1. Octet Training Part II: Kinetics on the Octet
Scott Zhou, North China FAS
MB: , Mar.20th, 2013

2. Agenda
Basic Kinetics
BLI Kinetics Workflow
BLI Kinetics Applications

3. Basic Kinetics

4. Intensity λ =ƒ(λ, ℓ) Biolayer Interferometry（BLI） 可实时检测到两个反射表面间距的改变
Relative Intensity
Wavelength (nm)
100%
Distance between the two reflecting surfaces = ℓ
The Octet will monitor change in wavelength shift over time. This real-time binding measurement can be used to calculate on and off rates, and ultimately concentration by plotting rates against concentration.
Octet可以实时检测由于表面厚度变化带来的波长偏移，这种实时检测到的变化可以用来计算结合及解离的速度，并且最终得到相应的浓度变化
nm shift
Intensity λ =ƒ(λ, ℓ)
Time

5. Introduction to Basic Kinetics
Definitions of ka, kd, KD, and kobs
Calculation of ka, kd, KD, and kobs
Concentration Dependent vs. Concentration Independent Parameters

6. Binding Kinetics: Overview
Our analysis follows a 1:1 binding model:
Ab + Ag Complex ka kd
ka = rate of association or “on-rate”; unit = M-1sec-1
kd = rate of disassociation or “off-rate”; unit = sec-1
Y=Y0+A(1-e-kobs*t)
Y=Y0+Ae-kd*t
Non-linear curve
fit of data 
kobs – kd
[Conc Ag (M)]
ka=
KD = kd ka
,unit = M
Note that ka (or kon) and KD (affinity value) are concentration dependent (M or molar in the units). Kd (or koff) is a concentration independent value. Kd can be used to screen crude samples relative to each other.

7. Real-Time Kinetics Provides Better Binding Information Over ELISA Techniques
ELISA methods only provide approximations of KD.
Different combinations of Kon and Koff can give the same affinity measurement.
These two compounds appear to have the same KD, however Example B has a 100x tighter dissociation than Example A. For this pharmaceutical application, a tighter dissociation would be preferred.
Washing steps in ELISA methods will remove weak antibody interactions, and thus are not feasible to use for characterization.
Association
Dissociation
KD = Affinty
Example A
1.00E+04
1.00E-02
1.00E-06
Example B
1.00E+02
1.00E-04

8. BLI Kinetics Workflow

9. Getting Started Computer & Instrument start-up Sensor handling
Loading of the instrument

10. Getting Started Turning On the system: Warming up the Octet
Turn on the computer.
Next, make sure the sensor and sample positions are clear inside the Octet and turn on the power supply.
Finally, launch the Octet Data Acquisition software. Octet will initialize and home the optics box.
Warming up the Octet
The Octet should be turned an hour prior to an experiment being run to allow for the lamp to warm up.
Setting up experiment
Use 200 µL sample in each well.
Pre-wet the sensors in buffer/media (time may vary depending on sensor – consult product insert).

11. Pre-wetting The Sensors
Correct
Incorrect
Be sure to get the corner of the 96 well plate under the lip of the sensor tray. If the 96 well plate is above the lip, the sensor tray will be out of alignment and the sensors will not be picked up by the optics box properly.
Keep the sensors in the pre-wet plate at all times when running an assay.

12. Inserting the Sensor Tray into the Sensor Tray Holder and the Sample Plate into the Sample Plate Holder 2 3 1 4
1-The sensor tray is “keyed” to fit into the holder in only one way (it looks vaguely like an arrow and it points into the instrument).
2-The Sample Plate holder is marked “A1” (Red Circle) which corresponds with well “A-1” of the 96 well plate.
3-Place the sample plate into the sample plate holder
4-Proper placement of the sensor tray and sample plate in the Octet.

