Transcript 分子相互作用2 Kinet

Octet Training
Part II: Kinetics on the Octet
Scott Zhou, North China FAS
MB:15810470035, Email:[email protected]
Mar.20th, 2013
Agenda

Basic Kinetics

BLI Kinetics Workflow

BLI Kinetics Applications
Basic Kinetics
Biolayer Interferometry（BLI）
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
Introduction to Basic Kinetics
• Definitions of ka, kd, KD, and kobs
• Calculation of ka, kd, KD, and kobs
• Concentration Dependent vs. Concentration Independent
Parameters
Binding Kinetics: Overview
Our analysis follows a 1:1 binding model:
Ab + Ag
ka
Complex
kd
Non-linear curve
fit of data 
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)
ka=
kobs – kd
[Conc Ag (M)]
Y=Y0+Ae-kd*t
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.
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.
Association
Dissociation
KD = Affinty
Example A
1.00E+04
1.00E-02
1.00E-06
Example B
1.00E+02
1.00E-04
1.00E-06
5.5
•
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.
4.5
example A
3.5
nm shift
•
example B
2.5
1.5
0.5
-0.5
0
100
200
300
400
Time (sec)
500
600
700
BLI Kinetics Workflow
Getting Started
• Computer & Instrument start-up
• Sensor handling
• Loading of the instrument
Getting Started
•
Turning On the system:
1. Turn on the computer.
2. Next, make sure the sensor and sample positions are clear inside the
Octet and turn on the power supply.
3. 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).
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.
Inserting the Sensor Tray into the Sensor Tray Holder and the Sample
Plate into the Sample Plate Holder
1
2
3
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.
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
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
NH2
(in solution)
Streptavidin
biosensor
NH2
Minimal
biotinylation
Immobilization
Biotin
Biotin–linker-NHS
Biotin
Streptavidin
biosensor
H2N
H2N
AR2G传感器的偶联：酸性环境，pH条件一般需优化
Amine Reactive
biosensor
Activation
Amine Reactive
biosensor
NH2
NH2
Immobilization
Amine Reactive
biosensor
NH2
EDC/NHS
H2N
NH2
NH2
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。
AHC, AMC，Anti-GST, Ni-NTA等传感器的偶联
Capture
protein
Analyze
kinetics
Association
Sample & concentration setting-important
•
Pre-test 1:
1.
2.
3.
4.
•
Relative high concentration(10*KD/100*KD).
Positive control.
Blank control.
Yes/No binding. Yes samples go next round detection.
Pre-test 2:
1. Yes samples go this round or KD test directly according to the researcher.
2. 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:
1. No less than 4 concentrations should be included in sample dilution series with
a dilution factor 2 or 3.
2. Positive/negative control.
3. Blank control.
4. Data quality determined by R/X square in addition to compare with other
source data.
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
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
1.
2.
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)。
SA, SSA, AR传感器的再生
Original
streptavidin
sensor surface
Binding of
biotinylated
receptor
Capture
protein
Association/
dissociation of
target protein
Analyze
kinetics
Target protein
removed
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。
AHC, AMC，Anti-His, Anti-GST传感器的再生
Original sensor
surface
Association and
dissociation of
target protein
Loading with IgG or Fc
containing ligand
Capture
protein
Analyze
kinetics
Sensor back to original
surface
