Label-Free Assays for High-Throughput Monoclonal Antibody ...

Label-Free Assays for High-Throughput 

Monoclonal Antibody Characterization 



Broadcast Date: Thursday, December 13, 2012 

Time: 2 pm ET, 11 am PT 

Sponsored by



Your Moderator 

Tamlyn Oliver 

Managing Editor 

Genetic Engineering & Biotechnology News



Sriram Kumaraswamy, Ph.D. 

Director, Applications 

Fortebio - A Division of Pall Life Sciences

Bio-layer Interferometry (BLI): 

Label-Free Technology for Rapid 

Protein Quantitation and Binding Kinetics 

Sriram Kumaraswamy, Ph.D., 

Global Director of Applications 

ForteBio, A Division of Pall Life Sciences

BLI Instruments for Rapid Protein Analysis 

Label-free protein analysis platform for easy, rapid 

quantitation, binding affinity and kinetics 

Octet RED384 

Octet QK384 

Octet RED96 

Octet QKe 

BLItz

BLI: One Platform, Many Capabilities 

• Direct 

• Sandwich 

• ELISA 

• mg/mL to sub-ng/mL 

Quantitation 

• Label-free k a , k d , K D 

• Proteins 

• Peptides, Oligos 

• Small molecules 

Kinetics

Shift (nm) 

Bio-layer Interferometry (BLI) Technology 

Time(s)

BLI Detects Broad Range of Analytes

ForteBio Biosensors and Assay Kits 

Biosensor 

AHQ 

AMQ 

FAB 

ProA 

ProG 

ProL 

SA 

HIS 

NTA 

GST 

AMC 

AHC 

AR2G 

APS 

SSA 

Kits/Methods 

Biosensor Functionality 

Anti-Human IgG Fc 

Anti-Murine IgG Fv 

Anti-Human Fab CH1 

Protein A 

Protein G 

Protein L 

Streptavidin 

Anti-Penta-HIS 

Ni-Tris NTA 

Anti-GST 

Anti-Murine IgG Fc Capture 

Anti-Human IgG Fc Capture 

Amine Reactive 2 nd Gen 

Aminopropylsilane 

Super Streptavidin 

Dip and Read Immunogenicity 

Dip and Read Residual Protein A 

Dip and Read HCP Custom Assay 

Custom biosensors can be made to 

specification by Fortebio

Sensors Move to Samples – No Microfluidics

Kinetic Characterization of Protein:Protein 

Interactions 

Kinetic information often reveals details of interactions missed 

in a purely affinity-based screen that are important in ranking 

and binning clones. 

Example: 

1) Tightest binding clones may not have the slowest off-rates 

Sample 

K D = 51 nM 

K D = 411 nM 

K D = 48 nM 

K D = 473 nM 

7 

slower 

off-rate 

8 

tighter 

affinity 

K D = 9 nM 

K D = 170 nM 

K D = 9 nM 

K D = 129 nM

BLI Instruments – Benefits For Everyone 

• Label-free measurement on native proteins and 

other biomolecules 

• Specific detection of protein of interest in crude 

samples 

• Simple assay technique (Dip and Read) 

• Higher throughput, microplate format 

• Multiple measurement capabilities in one system



Vishal Kamat, Ph.D. 

Scientist 

Therapeutic Proteins 

Regeneron Pharmaceuticals

Label-Free Epitope Binning Strategies Used in 

the Characterization of Fully Human Therapeutic 

Monoclonal Antibodies Isolated from 

VelocImmune® Mice 


Scientist, Therapeutic Proteins 

Regeneron Pharmaceuticals, Inc. 

Tarrytown, NY 

13 th December, 2012 

Confidential 

VISHAL KAMAT

Presentation Overview 

‣ Cross-Competition Assay Formats 

‣ Label-Free Technology Platforms 

‣ Advantages & Challenges of Different Platforms 

‣ Data Analysis 

‣ Interpretation of Results 

Confidential 

VISHAL KAMAT

‣ Regeneron is a fully integrated biopharmaceutical company that discovers, invents, develops, 

manufactures, and commercializes medicines for the treatment of serious medical conditions 

– Marketed Products: ARCALYST ® , EYLEA ® & ZALTRAP ® 

– Product Pipeline: 11 product candidates in various stages of clinical development 

‣ In 2012, Regeneron was Rated #1 among all global biopharmaceutical companies by Science magazine 

‣ Technology Platforms - VelociGene®, VelociMouse® 

‣ VelocImmune® Mice – Produces monoclonal antibodies with human variable region but preserving the 

mouse constant regions 

Confidential 

VISHAL KAMAT

Why is Epitope Binning Important? 

‣ Primary Screening: Survey different properties of therapeutic antibodies such as binding affinity, 

binding specificity & species cross-reactivity, functional properties, etc. 

‣ Epitope Binning: Potential to provide additional information to the “Classical” primary 

screening paradigm 

‣ Mode of Receptor Antagonism: Direct vs Steric 

Crystal Structure of EGFR ectodomain bound to Fab fragments of C225 (Cetuximab) and Matuzumab 

Fab-C225 

Fab-Matuzumab 

Fab-Matuzumab 

Confidential 

Kamat, V. (2010) “Combined Use of Multiple Monoclonal Antibodies Targeting Epidermal Growth 

Factor Receptor for Improved Cancer Therapeutics”, Ph.D. Drexel University 

VISHAL KAMAT

Typical Cross-Competition Assay Formats - (ELISA, Biacore, Octet, etc.) 

Classical 

Sandwich Assay 

Premix 

Assay 

In Tandem 

Assay 

• Need good quality homogeneous 

reagents 

• Antigen dissociation from mAb-1 

• Use anti-hFc or anti-mFc or anti-Fab 

surface to capture mAb-1. Need to 

quench unsaturated capture 

surface. 

• Use new sensor surface or optimize 

regeneration condition 

• Works for heterogeneous & dimeric 

antigens 

• Need lot of reagents 

mAb-1 (Saturating mAb) 

• Use anti-hFc or anti-mFc or anti-Fab 

surface to capture mAb-1. Need to 

quench unbound capture surface. 

mAb-2 (Competing mAb) 



Target / Antigen 

• Works for heterogeneous reagents 

• Tagged antigen or directly couple to 

the surface 

• Influenced by the size of antigen 

• Capturing the antigen might block the 

epitope for mAb binding 



Abdiche, Y. N. et al. (2012) J. Immunol. 