13. Biosensor selection According to application & sample type etc.
新开发了四种传感器
Anti-GST Biosensor: 用于含GST标签的蛋白
NTA Biosensor：用于含His标签的蛋白
Anti-human Fab-CH1：用于人Fab, F(ab’)2及Ab1~4
Anti-Flag biosensor：用于含Flag标签的蛋白

14. Ligand immobilization According to application & sample type etc.
（类）共价键偶联方式：结合稳定，无偶联特异性；要求偶联分子纯度高，浓度要求ug/ml级别；偶联的缓冲液不能含有氨基组分（如Tris），不能含有载体蛋白如BSA等（如有则需先替换），特别适合高亲和力检测。
SA, SSA传感器的偶联：中性环境
Minimal
biotinylation
(in solution)
Biotin–linker-NHS
NH2
H2N
Biotin
Immobilization
Streptavidin
biosensor
AR2G传感器的偶联：酸性环境，pH条件一般需优化
Activation
EDC/NHS
Immobilization
NH2
H2N
COOH
Amine Reactive
biosensor
NHS

15. Ligand immobilization According to application & sample type etc.
（类）亲和偶联方式：偶联特异性高，偶联结合力较共价键弱；对靶标样品纯度要求不高，粗样可直接用来偶联（如含靶标分子的培养液，裂解液，腹水等）；高亲和力相互作用推荐选择（类）共价键偶联方式。
Capture protein
Analyze kinetics
AHC, AMC，Anti-GST, Ni-NTA等传感器的偶联

16. Association Sample & concentration setting-important
Pre-test 1:
Relative high concentration(10*KD/100*KD).
Positive control.
Blank control.
Yes/No binding. Yes samples go next round detection.
Pre-test 2:
Yes samples go this round or KD test directly according to the researcher.
Find the proper concentration range(using a large dilution factor such as 10 or 5 to determine the highest and lowest concentration) if pre-test 2 performed.
KD test:
No less than 4 concentrations should be included in sample dilution series with a dilution factor 2 or 3.
Positive/negative control.
Data quality determined by R/X square in addition to compare with other source data.

17. Kinetics workflow 8 or 16 samples can be analyzed in parallel
Baseline
Baseline
Loading
Octet Biosensors
Buffer
Binding (nm)
Ligand-Biotin
Protein of Interest
Association
Dissociation
Using strepavidin coated biosensors:
Step 1 is Baseline- buffer only- want nice flat line, showing zero NSB
Step 2 load B-Ab- want gradual increase that eventually flattens out.
Step 3 is second basline to rid away any excess B-Ab
Step 4 is association step of your protein of interest
Step 5 is measure the dissociation of your protein of interest
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

18. Biosensor regeneration Depends on ligand & interaction characteristics etc
AHC, AMC,Anti-GST, Anti-His的再生
再生步骤：在仪器中在线完成，只需在微孔板中额外加一列再生缓冲液buffer。
传感器的再生条件：强酸，强碱，高盐缓冲液。
SA, SSA,AR的再生
SA,SSA,AR等传感器偶联靶标时，主要通过共价连接的方式，结合力强，再生条件不会将靶标洗脱下来，无需重新偶联（Loading）。
AHC等传感器主要通过特异的亲和将靶标分子偶联至传感器，结合力较共价键弱，再生条件下，靶标将会被洗脱下来，需重新偶联(Loading)。

19. SA, SSA, AR传感器的再生 再生效果跟偶联蛋白的稳定性有很大关系。
Original streptavidin sensor surface
Binding of biotinylated receptor
Association/ dissociation of target protein
Target protein removed
Capture protein
Analyze kinetics
Regenerate
再生效果跟偶联蛋白的稳定性有很大关系。
再生条件可根据传感器使用说明推荐的条件进行，也可实验室根据自身样品自行优化再生条件。
Regeneration of the Streptavidin biosensor refers to exposing the biosensor to a reagent that can effectively remove the target protein from the immobilized biotinylated receptor.
A good regeneration condition will remove all of the target protein without harming the activity of the immobilized receptor.