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。
NTA传感器的再生
Recharge with NiCl2
Original Nickel
NTA sensor
surface
Association/
dissociation or
quantitation of
analyte protein
Functional
surface target
captured
Capture HIStagged
protein
Analyze
kinetics
Target and NiCl2
removed
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)
BLI Kinetics Applications
小分子化合物筛选、亲和力测定
Small molecule screening-Novartis
Initial Screening of a Focused Library
Blue = target sensor
336 compounds screened and processed in one 384 well plate
Red = Control sensor
Hit confirmation
kon: 5E4 M-1s-1
koff: 7E-2 s-1
KD: 1.4 uM
Positive control
Negative control
CH3
H3C
Buffer
Non-binder
CH3
N
N
H3C
CH3
O
Non-binder
Non-binder
NH2
Aggregator
O
OH
O
N
OH
N
OH
O
S
Aggregator
Cl
O
NH2
Cl
S
kon: 2E5 M-1s-1
koff: 1E-1 s-1
KD: 613.5 nM
O
NH2
kon: 9E2 M-1s-1
koff: 1E-1 s-1
KD: 140.4 uM
O
N
NH2
O
Cl
O
S
Cl
NH2
kon: 2E5 M-1s-1
koff: 1E-1 s-1
KD: 597.8 nM
O
N
N
CH3
kon: 3E3 M-1s-1
koff: 8E-2 s-1
KD: 24.4 uM
Cl
CH3
F
S
O
NH2
O
kon: 8E5 M-1s-1
koff: 6E-2 s-1
KD: 73.9 nM
Steady-state analysis is in agreement with Kinetic analysis
Steady-state Analysis of Confirmed Hits
Furosemide
Steady state KD: 1.3 µM
Kinetic KD: 1.4 µM
Rack1-C6
Steady state KD: 630 nM
Kinetic KD: 613.5 nM
Rack 1-D10
Steady state KD: 130 µM
Kinetic KD: 140.4 µM
O
O
Cl
S
S
NH2
O
NH2
Cl
O
Rack1-F5
Steady state KD: 560 nM
Kinetic KD: 598 nM
O
Cl
O
S
NH2
Rack3-H5
Steady state KD: 26 µM
Kinetic KD: 24 µM
Cl
CH3
O
N
Cl
Rack7-H3
Steady state KD: 66 nM
Kinetic KD: 74 nM
N
CH3
F
S
O
NH2
O
Small molecule-Protein interaction
Each compound run in a titration series
of 5 concentrations in duplicate.
Furosemide（呋喃苯氨酸） (MW
330D)
Acetazolimide乙酰唑胺
(MW 222D)
All data from one walk away run
Sulpiride（硫苯酰胺）
(MW 341D)
Small molecule-Protein interaction
KD (M)
2.34E04
KD (M)
3.73E05
kon(1/Ms)
kdis(1/s)
3.09E+02
R^2
7.22E-02
kon(1/Ms)
kdis(1/s)
6.86E+03
0.913
R^2
2.56E-01
0.974
KD (M)
8.41E-06
KD (M)
1.04E04
kon(1/Ms)
1.76E+04
kon(1/Ms)
1.59E+03
kdis(1/s)
1.48E-01
kdis(1/s)
1.65E-01
R^2
0.981
R^2
0.985
Sensor type: SSA
Buffer: PBS + 1% DMSO
数据来自Beigene Inc.
KD (M)
3.62E06
kon(1/Ms
)
2.80E+04
kdis(1/s)
1.01E-01
R^2
0.980
KD (M)
1.00E-05
KD (M)
8.19E06
kon(1/Ms)
1.18E+04
kon(1/Ms)
7.84E+03
kdis(1/s)
R^2
1.18E-01
kdis(1/s
)
6.42E02
0.980
R^2
0.976
蛋白-蛋白、蛋白-DNA、蛋白-糖分子
Kinetics of Arabidopsis Proteins measured on Octet
WT
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
DNA-Binding Protein Kinetic Analysis on the Octet QK
BiotinDNA
Protein
Dissociation in Buffer
kd
ka
KD
4.78E-05
5.59E+0
4
8.55E-10
Binding of DNA-Binding Protein to Immobilized Biotinylated ssDNA
Rapid Analysis of Binding of Transcription Factors and Other
Promoter Elements to Specific DNA Sequences
Saccharide-Protein
Data from SWU from Glyconex,Taiwan
克隆筛选、抗体筛选、抗体-抗原亲和力
测定、抗体配对
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
Medium
The Octet can also be used to monitor
the purification process in addition to
screening of clones
Low
Blank
Media
Assay took less than 20 minutes to set
up and run on Octet QK
Antibody Screening
Baseline
Loading
Baseline
association
dissociation
buffer
Bio-Ag
buffer
Ab 2B
buffer
buffer
Bio-Ag
buffer
Ab 2D
buffer
buffer
Bio-Ag
buffer
Ab 1-1
buffer
buffer
Bio-Ag
buffer
Ab 1-2
buffer
buffer
Bio-Ag
buffer
Ab 1-4
buffer
buffer
Bio-Ag
buffer
Ab 2-1
buffer
buffer
Bio-Ag
buffer
Ab 2-2
buffer
buffer
Bio-Ag
buffer
Ab 2-3
buffer
Sensor type：SA
Sample：supernatant without dilution.
数据来自军事医学科学院.
