Samples

Report
Octet Training
Part III: Quantitation on the Octet
Scott Zhou, North China FAS
MB:15810470035, Email:[email protected]
Mar.20th, 2013
Agenda

BLI Quantitation Workflow

BLI Quantitation Applications
BLI Quantitation Workflow
Sandwich ELISA Assay Procedure
Typical read out could
be fluorescence or
luminescence
Bottom of well
(1)
(2)
(3)
(4)
(5)
Plate is coated with a capture antibody
Sample is added, and any antigen present binds to capture antibody
Detecting antibody is added, and binds to antigen
Enzyme-linked secondary antibody is added, and binds to detecting
antibody
Chromogen/substrate is added, and is converted by enzyme to
detectable form
Each step involves incubation time and wash steps in between. Manual ELISA
can be an all day assay for just a few plates. Multiple Steps add to higher CV
5-25%.
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
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
Octet Workflow for Quantitation
Anti-human IgG (Fc
specific), anti-murine
IgG (Fab’ specific), or
Protein A sensors
Standard samples of known
concentration used to
generate a standard curve
Unknown samples
Octet Automated Workflow for Quantitation
Calibrants
Binding (nm)
Anti-Human IgG, Anti-Murine or Protein A Biosensors
Test
Samples
•
•
•
Calibrants used to plot a binding rate vs
conc. calibration curve
The binding rates of test samples are
then measured and plotted on the
calibration curve to determine their
concentration
One sensor per one sample well; one
step assay with pre-made sensors.
Binding Rate
Time
Concentration
Octet Workflow for Quantitation with Regeneration
Octet Biosensors
Test
Samples
Binding (nm)
Standards
Time (sec)
•
•
•
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
Regen
Buffer/Neut.
Concentration
120
Lab Work Example – Quantitation Experiment
•
Set up the plate shown below:
1
2
3
4
A
1
700
10
500
B
3
500
10
C
10
300
D
30
E
10
11
…
…
Glycine pH2.0
SD
500
…
…
Glycine pH2.0
SD
10
500
…
…
Glycine pH2.0
SD
100
10
500
…
…
Glycine pH2.0
SD
100
30
10
500
…
…
Glycine pH2.0
SD
F
300
10
10
500
…
…
Glycine pH2.0
SD
G
500
3
10
500
…
…
Glycine pH2.0
SD
H
700
1
10
500
…
…
Glycine pH2.0
SD
Calibrators
•
•
•
•
Samples
5
6
7
8
9
Regeneration
Analyze using Protein A Biosensors and running the standard Protein A Protocol
Refer to Quick Start Q Assay pdf for further details of running Quantitation Assays
Generate a Quantitation Report
Calculate CVs of each of the 8 replicates of calibrators
12
Neutralization
Quantitation – Real-time binding curves
Rate of binding correlates to concentration
• Octet Binding Curve = rate of increase in optical
thickness as the sample binds to the sensor.
• Different protein concentrations result in different
binding curves
700 ug/mL
500 ug/mL
300 ug/mL
100 ug/mL
30 ug/mL
10 ug/mL
3 ug/mL
1 ug/mL
Rate of binding
is proportional to
concentration
BLI Quantitation Applications
Fc-fused protein Quantitation
Overview
•
•
•
•
•
