Scott Zhou, North China FASMB:15810470035, Email:scott_zhou@ap.pall.com

Mar.20th, 2013

Octet TrainingPart III: Quantitation on the Octet

Agenda

BLI Quantitation Workflow

BLI Quantitation Applications

BLI Quantitation Workflow

Sandwich ELISA Assay Procedure

Bottom of well

(1) Plate is coated with a capture antibody(2) Sample is added, and any antigen present binds to capture antibody(3) Detecting antibody is added, and binds to antigen(4) Enzyme-linked secondary antibody is added, and binds to detecting

antibody(5) Chromogen/substrate is added, and is converted by enzyme to

detectable form

Typical read out could be fluorescence or luminescence

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%.

Distance between the two reflecting

surfaces = ℓ

Intensity λ =ƒ(λ, ℓ)R

ela

tiv

e I

nte

ns

ity

Wavelength (nm)

100%

0

nm

sh

ift

Time

可实时检测到两个反射表面间距的改变

Biolayer Interferometry （ BLI ）

Biosensor selectionAccording 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 Ab1~44. 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 QuantitationAnti-Human IgG, Anti-Murine or Protein A Biosensors

Calibrants

TestSamples

Bin

din

g (

nm

)

Time

Bin

din

g R

ate

Concentration

• 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.

Octet Workflow for Quantitation with Regeneration

Standards

TestSamples

Bind

ing

(nm

)

Time (sec)

Bind

ing

Rate

Concentration

• 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

Octet Biosensors

120

Buffer/Neut.Regen

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 1 700 10 500 … … Glycine pH2.0 SD

B 3 500 10 500 … … Glycine pH2.0 SD

C 10 300 10 500 … … Glycine pH2.0 SD

D 30 100 10 500 … … Glycine pH2.0 SD

E 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

SamplesCalibrators Regeneration Neutralization

• Octet Binding Curve = rate of increase in optical thickness as the sample binds to the sensor.

• Different protein concentrations result in different binding curves

Quantitation – Real-time binding curvesRate of binding correlates to concentration

Rate of binding is proportional to concentration

700 ug/mL500 ug/mL300 ug/mL

100 ug/mL

30 ug/mL

10 ug/mL

3 ug/mL

1 ug/mL

BLI Quantitation Applications

Fc-fused protein QuantitationOverview

• 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

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.

Red curve was 100 fold diluted sup1.

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

Theo.con. Detected Calculated Mean SD CV% Re%

2000

2081.6 2081.6

2001.925 80.22341 4.007314 100.09632029.4 2029.4

2005.2 2005.2

1891.5 1891.5

500

504.1 504.1

500.125 4.535324 0.906838 100.025493.6 493.6

501.2 501.2

501.6 501.6

125

122.6 122.6

124.85 1.813836 1.452812 99.88125.2 125.2

124.6 124.6

127 127

31.25

31.3 31.3

31.2 0.141421 0.453274 99.8431.2 31.2

31 31

31.3 31.3

7.8125

9.26 9.26

9.375 0.110905 1.182991 1209.47 9.47

9.3 9.3

9.47 9.47

1.9531

2.76 2.76

2.735 0.070475 2.576767 140.03382.77 2.77

2.63 2.63

2.78 2.78

0.2441

0.0744 0.0744

0.110225 0.032226 29.23629 45.155670.1477 0.1477

0.0948 0.0948

0.124 0.124

0.061

0.1567 0.1567

0.17705 0.028779 16.25487 290.24590.1974 0.1974

Undefined Undefined

Undefined Undefined

Spiked std. Theo.con. DetectedCalculate

dMean SD CV% Re%

0.97656ug/ml Fc-pro

0.9766

1.48 1.48

1.5125 0.06994 4.624162 154.87410.97656ug/ml Fc-pro 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

3.9063

5.68 5.68

5.675 0.040415 0.71215 145.27813.9063ug/ml Fc-pro 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

15.625

17.9 17.9

17.75 0.173205 0.975803 113.615.625ug/ml Fc-pro 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

62.5

61.4 61.4

60.6 1.416569 2.337572 96.9662.5ug/ml Fc-pro 58.5 58.5

62.5ug/ml Fc-pro 61 61

62.5ug/ml Fc-pro 61.5 61.5

250ug/ml Fc-pro

250

294.3 294.3

294.875

1.76706 0.599257 117.95250ug/ml Fc-pro 297.5 297.5

250ug/ml Fc-pro 293.7 293.7

250ug/ml Fc-pro 294 294

1000ug/ml Fc-pro

1000

1124.6 1124.6

1137.125

22.23022 1.954949 113.71251000ug/ml Fc-pro 1118 1118

1000ug/ml Fc-pro 1137.8 1137.8

1000ug/ml Fc-pro 1168.1 1168.1

Std.curve Spiked Std.