20. AHC, AMC，Anti-His, Anti-GST传感器的再生
Association and dissociation of target protein
Original sensor surface
Loading with IgG or Fc containing ligand
Sensor back to original surface
Capture protein
Analyze kinetics
Regenerate
再生之后需重新Loading，之后再进行动力学检测。
与SA和AR不同，这类传感器再生之后，可偶联不同的靶标。
对偶联蛋白本身的稳定性要求不高。
The anti-human IgG capture biosensor can be regenerated back to the origin surface with a brief (5X5sec) exposure to 10 mM Glycine pH Regeneration of this biosensor restores the original surface so it can be reloaded with a hIgG (or Fc containing) capture protein. A good regeneration condition will remove all of the hIgG and target proteins without harming the activity of the original capture surface.

21. NTA传感器的再生
Recharge with NiCl2
Association/ dissociation or quantitation of analyte protein
Original Nickel NTA sensor surface
Functional surface target captured
Target and NiCl2 removed
Capture HIS-tagged protein
Analyze kinetics
Regenerate
与镍柱再生类似。
再生之后，较于AHC, AMC, Anti-GST等不同的是：需要对传感器Recharge (NiCl2)
Regeneration of the Streptavidin biosensor refers to exposing the biosensor to a reagent that can effectively remove the target protein from the immobilized biotinylated receptor.
A good regeneration condition will remove all of the target protein without harming the activity of the immobilized receptor.

22. BLI Kinetics Applications

23. 小分子化合物筛选、亲和力测定

24. Small molecule screening-Novartis
Initial Screening of a Focused Library 336 compounds screened and processed in one 384 well plate
Blue = target sensor
Red = Control sensor
单一浓度初筛结果,从336个小分子化合物进行初筛

25. Hit confirmation 加入阴阳对照后对10个Hit进行确定，其中5个表现出明显剂效关系 kon: 5E4 M-1s-1
Positive control
kon: 5E4 M-1s-1
koff: 7E-2 s-1
KD: 1.4 uM
Non-binder
Negative control
Buffer
Non-binder
Non-binder
Aggregator
kon: 2E5 M-1s-1
koff: 1E-1 s-1
KD: nM
kon: 9E2 M-1s-1
koff: 1E-1 s-1
KD: uM
Aggregator
加入阴阳对照后对10个Hit进行确定，其中5个表现出明显剂效关系
kon: 2E5 M-1s-1
koff: 1E-1 s-1
KD: nM
kon: 3E3 M-1s-1
koff: 8E-2 s-1
KD: uM
kon: 8E5 M-1s-1
koff: 6E-2 s-1
KD: nM

26. Steady-state Analysis of Confirmed Hits
Steady-state analysis is in agreement with Kinetic analysis
Steady-state Analysis of Confirmed Hits
Rack7-H3
Steady state KD: 66 nM
Kinetic KD: 74 nM
Rack3-H5
Steady state KD: 26 µM
Kinetic KD: 24 µM
Rack1-F5
Steady state KD: 560 nM
Kinetic KD: 598 nM
Rack 1-D10
Steady state KD: 130 µM
Kinetic KD: µM
Rack1-C6
Steady state KD: 630 nM
Kinetic KD: nM
Furosemide
Steady state KD: 1.3 µM
Kinetic KD: 1.4 µM
阳性对照及确证Hit的稳态模型数据，与筛选结果一致

27. Small molecule-Protein interaction
Furosemide（呋喃苯氨酸） (MW 330D)
Each compound run in a titration series of 5 concentrations in duplicate.
Acetazolimide乙酰唑胺
(MW 222D)
阳性参照分子选择
Sulpiride（硫苯酰胺）
(MW 341D)
All data from one walk away run