Samples: 14 hFc-fused proteins in supernatant 1
Standards: Fc-fused proteins with original conc. of 92.09mg/ml
Biosensors : Protein A biosensor
Octet platform: RED96
Other reagents & consumables : fresh medium, supernatant 2, PBS,
10mM pH1.5 glycine, Greiner 96-well micro-plate, pipettes , tips and etc.
• Goal: CV% & Re%, throughput and etc. as to ELISA
Workflow for the Fc-fused protein quantitation
•
•
•
•
•
•
Test for dilution factors
Dilute standards and unknowns with diluted supernatant
Enter sample information into software
Bind Fc-fused protein to protein A biosensor
Generate standard curve & Regenerate biosensors
Bind known concentrations of Fc-fused proteins to regenerated
sensor
• Bind unknown samples & Regenerate biosensors
• Interpolate samples from standard curve to determine active
concentration
Determine dilution factor
Red curve was 100 fold diluted sup1.
A-H: PBS, 0-, 10- and 100-fold diluted fresh medium with PBS and 0-,10-,100- &1000-fold diluted.
PBS as control while fresh medium and blank sup1 was diluted with PBS, and finally 100-fold
diluted sup 1 was chosen based on a balance of matrix effect and sensitivity as showed above.
Standard curve 1 in 100-fold diluted sup 1
•
•
•
•
•
Sensors regenerated 10 times.
Dynamic range setup: 2000ug/ml-0.061ug/ml(4-fold dilution series)
Spiked standard: 1000ug/ml-0.9766ug/ml (4-fold dilution series)
Unknowns: Dilute unknowns with diluted supernatant 1
Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine
Standard curve 1 determined in 100-fold diluted sup 1
• Analysis model: unweighted 4PL
• Effective Dynamic range setup: 2000ug/ml-7.8125ug/ml(4-fold dilution
series)
• Spiked standard: 1000ug/ml-15.625ug/ml (4-fold dilution series)
• Octet settings: 400rpm, 15s reading
Standard curve 1 in 100-fold diluted sup 1
Spiked Std.
Std.curve
Theo.con. Detected Calculated
2000
500
125
31.25
7.8125
1.9531
0.2441
0.061
2081.6
2081.6
2029.4
2029.4
2005.2
2005.2
1891.5
1891.5
504.1
504.1
493.6
493.6
501.2
501.2
501.6
501.6
122.6
122.6
125.2
125.2
124.6
124.6
127
127
31.3
31.3
31.2
31.2
Mean
SD
CV%
Re%
Spiked std.
Theo.con. Detected Calculated Mean
0.97656ug/ml Fc-pro
2001.925 80.22341 4.007314 100.0963
1.48
1.48
1.58
1.58
0.97656ug/ml Fc-pro
1.43
1.43
0.9766ug/ml Fc-pro
1.56
1.56
3.9063ug/ml Fc-pro
5.68
5.68
5.73
5.73
3.9063ug/ml Fc-pro
5.64
5.64
3.9063ug/ml Fc-pro
5.65
5.65
15.625ug/ml Fc-pro
17.9
17.9
17.8
17.8
15.625ug/ml Fc-pro
17.5
17.5
15.625ug/ml Fc-pro
17.8
17.8
62.5ug/ml Fc-pro
61.4
61.4
58.5
58.5
61
61
0.97656ug/ml Fc-pro
0.9766
SD
CV%
Re%
1.5125 0.06994 4.624162 154.8741
500.125 4.535324 0.906838 100.025
3.9063ug/ml Fc-pro
3.9063
124.85
31.2
1.813836 1.452812
0.141421 0.453274
99.88
99.84
15.625ug/ml Fc-pro
31
31
31.3
31.3
9.26
9.26
9.47
9.47
9.3
9.3
9.47
9.47
62.5ug/ml Fc-pro
2.76
2.76
62.5ug/ml Fc-pro
2.77
2.77
2.63
2.63
5.675 0.040415 0.71215 145.2781
15.625
9.375
2.735
0.110905 1.182991
120
0.070475 2.576767 140.0338
62.5
62.5ug/ml Fc-pro
