbent assy 酵素免疫 分析法

利用固體承載物,以及抗體抗原專一性,對於樣品

中的目標物進行檢測

利用最後呈色的量級進行定量

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 ELISA types 分類及原理

 ELISA Assay procedure 內容物與檢測介紹

Data analysis 數據分析

ELISA application ELISA 之應用

ELISA trouble shooting 疑難排除

Western Blot trouble shooting WB 疑難排除

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Indirect (間接式) ELISA

Direct (直接式) ELISA

Sandwich (雙抗夾心) ELISA

Competitive (競爭性) ELISA

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Anti-IgG

Primary Antibody (IgG) Antigen

Indicator

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Ag

Capture Antibody Detection Antibody

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Solid support Solid support

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ELISA types分類及原理

ELISA Assay procedure 內容物與檢測介紹

Data analysis 數據分析

ELISA application ELISA 之應用

ELISA trouble shooting 疑難排除

Western Blot trouble shooting WB 疑難排除

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Antibody/antigen coating (96-well) plate

Standards/calibrators (標準樣本)

Diluent (稀釋液)

Capture & detection antibody

Chromogen (顯色劑)

Stop solution (終止液)

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細胞培養液上清液, 血清

以離心去除非水溶性顆粒.

血漿

加入 citrate, EDTA 或 heparin 作為抗凝劑, 再離 心並收集上清液.

檢測前需再經過稀釋

樣本經前處理後應盡速檢測, 或分裝後保存於-20°C.

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以稀釋緩衝液將標準樣本進行系列稀釋

11

pg/mL 2000 1000 500 250 125 62.5 31.3 0

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清洗 包被

封閉

加入檢測抗體

呈色

吸光度測定 加入樣本/

標準品 樣本前處理 清洗

清洗 清洗 標準樣本

系列稀釋

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封閉非專一性蛋白質結合位點

Blocking Reagent:

1% BSA (Immunoassay Grade) 10% FBS

5% skim milk in PBS 5% serum

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清洗 包被

封閉

加入檢測抗體

呈色

吸光度測定 加入樣本/

標準品 樣本前處理 清洗

清洗 清洗 標準樣本

系列稀釋

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ELISA types分類及原理

 ELISA Assay procedure 內容物與檢測介紹

Data analysis 數據分析

ELISA application ELISA 之應用

ELISA trouble shooting 疑難排除

Western Blot trouble shooting WB 疑難排除

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Standard curve (標准曲線)

Precision (精確度)

Accuracy (准確度)

Sensitivity (靈敏度)

Specificity (特異性)

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www.abnova.com www.abnova.com 17 LipoDox conc. (ng/ml)

Con. 0 3.13 6.25 12.5 25 50 100 OD1 0.12 0.22 0.34 0.48 0.85 1.32 1.99 OD2 0.12 0.19 0.29 0.55 0.83 1.17 1.84 AVG 0.12 0.21 0.31 0.51 0.84 1.25 1.92 CV 1% 10% 11% 9% 2% 9% 6%

變異係數

(coefficient of variation) Sample CV < 15%

Blank CV < 20%

0.0 0.5 1.0 1.5 2.0

0 20 40 60 80 100 120

OD 450

conc. (ng/ml) Standard Curve

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Intra-assay (內部檢測) Precision (Precision within an assay)

Inter-assay (批間檢測) Precision (Precision between assays)

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樣本成分會影響吸亮度之測定

Recovery: 80-120%

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Limit of Detection (檢測限)

樣品中所含的目標蛋白可被檢測出之最低量或濃度.

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Metanephrine (甲基腎上腺素) ELISA Kit (KA1890)

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ELISA types分類及原理

 ELISA Assay procedure 內容物與檢測介紹

Data analysis 數據分析

ELISA application ELISA 之應用

ELISA trouble shooting 疑難排除

Western Blot trouble shooting WB 疑難排除

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Application

ELISPOT

Cell-Based ELISA Kit

Related product in Abnova

Ab Pair for ELISA (1000+)

ELISA Kit (2000+)

Cell-Based ELISA Kit (500+)

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適合檢測外泌性蛋白

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將細胞接種到各孔的底部上。將細胞固 定和封閉。

加入一抗和胞內之專一性抗原結合。

加入二抗。

HRP 酶將加入之 TMB 氧化。顏色由藍 變黃,加入酸將反應終止,再測量吸光 值 (450nm)。

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Cell-Based ELISA Western Blot

樣本類型

活細胞即可

(Suspension cells,

loosely attached cells, attached cells)

細胞裂解液

樣本前處理 不須額外處理 須將細胞裂解

檢測過程 快速 較費時

(Gel electrophoresis, Transfer)

檢測限 ng/mL mg/mL

應用 Qualitative/phosphory

-lation detection Qualitative/phosphoryl- ation detection

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ELISA types分類及原理

 ELISA Assay procedure 內容物與檢測介紹

Data analysis 數據分析

ELISA application ELISA 之應用

ELISA trouble shooting 疑難排除

Western Blot trouble shooting WB 疑難排除

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Possible Cause Solution The protein amount is added into

well insufficiently.

Ensure extract contains enough amount of proteins.

Incubation time and temperature is incorrect.

Ensure the incubation time and temperature described in the protocol are correctly followed.