Methods 382(1-2), pp. 101-116 

Confidential 

VISHAL KAMAT

Different Interaction Analysis Platforms in Regeneron 

Biacore 2000 

Biacore 3000 

Biacore T200 Biacore 4000 

Octet ® RED96 

Octet ® QK96 

KinExA ® 3200 

Luminex ® 

Octet ® 

QK384 

Confidential 

VISHAL KAMAT

SPR High Throughput Technology – Biacore 4000 

1 2 3 4 5 1 2 3 4 5 

1 2 3 4 5 1 2 3 4 5 

Flow Cell 1 Flow Cell 2 Flow Cell 3 Flow Cell 4 

Confidential 

VISHAL KAMAT

Cross-Competition Assay On Biacore 4000 

• First mAb (mAb-1) is 

immobilized on chip surface 

(16 mAbs immobilized) 

• Antigen was allowed to first 

bind to mAb-1 

• Second mAb (mAb-2) was 

later injected and binding 

response was measured in 

real time 

• Surface regenerated at the 

end of each cycle, repeated 

32 times 

Confidential 

VISHAL KAMAT

Challenges: Failure to Completely Regenerate mAb-1 Surface 

a 

c 

a – Complete Regeneration of Surface 

Antigen bound to mAb-1 was completely 

dissociated from mAb-1 surface all 

the way to the baseline 

c 

b 

c 

a 

b – Partial Regeneration of Surface 

Antigen bound to mAb-1 was partially 

dissociated from mAb-1 surface 

c – Poor Regeneration of Surface 

Antigen bound to mAb-1 was minimally 

dissociated from mAb-1 surface 

Confidential 

VISHAL KAMAT

Cross-Competition Cross-Competition on Octet – Optimization Octet of Regeneration Condition 

• No Regeneration: 

• 32x32 mAb cross-competition – 1024 anti-His Octet biosensors – Cost ~$7000 

• With Regeneration (16 regenerations per biosensor) : 

• 32x32 mAb cross-competition – 64 anti-His Octet biosensors – Cost ~$440 

3sec pulse of Gly pH2.0, 2x 

(16 Regeneration Cycles) 

Confidential 

VISHAL KAMAT

Challenges: Antigen Capture Using anti-His Sensors Depends on Protein 

All the antigen samples were prepared at 20ug/ml concentration 

Confidential 

VISHAL KAMAT

Schematic of Cross-Competition Assay On Octet 

D 

D 

Work Flow 

R 

R 

R 

R 

B 

B 

B 

B 

L 

L 

L 

L 

1 

1 

1 

1 

1 

2 

3 

4 

1. Regenerate Surface 

(3sec pulse of Gly pH2.0, 2x) 

2. Establish Stable Baseline 

3. Load Antigen 

Anti-Penta- 

His sensors 

R 

B 

L 

1 

5 

4. Saturate Antigen Surface 

with mAb-1 

5. Check Binding of mAb-2 

Confidential 

VISHAL KAMAT

32x32 Cross-Competition Experimental Set-up on QK384 

• Set-up two 384 well 

plates for a typical 

32x32 cross-competition 

– 16x32 MATRIX 

• Need to include a nonspecific 

mAb as a 

negative control 

• Can set-up at the most 

31x31 cross-competition 

Confidential 

VISHAL KAMAT

Raw Data for a Typical 384 Well Plate – 16x32 Cross-Competition 

ForteBio Data Analysis: Raw Data for a 16x32 Cross-Competition Assay 

Confidential 

VISHAL KAMAT

Baseline Drift Observed Following Sensor Regeneration 

Confidential 

VISHAL KAMAT

Development of Data Analysis Tool 

Sensorgrams from 64 anti-His Octet Sensors 

Open the result in Octet Data 

Analysis Software & Save Raw Data 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

Microsoft Excel Macro: 

Chops the long binding cycle with 

16 regenerations into individual 

cycles & saves data as text file 

Overlay of 1024 Sensorgrams 

Sensor 

Regeneration 

Scrubber 2.0c: 

Open the saved text file 

Antigen 

Capture 

mAb-1 

Binding 

mAb-2 

Binding 

Measure Binding Responses: 

Antigen captured 

mAb-1 binding 

mAb-2 binding 

Confidential 

VISHAL KAMAT

Data Analysis – Raw Data for 26x26 Cross-Competition 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 

mAb 

# 

Binding Respone of mAb-2 Binding to Antigen Pre-bound to mAb-1 (nm) 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.84 ± 0.12 1.20 ± 0.17 1 0.11 0.87 0.03 0.90 0.36 0.69 0.04 0.03 0.77 0.02 0.02 0.02 0.01 0.47 0.97 0.85 0.29 0.86 0.67 0.08 0.04 0.66 0.80 0.41 0.15 0.10 0.08 

0.88 ± 0.14 0.84 ± 0.12 2 1.24 0.02 1.06 0.87 0.70 0.78 0.07 0.08 0.73 0.07 0.08 0.03 0.06 0.53 0.96 0.86 0.39 0.93 0.07 0.13 0.10 0.71 0.81 0.54 0.19 0.44 0.04 

0.82 ± 0.11 0.88 ± 0.14 3 0.19 0.88 0.06 0.89 0.64 0.72 0.04 0.03 0.76 -0.03 0.04 0.00 0.05 0.48 0.93 0.81 0.34 0.83 0.71 0.06 0.02 0.63 0.78 0.41 0.16 0.07 0.05 

0.84 ± 0.11 0.78 ± 0.11 4 1.26 0.90 1.03 0.03 0.66 0.75 0.90 0.84 0.76 0.68 0.78 0.03 0.04 0.48 0.89 0.77 0.36 0.81 0.70 0.96 0.86 0.64 0.76 0.46 0.15 0.46 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 1.05 0.83 1.01 0.81 0.02 0.72 0.84 0.79 0.71 0.62 0.78 -0.01 0.00 0.42 0.92 0.79 0.31 0.82 0.69 0.89 0.85 0.61 0.70 0.41 0.14 0.38 -0.01 

0.83 ± 0.13 0.72 ± 0.11 6 1.23 0.85 1.05 0.87 0.70 0.04 0.85 0.81 0.78 0.63 0.79 0.02 0.04 0.05 0.95 0.82 0.02 0.22 0.08 0.91 0.78 0.05 0.76 0.03 0.19 0.39 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.22 0.02 0.02 0.89 0.69 0.76 0.02 0.02 0.81 0.01 0.01 0.01 0.01 0.49 0.98 0.84 0.36 0.82 0.72 0.02 0.02 0.62 0.77 0.39 0.17 0.05 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.24 0.02 0.07 0.90 0.70 0.77 0.04 0.03 0.88 0.05 0.06 0.04 0.04 0.50 1.02 0.87 0.37 0.96 0.74 0.07 0.04 0.70 0.83 0.45 0.23 0.10 0.07 