61.5
61.5
250ug/ml Fc-pro
294.3
294.3
297.5
297.5
293.7
293.7
250ug/ml Fc-pro
294
294
1000ug/ml Fc-pro
1124.6
1124.6
1118
1118
2.78
2.78
250ug/ml Fc-pro
0.0744
0.0744
250ug/ml Fc-pro
0.1477
0.1477
0.0948
0.0948
0.124
0.124
0.1567
0.1567
1000ug/ml Fc-pro
0.1974
0.1974
1000ug/ml Fc-pro
1137.8
1137.8
Undefined
Undefined
Undefined
Undefined
1000ug/ml Fc-pro
1168.1
1168.1
0.110225 0.032226 29.23629 45.15567
250
1000
0.17705 0.028779 16.25487 290.2459
17.75 0.173205 0.975803
113.6
60.6 1.416569 2.337572
96.96
294.87
1.76706 0.599257 117.95
5
1137.1
22.23022 1.954949 113.7125
25
Unknowns interpolated from standard curve 1 with 15s reading
compared to ELISA
Sample
1-100
1
2-100
2
3-100
3
4-100
4
5-100
5
6-100
6
7-100
7
8-100
8
9-100
9
10-100
10
ELISA
872.8
1220
unknown
704
1060.8
unknown
704
unknown
1280.8
872.8
11-100
12-100
15-100
16-100
1108
unknown
unknown
BLI
Detected
BLI Calculated
Re% as compared to ELISA
9.82
982
726.4
726.4
11.4
1140
700.1
700.1
112.5114574
83.2263978
93.44262295
57.3852459
10
1000
1168.1
1168.1
11.3
1130
791.1
791.1
12.3
1230
807.6
807.6
13.7
1370
823.3
823.3
11
1100
819.5
819.5
11.6
1160
851.3
851.3
14.4
1440
779.8
779.8
10.7
1070
720.4
720.4
11.2
1120
11.6
1160
3.28
328
3.36
336
160.5113636
112.3721591
115.9502262
76.13122172
156.25
116.40625
112.4297314
60.88382261
122.5939505
82.53895509
104.6931408
1-100~16-100 means 100-fold diluted samples while original samples
1-16 were tested meanwhile.
Standard curve 2 in 100-fold diluted sup 1
•
•
•
•
•
Sensors regenerated 10 times.
Dynamic range setup: 62.5ug/ml-0.0153ug/ml(2-fold dilution series)
Spiked standard: 50ug/ml-0.1593ug/ml (4-fold dilution series)
Unknowns: Dilute unknowns with diluted supernatant 1
Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine
Standard curve 2 determined in 100-fold diluted sup 1
• Analysis model: Linear point-to-point
• Effective Dynamic range setup: 62.5ug/ml-0.2441ug/ml(2-fold dilution
series)
• Spiked standard: 50ug/ml-0.7813ug/ml (4-fold dilution series)
• Octet settings: 400rpm, 120s reading
Theo.con. Detected Calc.con.
63.8
63.8
62.2
62.2
62.5
61.8
61.8
62.2
62.2
30.1
30.1
31.4
31.4
31.25
32.2
32.2
31.2
31.2
15.9
15.9
15.8
15.8
15.625
15.6
15.6
15.2
15.2
7.23
7.23
7.91
7.91
7.8125
8.06
8.06
7.89
7.89
3.95
3.95
3.93
3.93
3.90625
3.84
3.84
3.91
3.91
1.85
1.85
2
2
1.953125
2.03
2.03
1.93
1.93
0.995
0.995
0.9683 0.9683
0.976563
0.9291 0.9291
1.02
1.02
0.4408 0.4408
0.5036 0.5036
0.488281
0.5007 0.5007
0.5079 0.5079
0.2456 0.2456
0.2521 0.2521
0.244141
0.2211 0.2211
0.2588 0.2588
0.0804 0.0804
0.1441 0.1441
0.12207
0.1643 0.1643
0.1443 0.1443
0.0639 0.0639
0.0443 0.0443
0.061035
0.0389 0.0389
0.0672 0.0672
0.0298 0.0298
0.0264 0.0264
0.030518
0.0651 0.0651
0.0164 0.0164
0.0333 0.0333
0.0152 0.0152
0.015259
0.0148 0.0148
0.0242 0.0242
Mean
SD
CV%
Re%
62.5
0.887 1.419108
100
31.225
0.866 2.771959
99.92
15.625
0.310 1.981245
100
Standard curve 2 in 100-fold diluted sup 1
Std.curve
Spiked Std.