Unknowns interpolated from standard curve 1 with 15s reading compared to ELISA

Sample ELISA BLI Detected BLI Calculated Re% as compared to ELISA

1-100872.8

9.82 982 112.51145741 726.4 726.4 83.2263978

2-1001220

11.4 1140 93.442622952 700.1 700.1 57.3852459

3-100unknown

10 1000 　3 1168.1 1168.1 　

4-100704

11.3 1130 160.51136364 791.1 791.1 112.3721591

5-1001060.8

12.3 1230 115.95022625 807.6 807.6 76.13122172

6-100unknown

13.7 1370 　6 823.3 823.3 　

7-100704

11 1100 156.257 819.5 819.5 116.40625

8-100unknown

11.6 1160 　8 851.3 851.3 　

9-1001280.8

14.4 1440 112.42973149 779.8 779.8 60.88382261

10-100872.8

10.7 1070 122.593950510 720.4 720.4 82.53895509

11-100 11.2 1120 　12-100 1108 11.6 1160 104.693140815-100 unknown 3.28 328 　16-100 unknown 3.36 336 　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

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 59.2

59.15 0.070711 0.119545 118.350ug/ml Fc-pro 59.1 59.1

50ug/ml Fc-pro

12.5ug/ml Fc-pro12.5　

14.2 14.2

14.16667 0.251661 1.776432 113.333312.5ug/ml Fc-pro 13.9 13.9

12.5ug/ml Fc-pro 14.4 14.4

3.125ug/ml Fc-pro

3.125

3.48 3.48

3.426667 0.10116 2.952138 109.65333.125ug/ml Fc-pro 3.31 3.31

3.125ug/ml Fc-pro 3.49 3.49

0.78125ug/ml Fc-pro0.78125　

0.8571 0.8571

0.880767 0.023167 2.630355 112.73810.78125ug/ml Fc-pro 0.8818 0.8818

0.78125ug/ml Fc-pro 0.9034 0.9034

0.195313ug/ml Fc-pro

0.195313

0.2342 0.2342

0.275533 0.039714 14.41337 141.07310.195313ug/ml Fc-pro 0.3134 0.3134

0.195313ug/ml Fc-pro 0.279 0.279

Theo.con. Detected Calc.con. Mean SD CV% Re%

62.5

63.8 63.8

62.5 0.887 1.419108 10062.2 62.261.8 61.862.2 62.2

31.25

30.1 30.1

31.225 0.866 2.771959 99.9231.4 31.432.2 32.231.2 31.2

15.625

15.9 15.9

15.625 0.310 1.981245 10015.8 15.815.6 15.615.2 15.2

7.8125

7.23 7.23

7.7725 0.370 4.754429 99.4887.91 7.918.06 8.067.89 7.89

3.90625

3.95 3.95

3.9075 0.048 1.225115 100.0323.93 3.933.84 3.843.91 3.91

1.953125

1.85 1.85

1.9525 0.080 4.105306 99.9682 22.03 2.031.93 1.93

0.976563

0.995 0.995

0.9781 0.039 3.976498 100.15740.9683 0.96830.9291 0.92911.02 1.02

0.488281

0.4408 0.4408

0.48825 0.032 6.507182 99.99360.5036 0.50360.5007 0.50070.5079 0.5079

0.244141

0.2456 0.2456

0.2444 0.016 6.727338 100.10620.2521 0.25210.2211 0.22110.2588 0.2588

0.12207

0.0804 0.0804

0.133275 0.037 27.388 109.17890.1441 0.14410.1643 0.16430.1443 0.1443

0.061035

0.0639 0.0639

0.053575 0.014 26.25634 87.777280.0443 0.04430.0389 0.03890.0672 0.0672

0.030518

0.0298 0.0298

0.034425 0.021 61.6591 112.80410.0264 0.02640.0651 0.06510.0164 0.0164

0.015259

0.0333 0.0333

0.021875 0.009 40.07481 143.3580.0152 0.01520.0148 0.01480.0242 0.0242

Unknowns interpolated from standard curve 2 with 15s reading compared to ELISA

1-100~16-100 means 100-fold diluted samples.