0.84 ± 0.12 0.89 ± 0.12 9 1.23 0.76 1.07 0.89 0.67 0.73 0.90 0.91 0.04 0.68 0.84 0.05 0.02 0.49 0.94 0.86 0.34 0.02 0.74 1.01 0.91 0.67 0.04 0.54 0.18 0.31 0.03 

0.82 ± 0.12 0.78 ± 0.10 10 0.33 0.06 0.13 0.83 0.68 0.79 0.12 0.10 0.84 0.03 0.10 0.05 0.03 0.51 0.97 0.84 0.41 0.90 0.08 0.13 0.11 0.68 0.84 0.46 0.18 0.44 0.02 

0.84 ± 0.12 0.89 ± 0.13 11 0.21 0.01 0.04 0.85 0.66 0.75 0.01 0.03 0.72 0.01 0.03 0.04 0.04 0.49 0.95 0.85 0.33 0.88 0.73 0.01 0.02 0.66 0.81 0.46 0.19 0.05 0.02 

0.82 ± 0.13 0.02 ± 0.01 12 1.26 0.91 1.02 0.76 0.70 0.75 0.88 0.85 0.80 0.70 0.79 0.04 0.04 0.53 0.95 0.80 0.40 0.86 0.74 0.89 0.79 0.65 0.72 0.44 0.17 0.40 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 1.24 0.93 1.07 0.86 0.75 0.77 0.89 0.85 0.77 0.69 0.80 0.04 0.02 0.53 0.94 0.82 0.41 0.88 0.75 0.93 0.76 0.64 0.76 0.41 0.17 0.40 0.02 

0.83 ± 0.12 0.71 ± 0.10 14 1.28 0.89 1.04 0.88 0.72 0.09 0.87 0.85 0.83 0.67 0.81 0.01 0.00 0.04 0.92 0.83 -0.02 0.35 0.11 0.89 0.79 0.07 0.73 0.00 0.12 0.38 -0.02 

0.85 ± 0.13 0.78 ± 0.12 15 1.26 0.91 0.96 0.84 0.68 0.76 0.81 0.83 0.82 0.63 0.77 0.06 0.06 0.49 0.02 0.77 0.39 0.88 0.76 0.97 0.86 0.66 0.78 0.45 0.20 0.42 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 1.34 0.92 1.00 0.86 0.69 0.77 0.89 0.85 0.78 0.67 0.76 0.03 0.03 0.51 0.94 0.03 0.39 0.90 0.73 0.95 0.87 0.68 0.79 0.48 0.17 0.43 0.04 

0.82 ± 0.12 0.45 ± 0.06 17 1.27 0.87 0.92 0.78 0.66 0.29 0.79 0.80 0.80 0.64 0.68 0.04 0.00 0.18 0.88 0.77 0.07 0.44 0.31 0.87 0.79 0.26 0.76 0.13 0.15 0.37 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 1.22 0.82 0.97 0.82 0.64 0.15 0.85 0.84 0.06 0.59 0.78 0.03 0.02 0.11 0.94 0.78 0.02 0.01 0.69 0.94 0.88 0.06 0.07 0.02 0.16 0.40 -0.01 

0.80 ± 0.12 0.74 ± 0.11 19 1.23 0.11 1.02 0.80 0.64 0.06 0.81 0.78 0.75 0.09 0.78 0.05 0.01 0.03 0.85 0.77 0.04 0.83 0.02 0.91 0.77 0.06 0.73 0.02 0.16 0.38 0.03 

0.82 ± 0.13 0.88 ± 0.15 20 0.24 0.04 0.05 0.89 0.68 0.73 0.04 0.05 0.78 0.05 0.06 0.06 0.04 0.46 0.99 0.83 0.36 0.88 0.74 0.04 0.05 0.63 0.77 0.42 0.21 0.07 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.22 0.01 0.03 0.86 0.64 0.70 0.02 0.01 0.80 0.01 0.01 0.02 0.01 0.44 0.98 0.73 0.31 0.91 0.71 0.01 0.03 0.57 0.77 0.38 0.15 0.04 0.01 

0.86 ± 0.13 0.75 ± 0.11 22 1.25 0.86 1.00 0.84 0.69 0.07 0.82 0.81 0.80 0.66 0.75 0.01 0.04 0.05 0.93 0.83 -0.01 0.23 0.10 0.91 0.81 0.07 0.77 0.00 0.16 0.42 0.00 

0.82 ± 0.11 0.90 ± 0.13 23 1.25 0.83 0.98 0.87 0.67 0.75 0.91 0.88 0.04 0.68 0.82 0.04 0.04 0.46 1.00 0.81 0.35 0.02 0.73 0.97 0.91 0.66 0.05 0.51 0.19 0.35 0.02 

0.80 ± 0.12 0.56 ± 0.08 24 1.19 0.82 0.96 0.80 0.67 0.18 0.80 0.77 0.82 0.62 0.73 0.00 -0.01 0.10 0.89 0.70 0.02 0.33 0.20 0.84 0.74 0.16 0.74 0.07 0.13 0.38 0.00 

0.83 ± 0.12 0.27 ± 0.03 25 1.23 0.92 1.00 0.86 0.71 0.75 0.86 0.83 0.82 0.66 0.77 0.06 0.05 0.47 0.95 0.81 0.40 0.88 0.75 0.90 0.88 0.68 0.82 0.50 0.08 0.42 0.05 

0.81 ± 0.13 0.52 ± 0.09 26 0.23 0.81 0.03 0.84 0.68 0.73 0.05 0.04 0.68 0.63 0.04 0.02 0.02 0.49 0.96 0.77 0.35 0.88 0.65 0.05 0.03 0.62 0.65 0.43 0.16 0.05 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 1.28 0.95 1.03 0.86 0.72 0.74 0.89 0.85 0.81 0.69 0.81 0.05 0.04 0.51 0.95 0.77 0.40 0.86 0.74 0.88 0.81 0.61 0.71 0.46 0.18 0.39 0.03 

27 

(-) 