28. Small molecule-Protein interaction
KD (M)
kon(1/Ms)
kdis(1/s)
R^2
2.34E-04
3.09E+02
7.22E-02
0.913
KD (M)
kon(1/Ms)
kdis(1/s)
R^2
8.41E-06
1.76E+04
1.48E-01
0.981
KD (M)
kon(1/Ms)
kdis(1/s)
R^2
1.00E-05
1.18E+04
1.18E-01
0.980
KD (M)
kon(1/Ms)
kdis(1/s)
R^2
3.73E-05
6.86E+03
2.56E-01
0.974
KD (M)
kon(1/Ms)
kdis(1/s)
R^2
1.04E-04
1.59E+03
1.65E-01
0.985
KD (M)
kon(1/Ms)
kdis(1/s)
R^2
8.19E-06
7.84E+03
6.42E-02
0.976
Sensor type: SSA
Buffer: PBS + 1% DMSO
数据来自Beigene Inc.
KD (M)
kon(1/Ms)
kdis(1/s)
R^2
3.62E-06
2.80E+04
1.01E-01
0.980

29. 蛋白-蛋白、蛋白-DNA、蛋白-糖分子

30. Kinetics of Arabidopsis Proteins measured on OctetWT
Mutant
Despite gross changes in plant growth rate, Octet data demonstrates the kinetic parameters of the mutated kinase are unchanged from wild type.
Octet QK parameters:
Standard 96W plate
Anti-Murine biosensors
30C, 1000 RPM in MOPS buffer
Loaded anti-GST antibody to capture GST-BAK protein
15 minute association w/ BRI1
15 minute dissociation

31. DNA-Binding Protein Kinetic Analysis on the Octet QK
Biotin-
DNA
Protein
Dissociation in Buffer kd ka KD
4.78E-05
5.59E+04
8.55E-10
Binding buffer = 20 mM Tris, 150 mM KOAc, 10 mM MgOAc
Octet QK
Binding of DNA-Binding Protein to Immobilized Biotinylated ssDNA
Rapid Analysis of Binding of Transcription Factors and Other Promoter Elements to Specific DNA Sequences

32. Saccharide-Protein
Data from SWU from Glyconex,Taiwan

33. 克隆筛选、抗体筛选、抗体-抗原亲和力测定、抗体配对

34. Antibody Clone selection
Using the Anti-Murine Biosensor to Rank Order Clones
High
Binding of 7 antibody supernatants in DMEM media supplemented with 15% horse serum
Able to quickly bin into high, medium and low producing cell lines
The Octet can also be used to monitor the purification process in addition to screening of clones
Assay took less than 20 minutes to set up and run on Octet QK
Medium
Biosensors prewet in blank media
Low
Blank Media

35. Antibody Screening Loading association Sensor type：SA
Baseline
Loading
association
dissociation
buffer
Bio-Ag
Ab 2B
Ab 2D
Ab 1-1
Ab 1-2
Ab 1-4
Ab 2-1
Ab 2-2
Ab 2-3
Sensor type：SA
Sample：supernatant without dilution.
数据来自军事医学科学院.

36. Ab Screening & Order rank results
Sample ID
Response
kdis(1/s)
kdis Error
mAb 2B
0.4546
6.23E-03
1.41E-04
mAb 2D
0.4178
5.68E-03
1.61E-04
mono Ab 1-1
0.7806
6.19E-03
1.42E-04
mono Ab 1-2
1.0178
1.08E-02
2.39E-04
mono Ab 1-4
1.0235
7.25E-03
1.64E-04
mono Ab 2-1
0.4227
4.78E-03
3.03E-04
mono Ab 2-2
0.8906
5.24E-03
1.53E-04
mono Ab 2-3
0.993
1.36E-04
数据来自军事医学科学院.

37. Ab-Ag interaction Kinetic analysis of anti-influenza antibodies using a His-tagged Antigen
Octet RED
Detection of anti-influenza virus antibodies in diluted serum to a his-tagged HA antigen immobilized on Anti-Penta His Biosensors on the Octet RED
Carney, PJ et al. CLINICAL AND VACCINE IMMUNOLOGY, Sept. 2010, Vol. 17, No. 9

38. Screen for Antibody Specificity and Affinity Yes / No Binding Can be Visualized Quickly
detection 1
detection 2
detection 3
detection 4
B-capture 1
+ MCP-1
- MCP-1
B-capture 2
B-capture 3
B-capture 4
Buffer
B-capture 1
B-capture 2
B-capture 3
B-capture 4
MCP-1 (analyte)
Detection 1
Detection 2
Detection 3
Detection 4
A small diagnostic company needed a method to screen commercial Ab sandwich pairs against identified biomarkers to develop a bead-based diagnostic platform.
Plate layout was set up to screen 4 capture antibodies against 4 detection antibodies. 32 combinations were tested in 1 hour.
Slide 1 of 2.