Ab Screening & Order rank results
Sample ID
mAb 2B
mAb 2D
mono Ab 1-1
mono Ab 1-2
mono Ab 1-4
mono Ab 2-1
mono Ab 2-2
mono Ab 2-3
Respon
kdis
se
kdis(1/s) Error
0.4546 6.23E-03 1.41E-04
0.4178 5.68E-03 1.61E-04
0.7806 6.19E-03 1.42E-04
1.0178 1.08E-02 2.39E-04
1.0235 7.25E-03 1.64E-04
0.4227 4.78E-03 3.03E-04
0.8906 5.24E-03 1.53E-04
0.993 6.23E-03 1.36E-04
数据来自军事医学科学院.
Ab-Ag interaction
Kinetic analysis of anti-influenza antibodies using a His-tagged Antigen
Detection of anti-influenza virus antibodies in diluted
serum to a his-tagged HA antigen immobilized on AntiPenta His Biosensors on the Octet RED
Carney, PJ et al. CLINICAL AND VACCINE
IMMUNOLOGY, Sept. 2010, Vol. 17, No. 9
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
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
B-capture 2
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
B-capture 3
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
B-capture 4
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
+ MCP-1
- MCP-1
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.
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.
Capture
Ab1
Both selected pairs are specific for
MCP-1 and do not bind
MCP-2, MCP-3 or MCP-4
Detection
Ab 1
Detection
Ab2
Detection
Ab3
Detection
Ab4
+
-
-
-
Capture
Ab2
+
-
-
+
Capture
Ab3
+
-
-
+
Capture
Ab4
-
-
-
-
B capture 1
MCP-1
Detection 1
B capture 1
MCP- 2
Detection 1
B capture 1
MCP- 3
Detection 1
B capture 1
MCP- 4
Detection 1
B capture 3
MCP-1
Detection 1
B capture 3
MCP- 2
Detection 1
B capture 3
MCP- 3
Detection 1
B capture 3
MCP- 4
Detection 1
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）
Antibody-Virus
virus1
virus2
Anti-virus
antibody
virus3
Neg. virus
数据来自 中科院生物物理所。
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。
Protein- Q-dot
Immobilize protein on SA sensor surface and dilute quantum dot in solution as analyte
Data from Wuhan institute of virology,CAS
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.
Aggregation :protein-nanoparticle interaction
Pro-P1
Sample ID
P1
P1
P1
P1
P1
P1
P2
P2
P2
P2
P2
P2
KD (M)
2.95E-09
2.95E-09
2.95E-09
2.95E-09
2.95E-09
2.95E-09
1.22E-08
1.22E-08
1.22E-08
1.22E-08
1.22E-08
1.22E-08
kon(1/Ms)
8.07E+04
8.07E+04
8.07E+04
8.07E+04
8.07E+04
8.07E+04
1.39E+04
1.39E+04
1.39E+04
1.39E+04
1.39E+04
1.39E+04
kon Error
5.91E+02
5.91E+02
5.91E+02
5.91E+02
5.91E+02
5.91E+02
1.06E+02
1.06E+02
1.06E+02
1.06E+02
1.06E+02
1.06E+02
数据来自中科院高能所。
Pro-P2
kdis(1/s) kdis Error
2.38E-04 8.08E-06
2.38E-04 8.08E-06
2.38E-04 8.08E-06
2.38E-04 8.08E-06
2.38E-04 8.08E-06
2.38E-04 8.08E-06
1.71E-04 8.11E-06
1.71E-04 8.11E-06
1.71E-04 8.11E-06
1.71E-04 8.11E-06
1.71E-04 8.11E-06
1.71E-04 8.11E-06
kobs(1/s)
4.06E-02
2.04E-02
1.03E-02
5.28E-03
2.76E-03
1.50E-03
1.41E-02
7.14E-03
3.66E-03
1.91E-03
1.04E-03
6.06E-04
Full R^2
0.993833
0.993833
0.993833
0.993833
0.993833
0.993833
0.996337
0.996337
0.996337
0.996337
0.996337
0.996337
RNA-Protein
RNA immobilized to biosensors.
10uM
5uM
Bio-RNA
loading(10
nM)
Associated
with protein
2.5uM
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。
Protein-RNA
Protein immobilized to biosensors.