7.7725
0.370 4.754429
99.488
3.9075
0.048 1.225115
100.032
Spiked std.
1.9525
0.080 4.105306
99.968
Theo.con.
100.1574
99.9936
100.1062
109.1789
0.053575 0.014 26.25634
87.77728
0.034425 0.021 61.6591
112.8041
0.021875 0.009 40.07481
143.358
0.070711
0.119545
118.3
0.251661
1.776432 113.3333
0.10116
2.952138 109.6533
0.023167
2.630355 112.7381
0.039714
14.41337 141.0731
14.2
14.2
13.9
13.9 14.16667
14.4
14.4
3.48
3.48
3.31
3.31 3.426667
3.49
3.49
0.8571
0.8571
0.8818
0.8818 0.880767
0.78125ug/ml Fc-pro
0.9034
0.9034
0.195313ug/ml Fc-pro
0.2342
0.2342
0.3134
0.3134 0.275533
50
50ug/ml Fc-pro
12.5
12.5ug/ml Fc-pro
3.125
3.125ug/ml Fc-pro
0.195313ug/ml Fc-pro
0.133275 0.037 27.388
59.15
59.1
0.78125ug/ml Fc-pro
0.016 6.727338
Re%
59.2
0.78125ug/ml Fc-pro
0.2444
CV%
59.1
3.125ug/ml Fc-pro
0.032 6.507182
SD
59.2
3.125ug/ml Fc-pro
0.48825
Mean
50ug/ml Fc-pro
12.5ug/ml Fc-pro
0.039 3.976498
Calculated
50ug/ml Fc-pro
12.5ug/ml Fc-pro
0.9781
Detected
0.195313ug/ml Fc-pro
0.78125
0.195313
0.279
0.279
Unknowns interpolated from standard curve 2 with 15s reading
compared to ELISA
Sample
1-100
2-100
3-100
4-100
5-100
6-100
7-100
8-100
9-100
10-100
11-100
12-100
15-100
16-100
ELISA
872.8
1220
unknown
704
1060.8
unknown
704
unknown
1280.8
872.8
1108
unknown
unknown
BLI Detected
9.72
10.3
9.31
11.1
11.3
10.9
10.8
12.2
12.2
10.3
10.5
10.1
3.29
2.74
BLI Calculated
Re% as compared to ELISA
972
1030
931
1110
1130
1090
1080
1220
1220
1030
1050
1010
329
274
1-100~16-100 means 100-fold diluted samples.
111.3657
84.42623
157.6705
106.5234
153.4091
95.25297
118.011
91.15523
Summary
Method
Analysis model
1
2
3
4
5
6
7
sample
8
9
10
11
12
15
16
Test Conditions
Dynamic Range
ELISA
?
872.8
1220
unknown
704
1060.8
unknown
704
unknown
1280.8
872.8
Std. curve 1
4PL
5PL
982
945
1140
1100
1000
967
1130
1090
1230
1180
1370
1330
1100
1060
1160
1120
1440
1390
1070
1030
1120
1080
1108
1160
1120
unknown
328
308
unknown
336
316
?
400rpm,15s
?
2000ug/ml-7.8125ug/ml
Std. curve 2
Liner
972
1030
931
1110
1130
1090
1080
1220
1220
1030
1050
1010
329
274
400rpm,120s
62.5ug/ml-0.2441ug/ml
• A std curve for high concentration sample detection was determined as
std curve 1 as above.
• A std curve for low concentration sample detection was determined as
std curve 2 as above.
Conclusion
• A dilution factor of 100 for sup 1(samples in sup1) was determined
using PBS as control due to severe matrix effect of blank sup 1 as
well as fresh medium.
• A std curve with dynamic range 2000ug/ml-7.8125ug/ml (4-fold
dilution series, unweighted 4PL/5PL analysis) was developed for
high concentration samples under 400 rpm with 15s reading, and
samples were interpolated.
• More sensitive method was developed under 400 rpm with 120s300s reading and the dynamic range was 62.5ug/ml-0.2441ug/ml(2fold dilution series, Linear point-to-point analysis).
• Sensors could be regenerated well in 10mM pH1.5 glycine buffer.
Crude sample detection: quantitation
Case 1