39. Data Analysis Tools Can Quickly Identify Successful Pairs
16 antibody pairs screened, with and without MCP-1.
All 5 pairs confirmed specificity and did not bind without analyte.
Both selected pairs are specific for MCP-1 and do not bind
MCP-2, MCP-3 or MCP-4
Detection
Ab 1
Ab2
Ab3
Ab4
Capture
Ab1 + -
B capture 1
MCP-1
Detection 1
MCP- 2
MCP- 3
MCP- 4
B capture 3
Slide 2 of 2.

40. Cases Octet more preferable
抗体/蛋白-病毒亲和力测定
蛋白-细菌亲和力测定
蛋白-纳米颗粒亲和力测定
蛋白聚合(蛋白-纳米颗粒)
蛋白-RNA亲和力测定
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）

41. Antibody-Virus virus1 virus2 Anti-virus antibody virus3 Neg. virus
数据来自 中科院生物物理所。

42. Protein-bacteria interaction
steps: baseline-loading(bio-pro)-baseline-asso(E.Coli)-diss.(PBS)
Sensor type: SA
Buffer: PB + 0.5%BSA + 0.2% tween-20
数据来自中国农业大学。

43. Protein- Q-dot
Immobilize protein on SA sensor surface and dilute quantum dot in solution as analyte
Data from Wuhan institute of virology,CAS

44. Aggregation :protein-nanoparticle interaction
Object: bio-pro vs nanoparticle
Solution : Loading bio-pro + nanoparticle
Background: proteins form fibers in neutral solution, while in acidic solution the nanoparticle promotes fiber formation, both of which couldn’t be detected with ITC or SPR.
Buffer: pH3.0 HAc;
Outcome: good data.

45. Aggregation :protein-nanoparticle interaction
Pro-P1
Pro-P2
Sample ID
KD (M)
kon(1/Ms)
kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2 P1
2.95E-09
8.07E+04
5.91E+02
2.38E-04
8.08E-06
4.06E-02
2.04E-02
1.03E-02
5.28E-03
2.76E-03
1.50E-03 P2
1.22E-08
1.39E+04
1.06E+02
1.71E-04
8.11E-06
1.41E-02
7.14E-03
3.66E-03
1.91E-03
1.04E-03
6.06E-04
数据来自中科院高能所。

46. RNA-Protein RNA immobilized to biosensors.
10uM
5uM
Associated with protein
2.5uM
Bio-RNA loading(10nM)
1.25uM
0.625uM
KD=6.22E-07,R2=0.988
No NSB signal between 10uM protein and blank sensor without bio-RNA(data not showed)
Actually 5min is enough for association step
Compare with BIACORE, no need to regenerate sensors immobilized RNA.
数据来自于中科院上海生化与细胞所。

47. Protein-RNA Protein immobilized to biosensors. 数据来自军事医学科学院。 Conc. (nM)
KD (M)
kon(1/Ms)
kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2
200
2.97E-09
5.21E+05
4.41E+03
1.55E-03
9.22E-06
1.06E-01
100
5.37E-02 50
2.76E-02 25
1.46E-02
12.5
8.06E-03
数据来自军事医学科学院。

48. Membrane protein-protein interaction
Object: pro vs pro
Solution : Loading bio-pro + pro
Background: there is high percentage of detergents in membrane protein samples thus could not be measured on SPR-based system due to bubbles generated by the buffer.
Buffer: assay buffer containing detergents from customer;
Outcome: good data.

