Octet Training
Part I: The Octet System，
A Label-Free, Real-Time Detection System
Scott Zhou, North China FAS
MB:15810470035, Email:scott_zhou@ap.pall.com
Mar.20th, 2013
Agenda
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Label-free & BLI
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BLI Applications
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BLI Publications
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BLI Domestic Users
Label-free & Biolayer Interferometry（BLI）
Labeled：ABS,FI,FRET, Lumi, FP…
Label-free?
What is “Label-Free” ?
• Label-free is a technology platform that allows monitoring and
measuring direct molecular interactions without artificial labels.
Why “Label-free”?
• Bypass difficulties in label selection and design and avoid
artifacts due to the presence of chemical labels
• Eliminate use of selected ‘Probes” and the restriction on “probe”
binding sites
• Direct binding vs. competitive binding
• One-step assay
Biolayer Interferometry（BLI）—new generation label-free
什么是光干涉现象
Biolayer Interferometry（BLI）
干涉作用中的相互加强与相互抵消
a
b
Two waves in phase
 Constructive interference
http://en.wikipedia.org/wiki/Interference
Two waves 180° out of phase
 Destructive Interference
Biolayer Interferometry（BLI）技术原理
如何利用光干涉现象
White light
Relative Intensity
Constructive
Interference
100%
Partially
constructive
Interference
Internal Reference (reflection surface 1
0
Interface with Liquid (reflection surface 2)
Destructive
Wavelength波长
Interference
Biolayer Interferometry（BLI）技术原理
改变膜厚度对干涉光的影响
Relative Intensity
100%
0
Wavelength
Biolayer Interferometry（BLI）技术原理
两个反射表面间距的改变对干涉光强度的影响
Internal Reference
(reflection surface 1)
Distance
between the
two reflecting
surfaces =
ℓ
Interface with Liquid (reflection surface 2)
Relative Intensity
100%
0
Wavelength (nm)
ƒ(λ, ℓ)
Intensity λ =
Biolayer Interferometry（BLI）技术原理
可实时检测到两个反射表面间距的改变
Relative Intensity
100%
0
Wavelength (nm)
surfaces =
ℓ
ƒ(λ, ℓ)
Intensity λ =
nm shift
Distance
between the
two reflecting
Time
Biolayer Interferometry（BLI）技术原理
特殊的 BLI
•
Two-dimensional binding surface
结合在二维的表面
•
Biocompatible Matrix
- minimizes non-specific binding
具有生物亲和性的表面基团阵列
极小的非特异性结合
•
Uniform
一致性高
•
Non-denaturing
不会使结合上的蛋白或抗体变性
Biosensor
|---- 600 mm ----|
Biolayer Interferometry（BLI）
生物分子结合后的 BLI Biosensor
• 生物分子结合到传感器表面形成了一层生物膜
• 生物膜对透过传感器的光波造成干涉现象
• 干涉现象以相位移动的方式被检测，从而可以
检测结合到传感器表面分子数量的变化
Fortebio Octet 检测平台
Octet QK平台内部
Biosensor selection
According to application & sample type etc.
新开发了四种传感器
1. Anti-GST Biosensor: 用于含GST标签的蛋白
2. NTA Biosensor：用于含His标签的蛋白
3. Anti-human Fab-CH1：用于人Fab, F(ab’)2及Ab1~4
4. Anti-Flag biosensor：用于含Flag标签的蛋白
BLI Applications & Advantages
BLI Solution for Drug Development
Wide Fit Within Discovery and Development Process
Research
Assay development
Expression
monitoring
Clone selection and
ranking
Epitope mapping
Screening
Lead
Characterization
Preclinical and
Clinical
Manufacturing
and QC
Small molecule or
ligand screening
Affinity
measurement
Kinetic profiling of
immunotherapeutics
Production
monitoring
Kinetic screening of
hybridomas
On and off-rates
Rapid concentration
measurements
Process
optimization
Immunogenicity
Protein and small
molecule
therapeutics
Secondary hit
confirmation
Binding affinity
ranking
21CFR Compliant
Fast and accurate
concentration
measurements
Information Provided by Octet Platform
Affinity characterization
• Binding KD
• On and off rates
• Rank order
Protein Concentration
• Picogram to milligram detection range
Specificity of binding
• Yes / No binding screens
• Epitope binning / mapping
• Isotyping
BLI Versatility in Interaction Analysis
Bacteria细菌
Size
1000 nm
Cells细胞
200 nm
Bacteria细菌
75 nm
Virus病毒
MW
Virus病毒
Antibody – Antigen抗体-抗原
1,000,000
IgM免疫球蛋白M
Receptor – Ligand受体-配体
DNA - DNA
IgA免疫球蛋白A
7 nm
IgG, IgD, IgE
DNA - Protein
100,000
Antibody Fragments
抗体片段
Antigen – Fusion Protein融合入蛋白
Proteins蛋白质
Peptides肽
1 nm
0.1 nm
Nucleotides (DNA)
Small Molecules小分
子
Atoms原子
Antibody Fragment – Antigen抗体片段-抗原
Antibody – Peptide抗体-肽
1,000
150
1
Multiple Antibody Pairings多重抗体配对
Antibody抗体 - Small Molecule
Protein 蛋白- Small Molecule
Kinetics workflow
Octet Biosensors
Buffer
Ligand-Biotin
Protein of Interest
Binding (nm)
Baseline
Baseline
Loading
Association
Time
•
•
•
•
8 or 16 samples can be analyzed in parallel
Measure on rates and off rates
Data is displayed in real-time
Experimental protocols can be customized
Dissociation
Quantitation workflow
Octet Biosensors
Binding (nm)
Standards
Test
Samples
•
•
•
The binding rates of test samples
are measured and interpolated from
the standard curve to determine
concentration
96 samples analyzed in 15 - 30
minutes
Reuse of standard curve is optional
Binding Rate
Time (sec)
Concentration
120
BLI Applications
General Application
Cases only Octet works (IFC-free system)