Object: quantitate pro in supernatant
Solution : Pro A sensor with regeneration steps.(1000rpm,
ReadTime 120s)


Matrix: supernatant from CHO cells without centrifuge

Outcome: good data.
Method development with Pro A sensor
Std. curve in buffer with spike
Std. Spike
Original
(ug/ml)
Calculated
Mean
(ug/ml)
(ug/ml)
(4 replicates)
0.8382
0.8417
0.8278
0.8412
4.22
4.13
4.18
4.19
17
18.1
17.6
18
1
5
20
Std. Curve obtained in buffer
Calculated
(ug/ml,4 replicates)
Original
(ug/ml)
100.00
33.33
11.11
3.70
1.24
0.41
0.14
100.4
33.4
11.2
3.7
1.25
0.4201
0.1375
100.6
33.4
11.3
3.74
1.26
0.4158
0.1375
101
33.1
10.9
3.69
1.24
0.3973
0.1377
mean
(ug/ml)
98.1
33.4
11
3.69
1.19
0.4148
0.136
100.03
33.33
11.10
3.71
1.24
0.41
0.14
SD
1.31
0.15
0.18
0.02
0.03
0.01
0.00
CV%
1.31
0.45
1.64
0.64
2.52
2.44
0.58
Re%
100.03
99.98
100.00
100.14
100.41
100.92
100.13
SD
CV%
Re%
0.84
0.01 0.77 83.72
4.18
0.04 0.90 83.60
17.68
0.50 2.82 88.38
Method development with Pro A sensor
Std. curve in medium with spike
Std. Spike in medium
Original
(ug/ml)
Calculated
Mean
(ug/ml)
(ug/ml)
4 replicates
5
20
Std. Curve obtained in medium
Calculated
(ug/ml,4 replicates)
100.00
33.33
11.11
3.70
1.24
0.41
0.14
100.00
32.90
11.10
3.69
1.21
0.40
0.13
101.90
33.60
11.20
3.70
1.24
0.41
0.14
100.60
33.70
11.10
3.69
1.23
0.42
0.14
mean
(ug/ml)
97.50
33.20
11.10
3.73
1.26
0.42
0.14
100.00
33.35
11.13
3.70
1.24
0.41
0.14
SD
1.85
0.37
0.05
0.02
0.02
0.01
0.00
CV% Re%
0.88
0.86
0.87 0.01 1.15 86.81
0.86
0.87
4.43
4.46
4.47 0.07 1.54 89.40
4.42
4.57
18.60
18.60
18.58 0.29 1.55 92.88
18.20
18.90
1
Original
(ug/ml)
SD
CV%
1.85
1.11
0.45
0.51
1.69
2.69
1.56
Re%
100.00
100.06
100.23
100.07
100.41
101.61
100.15
Method development with Pro A sensor
regeneration
Samples calculated with loaded Std. curve obtained in medium
Regeneration
Original
(ug/ml)
33.30
11.10
10 times
3.33
1.11
33.30
11.10
20 times
3.33
1.11
33.30
11.10
30 times
3.33
1.11
Calculated
(ug/ml)
(duplicates)
32.20
31.90
10.50
10.80
3.26
3.26
1.09
1.10
31.80
31.20
10.30
10.80
3.26
3.32
1.13
1.12
31.80
30.70
10.30
10.70
3.23
3.27
1.09
1.11
Mean
(ug/ml)
32.05
10.65
SD
CV%
Re%
0.21
0.66
96.25
0.21
1.99
95.95
Plate No.
1
3.26
0.00
0.00
97.90
1.10
0.01
0.65
98.65
31.50
0.42
1.35
94.59
10.55
0.35
3.35
95.05
4
3.29
0.04
1.29
98.80
1.13
0.01
0.63
101.35
31.25
0.78
2.49
93.84
10.50
0.28
2.69
94.59
2
3.25
0.03
0.87
97.60
1.10
0.01
1.29
99.10
Method development with Pro A sensor
regeneration
Samples calculated with loaded Std. curve obtained in medium
Regeneration
Original
(ug/ml)
Calculated
(ug/ml)
Mean
(ug/ml)
SD
CV%
Re%
30.75
1.06
3.45
92.34
10.35
0.35
3.42
93.24
Plate No.
(duplicates)
33.30
11.10
40 times
3.33
1.11
33.30
11.10
50 times
3.33
1.11
33.30
11.10
60 times
3.33
1.11
31.50
30.00
10.10
10.60
3.18
3.25
1.12
1.11
30.90
29.70
9.85
10.30
3.16
3.19
1.07
1.08
30.30
28.90
9.69
10.20
3.11
3.18
1.11
1.09
3
3.22
0.05
1.54
96.55
1.12
0.01
0.63
100.45
30.30
0.85
2.80
90.99
10.08
0.32
3.16
90.77
3.18
0.02
0.67
95.35
1.08
0.01
0.66
96.85
29.60
0.99
3.34
88.89
9.95
0.36
3.63
89.59
3.15
0.05
1.57
94.44
1.10
0.01
1.29
99.10
2'(22)
3'(33)
Crude sample detection: quantitation
Case 2

Object: quantitate pro X in milk

Solution : AHC with regeneration steps.