49. Membrane protein-protein interaction
0 Ca2+
100nM Ca2+
Conc. (nM)
KD (M)
kon(1/Ms)
kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2
2.5
2.14E-08
2.75E+06
8.04E+04
5.89E-02
1.32E-03
6.58E-02 5
7.26E-02 10
8.64E-02 20
1.14E-01 40
1.69E-01 80
2.79E-01
Conc. (nM)
KD (M)
kon(1/Ms)
kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2
2.5
1.61E-08
3.66E+06
8.60E+04
5.88E-02
1.07E-03
6.79E-02 5
7.71E-02 10
9.54E-02 20
1.32E-01 40
2.05E-01 80
3.52E-01
100uM Ca2+
1mM Ca2+
Conc. (nM)
KD (M)
kon(1/Ms)
kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2
2.5
1.73E-08
2.84E+06
6.28E+04
4.93E-02
8.14E-04
5.64E-02 5
6.35E-02 10
7.77E-02 20
1.06E-01 40
1.63E-01 80
2.77E-01
Conc. (nM)
KD (M)
kon(1/Ms)
kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2
1.25
7.59E-09
3.33E+06
1.04E+05
2.53E-02
4.72E-04
2.94E-02
2.5
3.36E-02 5
4.19E-02 10
5.85E-02 20
9.18E-02
数据来自北大医学部。

50. 中药药理研究中的应用 研究背景: 中药成分复杂，光吸收法、荧光法等无法有效确证其细胞及活体水平研究结果；
利用传统SPR技术在原理上检测依赖于折光率变化，但很多碳水化合物与buffer折光率一致，不易引起折光率明显变化；进样模式上依赖于微流控系统，成分复杂容易造成系统堵塞；
BLI检测采用非标记技术、在微孔板中检测速度更快、通量更高，为复杂样品的确证提供有力工具。

51. 蛋白X与多糖相互作用 目的: 筛选蛋白X（含Fc片段）与不同多糖相互作用，确定其动力学参数； 样品信息：
多 糖：（包括阳性对照）12种，均由聚合度不同糖链构成，分子量未知，筛选浓度为1mg/ml及2mg/ml；
靶 标：蛋白X（含Fc段），总量50ug，以PBS配制成25ug/ml溶液；
传感器：AHC sensor.
Buffer：PBS

52. 蛋白X与多糖相互作用 结果：从12种多糖中初步筛选中发现3种可与蛋白X结合，阳性对照结合曲线可能因所用浓度过高所致。
将蛋白X固相化到AHC传感器上，检测浓度为1mg/ml及2mg/ml多糖，结合信号为阳性样品进一步采用浓度梯度确证.
Positive binding
Pos. Contr.: Positive binding?
结果：从12种多糖中初步筛选中发现3种可与蛋白X结合，阳性对照结合曲线可能因所用浓度过高所致。

53. 阳性对照动力学检测 Bio-Pro X：25ug/ml LPS： 15ug/ml~1.25ug/ml
成分复杂，摩尔浓度未知，仅拟合解离常数，Kd=0.255/s；
根据曲线快上快下特征，初步特测阳性对照中与蛋白X相互作用成分可能为低分子量物质，如小分子等。

54. 多糖动力学检测 Bio-Pro X：25ug/ml 多糖A： 0.5mg/ml~0.03mg/ml
成分复杂，摩尔浓度未知，假设分子量为180kDa，拟合所得KD=4.09nM；
根据曲线特征及信号大小，初步推测多糖A中与蛋白X相互作用成不会是低分子量物质。

55. Summary Workflow Keys & Optimization Select biosensors
Pre-wet sensors
Prepare samples
Pre-test 1(Yes/No binding, High concentration-10/100 fold KD, Pos.cont. Screening)
Pre-test 2(determine concentration dilution range)
KD test(no less than 4 conc.)
KD calculation
Keys & Optimization
Set proper control(pos. & neg. control)
Know KD from publications or other experiment and etc.
Assay buffer optimization(BSA & Tween-20)
Change sensor type

56. Pall ForteBio解决方案 Label-free Real time Fluidics-free
Fast，Accurate，Easy.