Sample ELISA BLI Detected BLI Calculated Re% as compared to ELISA

1-100 872.8 9.72 972 111.36572-100 1220 10.3 1030 84.426233-100 unknown 9.31 931 　4-100 704 11.1 1110 157.67055-100 1060.8 11.3 1130 106.52346-100 unknown 10.9 1090 　7-100 704 10.8 1080 153.40918-100 unknown 12.2 1220 　9-100 1280.8 12.2 1220 95.25297

10-100 872.8 10.3 1030 118.01111-100 　 10.5 1050 　12-100 1108 10.1 1010 91.1552315-100 unknown 3.29 329 　16-100 unknown 2.74 274 　

Method ELISA Std. curve 1 Std. curve 2Analysis model ? 4PL 5PL Liner

sample

1 872.8 982 945 9722 1220 1140 1100 10303 unknown 1000 967 9314 704 1130 1090 11105 1060.8 1230 1180 11306 unknown 1370 1330 10907 704 1100 1060 10808 unknown 1160 1120 12209 1280.8 1440 1390 122010 872.8 1070 1030 103011 　 1120 1080 105012 1108 1160 1120 101015 unknown 328 308 32916 unknown 336 316 274

Test Conditions ? 400rpm,15s 400rpm,120sDynamic Range ? 2000ug/ml-7.8125ug/ml 62.5ug/ml-0.2441ug/ml

Summary

• 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 120s-300s reading and the dynamic range was 62.5ug/ml-0.2441ug/ml(2-fold dilution series, Linear point-to-point analysis).

• Sensors could be regenerated well in 10mM pH1.5 glycine buffer.

Crude sample detection: quantitationCase 1

Object: quantitate pro in supernatantSolution : Pro A sensor with regeneration steps.(1000rpm,

ReadTime 120s)

Matrix: supernatant from CHO cells without centrifuge

Outcome: good data.

Method development with Pro A sensorStd. curve in buffer with spike

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 1.31 100.0333.33 33.4 33.4 33.1 33.4 33.33 0.15 0.45 99.9811.11 11.2 11.3 10.9 11 11.10 0.18 1.64 100.003.70 3.7 3.74 3.69 3.69 3.71 0.02 0.64 100.141.24 1.25 1.26 1.24 1.19 1.24 0.03 2.52 100.410.41 0.4201 0.4158 0.3973 0.4148 0.41 0.01 2.44 100.920.14 0.1375 0.1375 0.1377 0.136 0.14 0.00 0.58 100.13

Std. SpikeOriginal (ug/ml)

Calculated (ug/ml)

Mean

(ug/ml)SD CV% Re%

(4 replicates)

1

0.8382

0.84 0.01 0.77 83.720.84170.82780.8412

5

4.22

4.18 0.04 0.90 83.604.134.184.19

20

17

17.68 0.50 2.82 88.3818.117.6

18

Method development with Pro A sensorStd. curve in medium with spike

Std. Curve obtained in mediumOriginal (ug/ml)

Calculated (ug/ml,4 replicates)

mean (ug/ml) SD CV% Re%

100.00 100.00 101.90 100.60 97.50 100.00 1.85 1.85 100.0033.33 32.90 33.60 33.70 33.20 33.35 0.37 1.11 100.0611.11 11.10 11.20 11.10 11.10 11.13 0.05 0.45 100.233.70 3.69 3.70 3.69 3.73 3.70 0.02 0.51 100.071.24 1.21 1.24 1.23 1.26 1.24 0.02 1.69 100.410.41 0.40 0.41 0.42 0.42 0.41 0.01 2.69 101.610.14 0.13 0.14 0.14 0.14 0.14 0.00 1.56 100.15

Std. Spike in mediumOriginal (ug/ml)

Calculated (ug/ml)

