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

2. Agenda
BLI Quantitation Workflow
BLI Quantitation Applications

3. BLI Quantitation Workflow

4. Sandwich ELISA Assay Procedure
Typical read out could be fluorescence or luminescence
Bottom of well
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%.

5. 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

6. 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

7. 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
How does it work? This is the tip of a fiber. We coat the tip end with a special optical layer. The capturing molecule is then attached to the tip. The tip is dipped into the sample containing target molecule. The target molecule binds to the capture molecule, and two form a molecular layer. A white light is directed into the fiber. Two beams will be reflected to the back end. The first beam comes from the tip as a reference. The second light comes from the molecular layer. The difference of two beams will cause a spectrum color pattern as shown here. The phase is a function of the molecular layer thickness and corresponding to the number of molecules on the tip surface.

8. Octet Automated Workflow for Quantitation Anti-Human IgG, Anti-Murine or Protein A Biosensors
Calibrants
Binding (nm)
Test Samples
Time
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
Concentration

9. Octet Workflow for Quantitation with Regeneration
Octet Biosensors
Standards
Binding (nm)
Test Samples
Time (sec)
120
Regen
Buffer/Neut.
The binding rates of test samples are measured and interpolated from the standard curve to determine concentration
96 samples analyzed in minutes
Reuse of standard curve is optional
Binding Rate
Concentration 9

10. Lab Work Example – Quantitation Experiment
Set up the plate shown below:
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 1 2 3 4 5 6 7 8 9 10 11 12 A
700
500 …
Glycine pH2.0 SD B C
300 D 30
100 E F G H
Calibrators
Samples
Regeneration
Neutralization

11. 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
Rate of binding is proportional to concentration
700 ug/mL
500 ug/mL
300 ug/mL
100 ug/mL
30 ug/mL
10 ug/mL
3 ug/mL
1 ug/mL
The amount of IgG in a sample is determined by the initial rate of binding of the antibody to either the anti-human IgG or Protein A biosensor.
The graph here shows the rate of association of IgG from 1ug/ml to 700ug/ml

12. BLI Quantitation Applications

13. 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

14. 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

15. 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.

16. 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 ug/ml (4-fold dilution series)
Unknowns: Dilute unknowns with diluted supernatant 1
Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine

17. Standard curve 1 determined in 100-fold diluted sup 1
Analysis model: unweighted 4PL
Effective Dynamic range setup: 2000ug/ml ug/ml(4-fold dilution series)
Spiked standard: 1000ug/ml ug/ml (4-fold dilution series)
Octet settings: 400rpm, 15s reading

18. Standard curve 1 in 100-fold diluted sup 1
Std.curve
Spiked Std.
Theo.con.
Detected
Calculated
Mean SD
CV%
Re%
2000
2081.6
2029.4
2005.2
1891.5
500
504.1
493.6
501.2
501.6
125
122.6
124.85
99.88
125.2
124.6
127
31.25
31.3
31.2
99.84 31
7.8125
9.26
9.375
120
9.47
9.3
1.9531
2.76
2.735
2.77
2.63
2.78
0.2441
0.0744
0.1477
0.0948
0.124
0.061
0.1567
0.1974
Undefined
Spiked std.
Theo.con.
Detected
Calculated
Mean SD
CV%
Re%
ug/ml Fc-pro
0.9766
1.48
1.5125
1.58
1.43
0.9766ug/ml Fc-pro
1.56
3.9063ug/ml Fc-pro
3.9063
5.68
5.675
5.73
5.64
5.65
15.625ug/ml Fc-pro
15.625
17.9
17.75
113.6
17.8
17.5
62.5ug/ml Fc-pro
62.5
61.4
60.6
96.96
58.5 61
61.5
250ug/ml Fc-pro
250
294.3
117.95
297.5
293.7
294
1000ug/ml Fc-pro
1000
1124.6
1118
1137.8
1168.1

19. Re% as compared to ELISA
Unknowns interpolated from standard curve 1 with 15s reading compared to ELISA
Sample
ELISA
BLI Detected
BLI Calculated
Re% as compared to ELISA
1-100
872.8
9.82
982 1
726.4
2-100
1220
11.4
1140 2
700.1
3-100
unknown 10
1000 3
1168.1
4-100
704
11.3
1130 4
791.1
5-100
1060.8
12.3
1230 5
807.6
6-100
13.7
1370 6
823.3
7-100 11
1100
156.25 7
819.5
8-100
11.6
1160 8
851.3
9-100
1280.8
14.4
1440 9
779.8
10-100
10.7
1070
720.4
11-100
11.2
1120
12-100
1108
15-100
3.28
328
16-100
3.36
336
1-100~ means 100-fold diluted samples while original samples 1-16 were tested meanwhile.