Confidential 

VISHAL KAMAT

Data Analysis – Highlight Self-Self Competition 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 

mAb 

# 


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.84 ± 0.12 1.20 ± 0.17 1 0.11 0.87 0.03 0.90 0.36 0.69 0.04 0.03 0.77 0.02 0.02 0.02 0.01 0.47 0.97 0.85 0.29 0.86 0.67 0.08 0.04 0.66 0.80 0.41 0.15 0.10 0.08 

0.88 ± 0.14 0.84 ± 0.12 2 1.24 0.02 1.06 0.87 0.70 0.78 0.07 0.08 0.73 0.07 0.08 0.03 0.06 0.53 0.96 0.86 0.39 0.93 0.07 0.13 0.10 0.71 0.81 0.54 0.19 0.44 0.04 

0.82 ± 0.11 0.88 ± 0.14 3 0.19 0.88 0.06 0.89 0.64 0.72 0.04 0.03 0.76 -0.03 0.04 0.00 0.05 0.48 0.93 0.81 0.34 0.83 0.71 0.06 0.02 0.63 0.78 0.41 0.16 0.07 0.05 

0.84 ± 0.11 0.78 ± 0.11 4 1.26 0.90 1.03 0.03 0.66 0.75 0.90 0.84 0.76 0.68 0.78 0.03 0.04 0.48 0.89 0.77 0.36 0.81 0.70 0.96 0.86 0.64 0.76 0.46 0.15 0.46 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 1.05 0.83 1.01 0.81 0.02 0.72 0.84 0.79 0.71 0.62 0.78 -0.01 0.00 0.42 0.92 0.79 0.31 0.82 0.69 0.89 0.85 0.61 0.70 0.41 0.14 0.38 -0.01 

0.83 ± 0.13 0.72 ± 0.11 6 1.23 0.85 1.05 0.87 0.70 0.04 0.85 0.81 0.78 0.63 0.79 0.02 0.04 0.05 0.95 0.82 0.02 0.22 0.08 0.91 0.78 0.05 0.76 0.03 0.19 0.39 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.22 0.02 0.02 0.89 0.69 0.76 0.02 0.02 0.81 0.01 0.01 0.01 0.01 0.49 0.98 0.84 0.36 0.82 0.72 0.02 0.02 0.62 0.77 0.39 0.17 0.05 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.24 0.02 0.07 0.90 0.70 0.77 0.04 0.03 0.88 0.05 0.06 0.04 0.04 0.50 1.02 0.87 0.37 0.96 0.74 0.07 0.04 0.70 0.83 0.45 0.23 0.10 0.07 

0.84 ± 0.12 0.89 ± 0.12 9 1.23 0.76 1.07 0.89 0.67 0.73 0.90 0.91 0.04 0.68 0.84 0.05 0.02 0.49 0.94 0.86 0.34 0.02 0.74 1.01 0.91 0.67 0.04 0.54 0.18 0.31 0.03 

0.82 ± 0.12 0.78 ± 0.10 10 0.33 0.06 0.13 0.83 0.68 0.79 0.12 0.10 0.84 0.03 0.10 0.05 0.03 0.51 0.97 0.84 0.41 0.90 0.08 0.13 0.11 0.68 0.84 0.46 0.18 0.44 0.02 

0.84 ± 0.12 0.89 ± 0.13 11 0.21 0.01 0.04 0.85 0.66 0.75 0.01 0.03 0.72 0.01 0.03 0.04 0.04 0.49 0.95 0.85 0.33 0.88 0.73 0.01 0.02 0.66 0.81 0.46 0.19 0.05 0.02 

0.82 ± 0.13 0.02 ± 0.01 12 1.26 0.91 1.02 0.76 0.70 0.75 0.88 0.85 0.80 0.70 0.79 0.04 0.04 0.53 0.95 0.80 0.40 0.86 0.74 0.89 0.79 0.65 0.72 0.44 0.17 0.40 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 1.24 0.93 1.07 0.86 0.75 0.77 0.89 0.85 0.77 0.69 0.80 0.04 0.02 0.53 0.94 0.82 0.41 0.88 0.75 0.93 0.76 0.64 0.76 0.41 0.17 0.40 0.02 

0.83 ± 0.12 0.71 ± 0.10 14 1.28 0.89 1.04 0.88 0.72 0.09 0.87 0.85 0.83 0.67 0.81 0.01 0.00 0.04 0.92 0.83 -0.02 0.35 0.11 0.89 0.79 0.07 0.73 0.00 0.12 0.38 -0.02 

0.85 ± 0.13 0.78 ± 0.12 15 1.26 0.91 0.96 0.84 0.68 0.76 0.81 0.83 0.82 0.63 0.77 0.06 0.06 0.49 0.02 0.77 0.39 0.88 0.76 0.97 0.86 0.66 0.78 0.45 0.20 0.42 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 1.34 0.92 1.00 0.86 0.69 0.77 0.89 0.85 0.78 0.67 0.76 0.03 0.03 0.51 0.94 0.03 0.39 0.90 0.73 0.95 0.87 0.68 0.79 0.48 0.17 0.43 0.04 

0.82 ± 0.12 0.45 ± 0.06 17 1.27 0.87 0.92 0.78 0.66 0.29 0.79 0.80 0.80 0.64 0.68 0.04 0.00 0.18 0.88 0.77 0.07 0.44 0.31 0.87 0.79 0.26 0.76 0.13 0.15 0.37 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 1.22 0.82 0.97 0.82 0.64 0.15 0.85 0.84 0.06 0.59 0.78 0.03 0.02 0.11 0.94 0.78 0.02 0.01 0.69 0.94 0.88 0.06 0.07 0.02 0.16 0.40 -0.01 

0.80 ± 0.12 0.74 ± 0.11 19 1.23 0.11 1.02 0.80 0.64 0.06 0.81 0.78 0.75 0.09 0.78 0.05 0.01 0.03 0.85 0.77 0.04 0.83 0.02 0.91 0.77 0.06 0.73 0.02 0.16 0.38 0.03 

0.82 ± 0.13 0.88 ± 0.15 20 0.24 0.04 0.05 0.89 0.68 0.73 0.04 0.05 0.78 0.05 0.06 0.06 0.04 0.46 0.99 0.83 0.36 0.88 0.74 0.04 0.05 0.63 0.77 0.42 0.21 0.07 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.22 0.01 0.03 0.86 0.64 0.70 0.02 0.01 0.80 0.01 0.01 0.02 0.01 0.44 0.98 0.73 0.31 0.91 0.71 0.01 0.03 0.57 0.77 0.38 0.15 0.04 0.01 