Conc. (nM)
200
100
50
25
12.5
KD (M)
2.97E-09
2.97E-09
2.97E-09
2.97E-09
2.97E-09
kon(1/Ms) kon Error
5.21E+05 4.41E+03
5.21E+05 4.41E+03
5.21E+05 4.41E+03
5.21E+05 4.41E+03
5.21E+05 4.41E+03
kdis(1/s)
1.55E-03
1.55E-03
1.55E-03
1.55E-03
1.55E-03
数据来自军事医学科学院。
kdis Error
9.22E-06
9.22E-06
9.22E-06
9.22E-06
9.22E-06
kobs(1/s)
1.06E-01
5.37E-02
2.76E-02
1.46E-02
8.06E-03
Full R^2
0.989572
0.989572
0.989572
0.989572
0.989572
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.
Membrane protein-protein interaction
0 Ca2+
Conc. (nM) KD (M)
kon(1/Ms) kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2
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
0.934619
2.5
1.61E-08
3.66E+06
8.60E+04
5.88E-02
1.07E-03
6.79E-02
0.959003
5
2.14E-08
2.75E+06
8.04E+04
5.89E-02
1.32E-03
7.26E-02
0.934619
5
1.61E-08
3.66E+06
8.60E+04
5.88E-02
1.07E-03
7.71E-02
0.959003
10
2.14E-08
2.75E+06
8.04E+04
5.89E-02
1.32E-03
8.64E-02
0.934619
10
1.61E-08
3.66E+06
8.60E+04
5.88E-02
1.07E-03
9.54E-02
0.959003
20
2.14E-08
2.75E+06
8.04E+04
5.89E-02
1.32E-03
1.14E-01
0.934619
20
1.61E-08
3.66E+06
8.60E+04
5.88E-02
1.07E-03
1.32E-01
0.959003
40
2.14E-08
2.75E+06
8.04E+04
5.89E-02
1.32E-03
1.69E-01
0.934619
40
1.61E-08
3.66E+06
8.60E+04
5.88E-02
1.07E-03
2.05E-01
0.959003
80
2.14E-08
2.75E+06
8.04E+04
5.89E-02
1.32E-03
2.79E-01
0.934619
80
1.61E-08
3.66E+06
8.60E+04
5.88E-02
1.07E-03
3.52E-01
0.959003
1mM Ca2+
100uM Ca2+
Conc. (nM) KD (M)
2.5
5
1.73E-08
1.73E-08
kon(1/Ms)
2.84E+06
2.84E+06
kon Error
6.28E+04
6.28E+04
kdis(1/s)
4.93E-02
4.93E-02
kdis Error
8.14E-04
8.14E-04
kobs(1/s)
5.64E-02
6.35E-02
Full R^2
Conc. (nM)
KD (M)
kon(1/Ms) kon Error
kdis(1/s)
kdis Error
kobs(1/s)
Full R^2
0.959743
1.25 7.59E-09
3.33E+06 1.04E+05
2.53E-02
4.72E-04
2.94E-02
0.893988
0.959743
2.5 7.59E-09
3.33E+06 1.04E+05
2.53E-02
4.72E-04
3.36E-02
0.893988
3.33E+06 1.04E+05
2.53E-02
4.72E-04
4.19E-02
0.893988
10
1.73E-08
2.84E+06
6.28E+04
4.93E-02
8.14E-04
7.77E-02
0.959743
5 7.59E-09
20
1.73E-08
2.84E+06
6.28E+04
4.93E-02
8.14E-04
1.06E-01
0.959743
10 7.59E-09
3.33E+06 1.04E+05
2.53E-02
4.72E-04
5.85E-02
0.893988
40
1.73E-08
2.84E+06
6.28E+04
4.93E-02
8.14E-04
1.63E-01
0.959743
20 7.59E-09
3.33E+06 1.04E+05
2.53E-02
4.72E-04
9.18E-02
0.893988
80
1.73E-08
2.84E+06
6.28E+04
4.93E-02
8.14E-04
2.77E-01
0.959743
数据来自北大医学部。
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。
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
蛋白X与多糖相互作用
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.
Pos. Contr.: Positive
binding?
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。
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。
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。
Summary
Workflow
Keys & Optimization
•
Select biosensors
•
Set proper control(pos. & neg. control)
•
Pre-wet sensors
•
•
Prepare samples
Know KD from publications or other
experiment and etc.
•
Assay buffer optimization(BSA & Tween-20)
•
Change sensor type
•
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
Pall ForteBio解决方案
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