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
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
•
•
•
Ag
Capture Antibody Detection Antibody
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Solid support Solid support
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)
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
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
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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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
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%
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)。
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
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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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.
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
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
•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
boiling
原態蛋白質 線性變性蛋白質
Sodium Dodecyl Sulphate (SDS)
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)
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
83
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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