0.86 ± 0.13 0.75 ± 0.11 22 1.25 0.86 1.00 0.84 0.69 0.07 0.82 0.81 0.80 0.66 0.75 0.01 0.04 0.05 0.93 0.83 -0.01 0.23 0.10 0.91 0.81 0.07 0.77 0.00 0.16 0.42 0.00 

0.82 ± 0.11 0.90 ± 0.13 23 1.25 0.83 0.98 0.87 0.67 0.75 0.91 0.88 0.04 0.68 0.82 0.04 0.04 0.46 1.00 0.81 0.35 0.02 0.73 0.97 0.91 0.66 0.05 0.51 0.19 0.35 0.02 

0.80 ± 0.12 0.56 ± 0.08 24 1.19 0.82 0.96 0.80 0.67 0.18 0.80 0.77 0.82 0.62 0.73 0.00 -0.01 0.10 0.89 0.70 0.02 0.33 0.20 0.84 0.74 0.16 0.74 0.07 0.13 0.38 0.00 

0.83 ± 0.12 0.27 ± 0.03 25 1.23 0.92 1.00 0.86 0.71 0.75 0.86 0.83 0.82 0.66 0.77 0.06 0.05 0.47 0.95 0.81 0.40 0.88 0.75 0.90 0.88 0.68 0.82 0.50 0.08 0.42 0.05 

0.81 ± 0.13 0.52 ± 0.09 26 0.23 0.81 0.03 0.84 0.68 0.73 0.05 0.04 0.68 0.63 0.04 0.02 0.02 0.49 0.96 0.77 0.35 0.88 0.65 0.05 0.03 0.62 0.65 0.43 0.16 0.05 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 1.28 0.95 1.03 0.86 0.72 0.74 0.89 0.85 0.81 0.69 0.81 0.05 0.04 0.51 0.95 0.77 0.40 0.86 0.74 0.88 0.81 0.61 0.71 0.46 0.18 0.39 0.03 

27 

(-) 

Self-Competition 

Confidential 

VISHAL KAMAT

Data Analysis – Highlight Negative Control mAb 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 

mAb 

# 


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.84 ± 0.12 1.20 ± 0.17 1 0.11 0.87 0.03 0.90 0.36 0.69 0.04 0.03 0.77 0.02 0.02 0.02 0.01 0.47 0.97 0.85 0.29 0.86 0.67 0.08 0.04 0.66 0.80 0.41 0.15 0.10 0.08 

0.88 ± 0.14 0.84 ± 0.12 2 1.24 0.02 1.06 0.87 0.70 0.78 0.07 0.08 0.73 0.07 0.08 0.03 0.06 0.53 0.96 0.86 0.39 0.93 0.07 0.13 0.10 0.71 0.81 0.54 0.19 0.44 0.04 

0.82 ± 0.11 0.88 ± 0.14 3 0.19 0.88 0.06 0.89 0.64 0.72 0.04 0.03 0.76 -0.03 0.04 0.00 0.05 0.48 0.93 0.81 0.34 0.83 0.71 0.06 0.02 0.63 0.78 0.41 0.16 0.07 0.05 

0.84 ± 0.11 0.78 ± 0.11 4 1.26 0.90 1.03 0.03 0.66 0.75 0.90 0.84 0.76 0.68 0.78 0.03 0.04 0.48 0.89 0.77 0.36 0.81 0.70 0.96 0.86 0.64 0.76 0.46 0.15 0.46 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 1.05 0.83 1.01 0.81 0.02 0.72 0.84 0.79 0.71 0.62 0.78 -0.01 0.00 0.42 0.92 0.79 0.31 0.82 0.69 0.89 0.85 0.61 0.70 0.41 0.14 0.38 -0.01 

0.83 ± 0.13 0.72 ± 0.11 6 1.23 0.85 1.05 0.87 0.70 0.04 0.85 0.81 0.78 0.63 0.79 0.02 0.04 0.05 0.95 0.82 0.02 0.22 0.08 0.91 0.78 0.05 0.76 0.03 0.19 0.39 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.22 0.02 0.02 0.89 0.69 0.76 0.02 0.02 0.81 0.01 0.01 0.01 0.01 0.49 0.98 0.84 0.36 0.82 0.72 0.02 0.02 0.62 0.77 0.39 0.17 0.05 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.24 0.02 0.07 0.90 0.70 0.77 0.04 0.03 0.88 0.05 0.06 0.04 0.04 0.50 1.02 0.87 0.37 0.96 0.74 0.07 0.04 0.70 0.83 0.45 0.23 0.10 0.07 

0.84 ± 0.12 0.89 ± 0.12 9 1.23 0.76 1.07 0.89 0.67 0.73 0.90 0.91 0.04 0.68 0.84 0.05 0.02 0.49 0.94 0.86 0.34 0.02 0.74 1.01 0.91 0.67 0.04 0.54 0.18 0.31 0.03 

0.82 ± 0.12 0.78 ± 0.10 10 0.33 0.06 0.13 0.83 0.68 0.79 0.12 0.10 0.84 0.03 0.10 0.05 0.03 0.51 0.97 0.84 0.41 0.90 0.08 0.13 0.11 0.68 0.84 0.46 0.18 0.44 0.02 

0.84 ± 0.12 0.89 ± 0.13 11 0.21 0.01 0.04 0.85 0.66 0.75 0.01 0.03 0.72 0.01 0.03 0.04 0.04 0.49 0.95 0.85 0.33 0.88 0.73 0.01 0.02 0.66 0.81 0.46 0.19 0.05 0.02 

0.82 ± 0.13 0.02 ± 0.01 12 1.26 0.91 1.02 0.76 0.70 0.75 0.88 0.85 0.80 0.70 0.79 0.04 0.04 0.53 0.95 0.80 0.40 0.86 0.74 0.89 0.79 0.65 0.72 0.44 0.17 0.40 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 1.24 0.93 1.07 0.86 0.75 0.77 0.89 0.85 0.77 0.69 0.80 0.04 0.02 0.53 0.94 0.82 0.41 0.88 0.75 0.93 0.76 0.64 0.76 0.41 0.17 0.40 0.02 

0.83 ± 0.12 0.71 ± 0.10 14 1.28 0.89 1.04 0.88 0.72 0.09 0.87 0.85 0.83 0.67 0.81 0.01 0.00 0.04 0.92 0.83 -0.02 0.35 0.11 0.89 0.79 0.07 0.73 0.00 0.12 0.38 -0.02 