Inadequate reagent volumes or improper dilution

Check pipettes and ensure correct preparation

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www.abnova.com www.abnova.com Possible Cause Solution

Poor blocking Try a different blocking agent.

Contaminated wash buffer Make fresh wash buffer Insufficient washing Increase number of washes

Increase time of soaking between in wash Incubation time and

temperature is incorrect.

Ensure the incubation time and temperature described in the protocol are correctly followed.

Primary and secondary antibodies

Try decreasing the antibody concentration and/or the incubation period.

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Possible Cause Solution Reagents not fresh or not at

the correct pH

Use freshly prepared solutions and reagents.

Reagent added in incorrect order, or incorrectly prepared

Review protocol and check the concentration of reagents.

Bubbles in the wells Make sure you remove air bubbles within wells.

Uneven temperature around work surface

Avoid incubating plate in areas where environmental conditions vary, and use plate sealer

Filth at the bottom of 96-well plate

Make sure the surface is clean before adding the reagents into wells

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ELISA types分類及原理

 ELISA Assay procedure 內容物與檢測介紹

Data analysis 數據分析

ELISA application ELISA 之應用

ELISA trouble shooting 疑難排除

Western Blot trouble shooting 疑難排除

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利用膠體電泳法分離樣品中的蛋白質、轉漬到膜上

後,使用對目標蛋白具專一性的抗體進行蛋白質偵

測。

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SDS-PAGE

(SDS-polyacrylamide gel electrophoresis)

|

Transfer

|

Blocking

|

Primary and Secondary Detection Reagents

|

Enzyme Substrates

| Film

PVDF Nitrocellulose

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•SDS-PAGE 系統中,樣本分子的泳動率,

僅取決於其分子量,而與原來分子所帶的電荷無關

O-S-O-(CH2)11-CH3 極性部分

O

O

非極性部分

Sodium Dodecyl Sulphate (SDS)

SDS

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boiling

原態蛋白質 線性變性蛋白質

Sodium Dodecyl Sulphate (SDS)

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帶電分子在電場作用下,向著與其電性相反的電極移動,稱為電泳

Electrophoresis (電泳)

泳動率 (v) ~ 外加電壓 (E) × 分子淨電荷 (q)

分子泳動時與介質間之摩擦力 (f)

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利用一個分子篩來分離大小不同的蛋白質

PAGE

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封閉膜上非專一性蛋白質結合位點

減少抗體非專一性結合,進而減低背景干擾

Blocking Reagent:

1% BSA (Immunoassay Grade) 10% FBS

5% skim milk in PBS 5% serum

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12.5 % SDS PAGE 175

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62 47.5

32.5

25

marker 1 2 3 4 5 6

Current BCG: 50,45,1.00 Applied BCG: NONE Exposure: 0.00 Accumul ated: 0 12/ 17/2003 02: 55:43. 593 P M

F i le Name: C:\Document s and S ettings\level\桌面\user\昌維\1217-cbr.tif Image S ignature: F 3C1-7779-9562-A1B6-4F 32-ADEF -2792-3EAA

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Weak or no signal 無信號

High background 高背景

Multiple bands 多帶現象

Speckled or Dotted Appearance 出現不均勻的斑點

White bands on a black blot 出現白色條帶

Smile effect of the bands 條帶“微笑”效應

Bands Smeared, Bands not Sharp 條帶不銳利清晰

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Weak or No Signal 無信號

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Weak or No Signal 無信號

Possible Cause Solution

Insufficient antigen Load at least 20-30 μg protein per lane; Use protease inhibitors.

The 1st Ab and the 2nd Ab are not

compatible Use 2nd Ab that was raised against the species in which the 1st Ab was raised.

2nd Ab was inhibited by sodium

azide Do not use sodium azide together

with HRP-conjugated antibodies.

Detection system Check that the detection reagents are being stored correctly and used as recommended.

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High Background 高背景

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High Background 高背景

Possible Cause Solution

Poor blocking Try a different blocking agent (5%

non-fat dry milk, 3% BSA, or normal serum).

Insufficient washing Washing should be thorough at each step.

Choice of membrane Nitrocellulose membrane is

considered to give less background than PVDF.

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Multiple Bands 多帶現象

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Multiple Bands 多帶現象

Possible Cause Solution

Post-translation modification (acetylation, methylation,

phosphorylation, glycosylation)

Examine the literature and use an agent to de-phosphorylate, de- glycosylate.

SDS caused nonspecific binding Wash blots after transfer. Do not use SDS during immunoassay procedure.

Primary and secondary antibodies Try decreasing the antibody

concentration and/or the incubation period.

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Speckled or Dotted Appearance 不均勻的斑點

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Speckled or Dotted Appearance 不均勻的斑點

Possible Cause Solution

Black Spots Filter or change a new blocking reagent to avoid the antibodies are binding to blocking reagent.

White Spots Make sure you remove air bubbles

when preparing the gel for transfer.

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Other question

Smile effect of the bands Bands Smeared

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Problem Possible Cause Solution

Smile effect of the

bands Migration is too fast or

too hot Slow down the

migration or run the gel in the cold room or on ice.

Bands Smeared,

Bands not Sharp Bands smeared due to hot

gel, high voltage Decrease voltage, and prepare new running buffer.

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