Matrix: 100 fold dilution milk

Outcome: good data.
BLI vs ELISA
ELISA, R2=0.98
RED96,R2=0.99977
Standard curve obtained in milk with Octet RED96
Original
ug/ml
100
50
25
12.5
6.25
3.13
1.56
Caculated
ug/ml
99.5
100.5
52.1
48.7
25.7
24.3
13.8
11.4
6.1
6.4
3.3
2.96
1.82
1.37
Mean
ug/ml
100.00
50.40
25.00
12.60
6.25
3.13
1.60
SD
0.71
2.40
0.99
1.70
0.21
0.24
0.32
Sensor type: AHC with regeneration
Matrix: 100 fold diluted milk
数据来自中国农业大学。
CV%
0.71
4.77
3.96
3.47
3.39
7.68
9.95
Re%
100.00
100.80
100.00
100.80
100.00
100.00
102.24
BLI vs ELISA
Sample
ELISA(mg/ml) SD
090216-beestings
090216-1 month
090216-2 months
090216-3 months
090216-4 months
2.54
3.83
1.15
0.5
0.4
0.1
0.1
0.02
0.004
0.02
CV%
Fortebio(mg/ml)
SD
CV%
5.7
1.7
1.7
0.7
5.5
2.25
4.74
1.075
0.426
0.6415
0.2
1
0.4
0.1
0.1
2
7
3
2
2
 Std. curve: ELISA R2=0.98; Fortebio R2=0.99977.
 CV<10%;
 Re%:84-118%
Sensor type: AHC with regeneration
Matrix: 100 fold diluted milk
数据来自中国农业大学。
Assay Protocols for Increasing Sensitivity on the Octet
1st Step : 2nd Step : 3rd Step
1-Step
Amplification
sensor ||-Capture : Analyte
High Speed Mixing
Longer Incubation
2-Step
sensor ||-Capture : Analyte : Ab
High Speed Mixing
Longer Incubation
2nd reagent
3-Step
sensor ||-Capture : Analyte : Ab-Enz : 1) Substrate
High Speed Mixing
2) anti-Ab-Mass Longer Incubation
2nd reagent
• Last steps are measured on-line to obtain signal
• Longer incubation at the 1st step allows signal amplification
• 1st and 2nd steps can be done off-line to shorten the steps
PPT Substrate
Special Conjugate
Qualifying Concentration – Which Assay Method
Fits Customer Need
Higher sensitivity
Example : Clone Screening Using Sandwich Assay
Anti-hIgG
(fc)
biosensor
YYYYYY
Y Y Y
Y Y Y
hIgG; on Octet or
offline longer
incubation
Anti-hIgG
Ab 2 min
incubation
1: hIgG
2: Anti-hIgG Ab
2nd reagent format does NOT require wash step  simple and easy protocol
2-Step Clone Selection assay using Octet QK
Run Condition
(sensitivity down to 156 pg/mL of HIgG)
- O/N
- 500 rpm
- 2nd Ab = Special Conj.
ng/mL
10
5
2.5
1.25
0.625
0.313
0.156
0
ELISA Conversion Flow Chart
Obtain Assay Requirements and Existing ELISA Format (customer input)
Select Sensor Type (immobilization mode)
Select Assay Format (sensitivity & throughput)
Validating Assay Format
Managing NSB & Matrix Effect
Optimize Reagent Formulation
Further optimization if not meeting the spec.
(modifying configuration to increase specific signal and reduce NSB)
Summary
Workflow
• Test for dilution factors
• Dilute standards and unknowns with diluted
supernatant
• Enter sample information into software
• Bind Standard to pre-wetted biosensor
• Generate standard curve & Regenerate
biosensors
• Bind known concentration samples to
regenerated sensor
• Regenerate biosensors
• Bind unknown samples
• Interpolate samples from standard
curve(different models & different timewindows) to determine active concentration
• Calculate CV%, Re% of standard curve and
spiked samples and determine proper
standard curve
Optimization
• Shaking Speed( much higher more
sensitive)
• Detection time(longer more sensitive)
• Regeneration pH(sometimes need scouting)
• Data Models(try different models)
Pall ForteBio解决方案

Label-free

Real time

Fluidics-free

Fast,Accurate,Easy.
www.fortebio.com

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