0.85 ± 0.13 0.78 ± 0.12 15 1.26 0.91 0.96 0.84 0.68 0.76 0.81 0.83 0.82 0.63 0.77 0.06 0.06 0.49 0.02 0.77 0.39 0.88 0.76 0.97 0.86 0.66 0.78 0.45 0.20 0.42 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 1.34 0.92 1.00 0.86 0.69 0.77 0.89 0.85 0.78 0.67 0.76 0.03 0.03 0.51 0.94 0.03 0.39 0.90 0.73 0.95 0.87 0.68 0.79 0.48 0.17 0.43 0.04 

0.82 ± 0.12 0.45 ± 0.06 17 1.27 0.87 0.92 0.78 0.66 0.29 0.79 0.80 0.80 0.64 0.68 0.04 0.00 0.18 0.88 0.77 0.07 0.44 0.31 0.87 0.79 0.26 0.76 0.13 0.15 0.37 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 1.22 0.82 0.97 0.82 0.64 0.15 0.85 0.84 0.06 0.59 0.78 0.03 0.02 0.11 0.94 0.78 0.02 0.01 0.69 0.94 0.88 0.06 0.07 0.02 0.16 0.40 -0.01 

0.80 ± 0.12 0.74 ± 0.11 19 1.23 0.11 1.02 0.80 0.64 0.06 0.81 0.78 0.75 0.09 0.78 0.05 0.01 0.03 0.85 0.77 0.04 0.83 0.02 0.91 0.77 0.06 0.73 0.02 0.16 0.38 0.03 

0.82 ± 0.13 0.88 ± 0.15 20 0.24 0.04 0.05 0.89 0.68 0.73 0.04 0.05 0.78 0.05 0.06 0.06 0.04 0.46 0.99 0.83 0.36 0.88 0.74 0.04 0.05 0.63 0.77 0.42 0.21 0.07 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.22 0.01 0.03 0.86 0.64 0.70 0.02 0.01 0.80 0.01 0.01 0.02 0.01 0.44 0.98 0.73 0.31 0.91 0.71 0.01 0.03 0.57 0.77 0.38 0.15 0.04 0.01 

0.86 ± 0.13 0.75 ± 0.11 22 1.25 0.86 1.00 0.84 0.69 0.07 0.82 0.81 0.80 0.66 0.75 0.01 0.04 0.05 0.93 0.83 -0.01 0.23 0.10 0.91 0.81 0.07 0.77 0.00 0.16 0.42 0.00 

0.82 ± 0.11 0.90 ± 0.13 23 1.25 0.83 0.98 0.87 0.67 0.75 0.91 0.88 0.04 0.68 0.82 0.04 0.04 0.46 1.00 0.81 0.35 0.02 0.73 0.97 0.91 0.66 0.05 0.51 0.19 0.35 0.02 

0.80 ± 0.12 0.56 ± 0.08 24 1.19 0.82 0.96 0.80 0.67 0.18 0.80 0.77 0.82 0.62 0.73 0.00 -0.01 0.10 0.89 0.70 0.02 0.33 0.20 0.84 0.74 0.16 0.74 0.07 0.13 0.38 0.00 

0.83 ± 0.12 0.27 ± 0.03 25 1.23 0.92 1.00 0.86 0.71 0.75 0.86 0.83 0.82 0.66 0.77 0.06 0.05 0.47 0.95 0.81 0.40 0.88 0.75 0.90 0.88 0.68 0.82 0.50 0.08 0.42 0.05 

0.81 ± 0.13 0.52 ± 0.09 26 0.23 0.81 0.03 0.84 0.68 0.73 0.05 0.04 0.68 0.63 0.04 0.02 0.02 0.49 0.96 0.77 0.35 0.88 0.65 0.05 0.03 0.62 0.65 0.43 0.16 0.05 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 1.28 0.95 1.03 0.86 0.72 0.74 0.89 0.85 0.81 0.69 0.81 0.05 0.04 0.51 0.95 0.77 0.40 0.86 0.74 0.88 0.81 0.61 0.71 0.46 0.18 0.39 0.03 

27 

(-) 


Confidential 

VISHAL KAMAT 

Negative Control

Data Analysis – Highlight Cells with Low Binding Response 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 

Confidential 

mAb 

# 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.84 ± 0.12 1.20 ± 0.17 1 0.11 0.87 0.03 0.90 0.36 0.69 0.04 0.03 0.77 0.02 0.02 0.02 0.01 0.47 0.97 0.85 0.29 0.86 0.67 0.08 0.04 0.66 0.80 0.41 0.15 0.10 0.08 

0.88 ± 0.14 0.84 ± 0.12 2 1.24 0.02 1.06 0.87 0.70 0.78 0.07 0.08 0.73 0.07 0.08 0.03 0.06 0.53 0.96 0.86 0.39 0.93 0.07 0.13 0.10 0.71 0.81 0.54 0.19 0.44 0.04 

0.82 ± 0.11 0.88 ± 0.14 3 0.19 0.88 0.06 0.89 0.64 0.72 0.04 0.03 0.76 -0.03 0.04 0.00 0.05 0.48 0.93 0.81 0.34 0.83 0.71 0.06 0.02 0.63 0.78 0.41 0.16 0.07 0.05 

0.84 ± 0.11 0.78 ± 0.11 4 1.26 0.90 1.03 0.03 0.66 0.75 0.90 0.84 0.76 0.68 0.78 0.03 0.04 0.48 0.89 0.77 0.36 0.81 0.70 0.96 0.86 0.64 0.76 0.46 0.15 0.46 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 1.05 0.83 1.01 0.81 0.02 0.72 0.84 0.79 0.71 0.62 0.78 -0.01 0.00 0.42 0.92 0.79 0.31 0.82 0.69 0.89 0.85 0.61 0.70 0.41 0.14 0.38 -0.01 

0.83 ± 0.13 0.72 ± 0.11 6 1.23 0.85 1.05 0.87 0.70 0.04 0.85 0.81 0.78 0.63 0.79 0.02 0.04 0.05 0.95 0.82 0.02 0.22 0.08 0.91 0.78 0.05 0.76 0.03 0.19 0.39 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.22 0.02 0.02 0.89 0.69 0.76 0.02 0.02 0.81 0.01 0.01 0.01 0.01 0.49 0.98 0.84 0.36 0.82 0.72 0.02 0.02 0.62 0.77 0.39 0.17 0.05 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.24 0.02 0.07 0.90 0.70 0.77 0.04 0.03 0.88 0.05 0.06 0.04 0.04 0.50 1.02 0.87 0.37 0.96 0.74 0.07 0.04 0.70 0.83 0.45 0.23 0.10 0.07 