20. Standard curve 2 in 100-fold diluted sup 1
Sensors regenerated 10 times.
Dynamic range setup: 62.5ug/ml ug/ml(2-fold dilution series)
Spiked standard: 50ug/ml ug/ml (4-fold dilution series)
Unknowns: Dilute unknowns with diluted supernatant 1
Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine

21. Standard curve 2 determined in 100-fold diluted sup 1
Analysis model: Linear point-to-point
Effective Dynamic range setup: 62.5ug/ml ug/ml(2-fold dilution series)
Spiked standard: 50ug/ml ug/ml (4-fold dilution series)
Octet settings: 400rpm, 120s reading

22. Standard curve 2 in 100-fold diluted sup 1
Theo.con.
Detected
Calc.con.
Mean SD
CV%
Re%
62.5
63.8
0.887
100
62.2
61.8
31.25
30.1
31.225
0.866
99.92
31.4
32.2
31.2
15.625
15.9
0.310
15.8
15.6
15.2
7.8125
7.23
7.7725
0.370
99.488
7.91
8.06
7.89
3.95
3.9075
0.048
3.93
3.84
3.91
1.85
1.9525
0.080
99.968 2
2.03
1.93
0.995
0.9781
0.039
0.9683
0.9291
1.02
0.4408
0.032
0.5036
0.5007
0.5079
0.2456
0.2444
0.016
0.2521
0.2211
0.2588
0.0804
0.037
27.388
0.1441
0.1643
0.1443
0.0639
0.014
0.0443
0.0389
0.0672
0.0298
0.021
0.0264
0.0651
0.0164
0.0333
0.009
0.0152
0.0148
0.0242
Standard curve 2 in 100-fold diluted sup 1
Std.curve
Spiked Std.
Spiked std.
Theo.con.
Detected
Calculated
Mean SD
CV%
Re%
50ug/ml Fc-pro 50
59.2
59.15
118.3
59.1
12.5ug/ml Fc-pro
12.5
14.2
13.9
14.4
3.125ug/ml Fc-pro
3.125
3.48
3.31
3.49
ug/ml Fc-pro
0.8571
0.8818
0.9034
ug/ml Fc-pro
0.2342
0.3134
0.279

23. Re% as compared to ELISA
Unknowns interpolated from standard curve 2 with 15s reading compared to ELISA
Sample
ELISA
BLI Detected
BLI Calculated
Re% as compared to ELISA
1-100
872.8
9.72
972
2-100
1220
10.3
1030
3-100
unknown
9.31
931
4-100
704
11.1
1110
5-100
1060.8
11.3
1130
6-100
10.9
1090
7-100
10.8
1080
8-100
12.2
9-100
1280.8
10-100
11-100
10.5
1050
12-100
1108
10.1
1010
15-100
3.29
329
16-100
2.74
274
1-100~ means 100-fold diluted samples.

24. Summary
Method
ELISA
Std. curve 1
Std. curve 2
Analysis model ?
4PL
5PL
Liner
sample 1
872.8
982
945
972 2
1220
1140
1100
1030 3
unknown
1000
967
931 4
704
1130
1090
1110 5
1060.8
1230
1180 6
1370
1330 7
1060
1080 8
1160
1120 9
1280.8
1440
1390 10
1070 11
1050 12
1108
1010 15
328
308
329 16
336
316
274
Test Conditions
400rpm,15s
400rpm,120s
Dynamic Range
2000ug/ml ug/ml
62.5ug/ml ug/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.

25. 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 ug/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 120s-300s reading and the dynamic range was 62.5ug/ml ug/ml(2-fold dilution series, Linear point-to-point analysis).
Sensors could be regenerated well in 10mM pH1.5 glycine buffer.

26. 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.

27. Std. Curve obtained in buffer
Method development with Pro A sensor
Std. curve in buffer with spike
Std. Spike
Original (ug/ml)
Calculated (ug/ml)
Mean (ug/ml) SD
CV%
Re%
(4 replicates) 1
0.8382
0.84
0.01
0.77
83.72
0.8417
0.8278
0.8412 5
4.22
4.18
0.04
0.90
83.60
4.13
4.19 20 17
17.68
0.50
2.82
88.38
18.1
17.6 18
Std. Curve obtained in buffer
Original (ug/ml)
Calculated
(ug/ml,4 replicates)
mean (ug/ml) SD
CV%
Re%
100.00
100.4
100.6
101
98.1
100.03
1.31
33.33
33.4
33.1
0.15
0.45
99.98
11.11
11.2
11.3
10.9 11
11.10
0.18
1.64
3.70
3.7
3.74
3.69
3.71
0.02
0.64
100.14
1.24
1.25
1.26
1.19
0.03
2.52
100.41
0.41
0.4201
0.4158
0.3973
0.4148
0.01
2.44
100.92
0.14
0.1375
0.1377
0.136
0.00
0.58
100.13