Mean

(ug/ml)SD CV% Re%

4 replicates

1

0.88

0.87 0.01 1.15 86.810.860.860.87

5

4.43

4.47 0.07 1.54 89.404.464.424.57

20

18.60

18.58 0.29 1.55 92.8818.6018.2018.90

Method development with Pro A sensorregeneration

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 0.21 1.99 95.95 10.80

3.33 3.26

3.26 0.00 0.00 97.90 3.26

1.11 1.09

1.10 0.01 0.65 98.65 1.10

20 times

33.30 31.80

31.50 0.42 1.35 94.59

4

31.20

11.10 10.30

10.55 0.35 3.35 95.05 10.80

3.33 3.26

3.29 0.04 1.29 98.80 3.32

1.11 1.13

1.13 0.01 0.63 101.35 1.12

30 times

33.30 31.80

31.25 0.78 2.49 93.84

2

30.70

11.10 10.30

10.50 0.28 2.69 94.59 10.70

3.33 3.23

3.25 0.03 0.87 97.60 3.27

1.11 1.09

1.10 0.01 1.29 99.10 1.11

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

1.12 0.01 0.63 100.45 1.11

50 times

33.30 30.90

30.30 0.85 2.80 90.99

2'(22)

29.70

11.10 9.85

10.08 0.32 3.16 90.77 10.30

3.33 3.16

3.18 0.02 0.67 95.35 3.19

1.11 1.07

1.08 0.01 0.66 96.85 1.08

60 times

33.30 30.30

29.60 0.99 3.34 88.89

3'(33)

28.90

11.10 9.69

9.95 0.36 3.63 89.59 10.20

3.33 3.11

3.15 0.05 1.57 94.44 3.18

1.11 1.11

1.10 0.01 1.29 99.10 1.09

Method development with Pro A sensorregeneration

Crude sample detection: quantitationCase 2

Object: quantitate pro X in milkSolution : AHC with regeneration steps.

Matrix: 100 fold dilution milk

Outcome: good data.

RED96,R2=0.99977 ELISA, R2=0.98

BLI vs ELISA

Standard curve obtained in milk with Octet RED96

Original Caculated MeanSD CV% Re%

ug/ml ug/ml ug/ml100 99.5 100.5 100.00 0.71 0.71 100.00

50 52.1 48.7 50.40 2.40 4.77 100.80 25 25.7 24.3 25.00 0.99 3.96 100.00

12.5 13.8 11.4 12.60 1.70 3.47 100.80 6.25 6.1 6.4 6.25 0.21 3.39 100.00 3.13 3.3 2.96 3.13 0.24 7.68 100.00 1.56 1.82 1.37 1.60 0.32 9.95 102.24

Sensor type: AHC with regenerationMatrix: 100 fold diluted milk数据来自中国农业大学。

Sample ELISA （ mg/ml ） SD CV% Fortebio(mg/ml) SD CV%

090216-beestings 2.54 0.1 5.7 2.25 0.2 2090216-1 month 3.83 0.1 1.7 4.74 1 7090216-2 months 1.15 0.02 1.7 1.075 0.4 3090216-3 months 0.5 0.004 0.7 0.426 0.1 2090216-4 months 0.4 0.02 5.5 0.6415 0.1 2

Std. curve: ELISA R2=0.98; Fortebio R2=0.99977. CV<10%； Re%： 84-118%

BLI vs ELISA

Sensor type: AHC with regenerationMatrix: 100 fold diluted milk数据来自中国农业大学。

Assay Protocols for Increasing Sensitivity on the Octet

||-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

||-Capture : Analyte High Speed Mixing

Longer Incubation

sensor1-Step

||-Capture : Analyte : Ab High Speed Mixing

Longer Incubation

2nd reagent

sensor2-Step

sensor3-Step

1st Step : 2nd Step : 3rd Step Amplification

Special Conjugate

Qualifying Concentration – Which Assay Method Fits Customer Need

Higher sensitivityExample : Clone Screening Using Sandwich Assay

2nd reagent format does NOT require wash step simple and easy protocol

YYYYYY

YY Y

2: Anti-hIgG Ab1: hIgG

Anti-hIgG (fc)

biosensor

Anti-hIgG Ab 2 min

incubation

YY Y hIgG; on Octet or offline longer

incubation

2-Step Clone Selection assay using Octet QK (sensitivity down to 156 pg/mL of HIgG)

ng/mL

10

5

2.5

1.25

0.6250.3130.1560

Run Condition

- O/N

- 500 rpm

- 2nd Ab = Special Conj.

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 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)

Pall ForteBio 解决方案

Label-free

Real time

Fluidics-free

Fast ， Accurate ， Easy.

www.fortebio.com