0.84 ± 0.12 0.89 ± 0.12 9 1.23 0.76 1.07 0.89 0.67 0.73 0.90 0.91 0.04 0.68 0.84 0.05 0.02 0.49 0.94 0.86 0.34 0.02 0.74 1.01 0.91 0.67 0.04 0.54 0.18 0.31 0.03 

0.82 ± 0.12 0.78 ± 0.10 10 0.33 0.06 0.13 0.83 0.68 0.79 0.12 0.10 0.84 0.03 0.10 0.05 0.03 0.51 0.97 0.84 0.41 0.90 0.08 0.13 0.11 0.68 0.84 0.46 0.18 0.44 0.02 

0.84 ± 0.12 0.89 ± 0.13 11 0.21 0.01 0.04 0.85 0.66 0.75 0.01 0.03 0.72 0.01 0.03 0.04 0.04 0.49 0.95 0.85 0.33 0.88 0.73 0.01 0.02 0.66 0.81 0.46 0.19 0.05 0.02 

0.82 ± 0.13 0.02 ± 0.01 12 1.26 0.91 1.02 0.76 0.70 0.75 0.88 0.85 0.80 0.70 0.79 0.04 0.04 0.53 0.95 0.80 0.40 0.86 0.74 0.89 0.79 0.65 0.72 0.44 0.17 0.40 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 1.24 0.93 1.07 0.86 0.75 0.77 0.89 0.85 0.77 0.69 0.80 0.04 0.02 0.53 0.94 0.82 0.41 0.88 0.75 0.93 0.76 0.64 0.76 0.41 0.17 0.40 0.02 

0.83 ± 0.12 0.71 ± 0.10 14 1.28 0.89 1.04 0.88 0.72 0.09 0.87 0.85 0.83 0.67 0.81 0.01 0.00 0.04 0.92 0.83 -0.02 0.35 0.11 0.89 0.79 0.07 0.73 0.00 0.12 0.38 -0.02 

0.85 ± 0.13 0.78 ± 0.12 15 1.26 0.91 0.96 0.84 0.68 0.76 0.81 0.83 0.82 0.63 0.77 0.06 0.06 0.49 0.02 0.77 0.39 0.88 0.76 0.97 0.86 0.66 0.78 0.45 0.20 0.42 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 1.34 0.92 1.00 0.86 0.69 0.77 0.89 0.85 0.78 0.67 0.76 0.03 0.03 0.51 0.94 0.03 0.39 0.90 0.73 0.95 0.87 0.68 0.79 0.48 0.17 0.43 0.04 

0.82 ± 0.12 0.45 ± 0.06 17 1.27 0.87 0.92 0.78 0.66 0.29 0.79 0.80 0.80 0.64 0.68 0.04 0.00 0.18 0.88 0.77 0.07 0.44 0.31 0.87 0.79 0.26 0.76 0.13 0.15 0.37 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 1.22 0.82 0.97 0.82 0.64 0.15 0.85 0.84 0.06 0.59 0.78 0.03 0.02 0.11 0.94 0.78 0.02 0.01 0.69 0.94 0.88 0.06 0.07 0.02 0.16 0.40 -0.01 

0.80 ± 0.12 0.74 ± 0.11 19 1.23 0.11 1.02 0.80 0.64 0.06 0.81 0.78 0.75 0.09 0.78 0.05 0.01 0.03 0.85 0.77 0.04 0.83 0.02 0.91 0.77 0.06 0.73 0.02 0.16 0.38 0.03 

0.82 ± 0.13 0.88 ± 0.15 20 0.24 0.04 0.05 0.89 0.68 0.73 0.04 0.05 0.78 0.05 0.06 0.06 0.04 0.46 0.99 0.83 0.36 0.88 0.74 0.04 0.05 0.63 0.77 0.42 0.21 0.07 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.22 0.01 0.03 0.86 0.64 0.70 0.02 0.01 0.80 0.01 0.01 0.02 0.01 0.44 0.98 0.73 0.31 0.91 0.71 0.01 0.03 0.57 0.77 0.38 0.15 0.04 0.01 

0.86 ± 0.13 0.75 ± 0.11 22 1.25 0.86 1.00 0.84 0.69 0.07 0.82 0.81 0.80 0.66 0.75 0.01 0.04 0.05 0.93 0.83 -0.01 0.23 0.10 0.91 0.81 0.07 0.77 0.00 0.16 0.42 0.00 

0.82 ± 0.11 0.90 ± 0.13 23 1.25 0.83 0.98 0.87 0.67 0.75 0.91 0.88 0.04 0.68 0.82 0.04 0.04 0.46 1.00 0.81 0.35 0.02 0.73 0.97 0.91 0.66 0.05 0.51 0.19 0.35 0.02 

0.80 ± 0.12 0.56 ± 0.08 24 1.19 0.82 0.96 0.80 0.67 0.18 0.80 0.77 0.82 0.62 0.73 0.00 -0.01 0.10 0.89 0.70 0.02 0.33 0.20 0.84 0.74 0.16 0.74 0.07 0.13 0.38 0.00 

0.83 ± 0.12 0.27 ± 0.03 25 1.23 0.92 1.00 0.86 0.71 0.75 0.86 0.83 0.82 0.66 0.77 0.06 0.05 0.47 0.95 0.81 0.40 0.88 0.75 0.90 0.88 0.68 0.82 0.50 0.08 0.42 0.05 

0.81 ± 0.13 0.52 ± 0.09 26 0.23 0.81 0.03 0.84 0.68 0.73 0.05 0.04 0.68 0.63 0.04 0.02 0.02 0.49 0.96 0.77 0.35 0.88 0.65 0.05 0.03 0.62 0.65 0.43 0.16 0.05 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 1.28 0.95 1.03 0.86 0.72 0.74 0.89 0.85 0.81 0.69 0.81 0.05 0.04 0.51 0.95 0.77 0.40 0.86 0.74 0.88 0.81 0.61 0.71 0.46 0.18 0.39 0.03 



VISHAL KAMAT 

Low mAb Binding 

Response (

Data Analysis – Highlight Cells with Poor mAb-1 Binding 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 

Confidential 

mAb 

# 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.84 ± 0.12 1.20 ± 0.17 1 0.11 0.87 0.03 0.90 0.36 0.69 0.04 0.03 0.77 0.02 0.02 0.02 0.01 0.47 0.97 0.85 0.29 0.86 0.67 0.08 0.04 0.66 0.80 0.41 0.15 0.10 0.08 