28. Std. Curve obtained in medium
Method development with Pro A sensor
Std. curve in medium with spike
Std. Spike in medium
Original (ug/ml)
Calculated (ug/ml)
Mean (ug/ml) SD
CV%
Re%
4 replicates 1
0.88
0.87
0.01
1.15
86.81
0.86 5
4.43
4.47
0.07
1.54
89.40
4.46
4.42
4.57 20
18.60
18.58
0.29
1.55
92.88
18.20
18.90
Std. Curve obtained in medium
Original (ug/ml)
Calculated
(ug/ml,4 replicates)
mean (ug/ml) SD
CV%
Re%
100.00
101.90
100.60
97.50
1.85
33.33
32.90
33.60
33.70
33.20
33.35
0.37
1.11
100.06
11.11
11.10
11.20
11.13
0.05
0.45
100.23
3.70
3.69
3.73
0.02
0.51
100.07
1.24
1.21
1.23
1.26
1.69
100.41
0.41
0.40
0.42
0.01
2.69
101.61
0.14
0.13
0.00
1.56
100.15

29. Samples calculated with loaded Std. curve obtained in medium
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%
Plate No.
(duplicates)
10 times
33.30
32.20
32.05
0.21
0.66
96.25 1
31.90
11.10
10.50
10.65
1.99
95.95
10.80
3.33
3.26
0.00
97.90
1.11
1.09
1.10
0.01
0.65
98.65
20 times
31.80
31.50
0.42
1.35
94.59 4
31.20
10.30
10.55
0.35
3.35
95.05
3.29
0.04
1.29
98.80
3.32
1.13
0.63
101.35
1.12
30 times
31.25
0.78
2.49
93.84 2
30.70
0.28
2.69
10.70
3.23
3.25
0.03
0.87
97.60
3.27
99.10

30. Samples calculated with loaded Std. curve obtained in medium
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%
Plate No.
(duplicates)
40 times
33.30
31.50
30.75
1.06
3.45
92.34 3
30.00
11.10
10.10
10.35
0.35
3.42
93.24
10.60
3.33
3.18
3.22
0.05
1.54
96.55
3.25
1.11
1.12
0.01
0.63
100.45
50 times
30.90
30.30
0.85
2.80
90.99
2'(22)
29.70
9.85
10.08
0.32
3.16
90.77
10.30
0.02
0.67
95.35
3.19
1.07
1.08
0.66
96.85
60 times
29.60
0.99
3.34
88.89
3'(33)
28.90
9.69
9.95
0.36
3.63
89.59
10.20
3.11
3.15
1.57
94.44
1.10
1.29
99.10
1.09

31. 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.

32. Standard curve obtained in milk with Octet RED96
BLI vs ELISA
ELISA, R2=0.98
RED96,R2=
Standard curve obtained in milk with Octet RED96
Original
Caculated
Mean SD
CV%
Re%
ug/ml
100
99.5
100.5
100.00
0.71 50
52.1
48.7
50.40
2.40
4.77
100.80 25
25.7
24.3
25.00
0.99
3.96
12.5
13.8
11.4
12.60
1.70
3.47
6.25
6.1
6.4
0.21
3.39
3.13
3.3
2.96
0.24
7.68
1.56
1.82
1.37
1.60
0.32
9.95
102.24
Sensor type: AHC with regeneration
Matrix: 100 fold diluted milk
数据来自中国农业大学。

33. BLI vs ELISA Sample ELISA（mg/ml） SD CV% Fortebio(mg/ml) SD CV%
beestings
month
months
months
months
Std. curve: ELISA R2=0.98; Fortebio R2=
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。

34. Assay Protocols for Increasing Sensitivity on the Octet
1st Step : 2nd Step : 3rd Step Amplification
1-Step
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
PPT Substrate
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
Special Conjugate

35. Qualifying Concentration – Which Assay Method Fits Customer Need

36. Example : Clone Screening Using Sandwich Assay
Higher sensitivity
Example : Clone Screening Using Sandwich Assay Y
2: Anti-hIgG Ab
1: hIgG
Anti-hIgG (fc) biosensor
Anti-hIgG Ab 2 min incubation
hIgG; on Octet or offline longer incubation
2nd reagent format does NOT require wash step  simple and easy protocol 36

37. 2-Step Clone Selection assay using Octet QK (sensitivity down to 156 pg/mL of HIgG)
Run Condition
O/N
500 rpm
2nd Ab = Special Conj.
ng/mL 10 5
2.5
1.25
0.625
0.313
0.156

38. 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)

39. 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 time-windows) 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)

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