蛋白-蛋白/DNA

蛋白-糖分子

抗体/蛋白-病毒亲和力测定

克隆筛选
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蛋白-细菌亲和力测定
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抗体筛选
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蛋白-纳米颗粒亲和力测定

抗体抗原亲和力测定

蛋白聚合(蛋白-纳米颗粒)

抗体配对

蛋白-RNA亲和力测定

ADA（anti-drug-antibody）研究

膜蛋白亲和力测定（蛋白-蛋白）

定量测定（粗制样品）

中药研究 （蛋白-多糖）
生物膜干涉技术检测分子间相互作用的优点
• 通量高, 速度快
• 浓度测定比ELISA和HPLC快
• 样本动力学筛选比SPR方法更快
• 无需样本制备或稀释
• 容易使用
• 简便易学
• 一次性的检测器，无微流控系统
• 无需维护，无需清洁，无需预热
• 对样品要求宽松
• 样本适应性强，粗样本也能轻松检测
• 更广泛的溶剂耐受性(DMSO 10%)
• 检测通量的灵活
• 使用成本低廉
• Biosonsor可重复使用
• 无微流控系统，无需维护成本
BLI Publications
Rapid Rise in Publications Citing Octet
Publications Citing Octet Platform
200
180
160
140
120
100
80
60
40
20
0
195
141
75
55
0 2
2006
2
6
2007
7
16
2008
Peer-reviewed Publications
48
16
2009
2010 2011/07
All Patents and Publications
Since the first Octet system shipment in late 2005, more than 100
articles citing Octet systems have been published in a span of
less than 5 years
2012 high IF papers issued till Aug.
国内用户
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军事医学科学院三所
军事医学科学院八所(2台)
清华大学
中科院遗传与发育研究所
北京药用植物研究所
中国药品生物制品检定所
神州细胞生物技术有限公司
中国农业大学
吉林大学
Novo Nordisk中国研发中心
天津商业大学
甘肃健顺生物科技有限公司
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葛兰素史克(GSK) 中国研发中心
药明康德医药开发有限公司
厦门大学
江苏泰康生物医药
中国科学院武汉病毒研究所
中科院生化细胞研究所
上海百迈博制药有限公司
上海睿智化学研究有限公司
江苏太平洋美诺克生物药业有限公司
江西合信康宁生物医药有限公司
上海Newbio
四川康弘药业
浙江医学研究院
Pall ForteBio解决方案

Label-free

Real time

Fluidics-free

Fast，Accurate，Easy.
www.fortebio.com