0.88 ± 0.14 0.84 ± 0.12 2 1.24 0.02 1.06 0.87 0.70 0.78 0.07 0.08 0.73 0.07 0.08 0.03 0.06 0.53 0.96 0.86 0.39 0.93 0.07 0.13 0.10 0.71 0.81 0.54 0.19 0.44 0.04 

0.82 ± 0.11 0.88 ± 0.14 3 0.19 0.88 0.06 0.89 0.64 0.72 0.04 0.03 0.76 -0.03 0.04 0.00 0.05 0.48 0.93 0.81 0.34 0.83 0.71 0.06 0.02 0.63 0.78 0.41 0.16 0.07 0.05 

0.84 ± 0.11 0.78 ± 0.11 4 1.26 0.90 1.03 0.03 0.66 0.75 0.90 0.84 0.76 0.68 0.78 0.03 0.04 0.48 0.89 0.77 0.36 0.81 0.70 0.96 0.86 0.64 0.76 0.46 0.15 0.46 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 1.05 0.83 1.01 0.81 0.02 0.72 0.84 0.79 0.71 0.62 0.78 -0.01 0.00 0.42 0.92 0.79 0.31 0.82 0.69 0.89 0.85 0.61 0.70 0.41 0.14 0.38 -0.01 

0.83 ± 0.13 0.72 ± 0.11 6 1.23 0.85 1.05 0.87 0.70 0.04 0.85 0.81 0.78 0.63 0.79 0.02 0.04 0.05 0.95 0.82 0.02 0.22 0.08 0.91 0.78 0.05 0.76 0.03 0.19 0.39 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.22 0.02 0.02 0.89 0.69 0.76 0.02 0.02 0.81 0.01 0.01 0.01 0.01 0.49 0.98 0.84 0.36 0.82 0.72 0.02 0.02 0.62 0.77 0.39 0.17 0.05 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.24 0.02 0.07 0.90 0.70 0.77 0.04 0.03 0.88 0.05 0.06 0.04 0.04 0.50 1.02 0.87 0.37 0.96 0.74 0.07 0.04 0.70 0.83 0.45 0.23 0.10 0.07 

0.84 ± 0.12 0.89 ± 0.12 9 1.23 0.76 1.07 0.89 0.67 0.73 0.90 0.91 0.04 0.68 0.84 0.05 0.02 0.49 0.94 0.86 0.34 0.02 0.74 1.01 0.91 0.67 0.04 0.54 0.18 0.31 0.03 

0.82 ± 0.12 0.78 ± 0.10 10 0.33 0.06 0.13 0.83 0.68 0.79 0.12 0.10 0.84 0.03 0.10 0.05 0.03 0.51 0.97 0.84 0.41 0.90 0.08 0.13 0.11 0.68 0.84 0.46 0.18 0.44 0.02 

0.84 ± 0.12 0.89 ± 0.13 11 0.21 0.01 0.04 0.85 0.66 0.75 0.01 0.03 0.72 0.01 0.03 0.04 0.04 0.49 0.95 0.85 0.33 0.88 0.73 0.01 0.02 0.66 0.81 0.46 0.19 0.05 0.02 

0.82 ± 0.13 0.02 ± 0.01 12 1.26 0.91 1.02 0.76 0.70 0.75 0.88 0.85 0.80 0.70 0.79 0.04 0.04 0.53 0.95 0.80 0.40 0.86 0.74 0.89 0.79 0.65 0.72 0.44 0.17 0.40 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 1.24 0.93 1.07 0.86 0.75 0.77 0.89 0.85 0.77 0.69 0.80 0.04 0.02 0.53 0.94 0.82 0.41 0.88 0.75 0.93 0.76 0.64 0.76 0.41 0.17 0.40 0.02 

0.83 ± 0.12 0.71 ± 0.10 14 1.28 0.89 1.04 0.88 0.72 0.09 0.87 0.85 0.83 0.67 0.81 0.01 0.00 0.04 0.92 0.83 -0.02 0.35 0.11 0.89 0.79 0.07 0.73 0.00 0.12 0.38 -0.02 

0.85 ± 0.13 0.78 ± 0.12 15 1.26 0.91 0.96 0.84 0.68 0.76 0.81 0.83 0.82 0.63 0.77 0.06 0.06 0.49 0.02 0.77 0.39 0.88 0.76 0.97 0.86 0.66 0.78 0.45 0.20 0.42 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 1.34 0.92 1.00 0.86 0.69 0.77 0.89 0.85 0.78 0.67 0.76 0.03 0.03 0.51 0.94 0.03 0.39 0.90 0.73 0.95 0.87 0.68 0.79 0.48 0.17 0.43 0.04 

0.82 ± 0.12 0.45 ± 0.06 17 1.27 0.87 0.92 0.78 0.66 0.29 0.79 0.80 0.80 0.64 0.68 0.04 0.00 0.18 0.88 0.77 0.07 0.44 0.31 0.87 0.79 0.26 0.76 0.13 0.15 0.37 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 1.22 0.82 0.97 0.82 0.64 0.15 0.85 0.84 0.06 0.59 0.78 0.03 0.02 0.11 0.94 0.78 0.02 0.01 0.69 0.94 0.88 0.06 0.07 0.02 0.16 0.40 -0.01 

0.80 ± 0.12 0.74 ± 0.11 19 1.23 0.11 1.02 0.80 0.64 0.06 0.81 0.78 0.75 0.09 0.78 0.05 0.01 0.03 0.85 0.77 0.04 0.83 0.02 0.91 0.77 0.06 0.73 0.02 0.16 0.38 0.03 

0.82 ± 0.13 0.88 ± 0.15 20 0.24 0.04 0.05 0.89 0.68 0.73 0.04 0.05 0.78 0.05 0.06 0.06 0.04 0.46 0.99 0.83 0.36 0.88 0.74 0.04 0.05 0.63 0.77 0.42 0.21 0.07 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.22 0.01 0.03 0.86 0.64 0.70 0.02 0.01 0.80 0.01 0.01 0.02 0.01 0.44 0.98 0.73 0.31 0.91 0.71 0.01 0.03 0.57 0.77 0.38 0.15 0.04 0.01 

0.86 ± 0.13 0.75 ± 0.11 22 1.25 0.86 1.00 0.84 0.69 0.07 0.82 0.81 0.80 0.66 0.75 0.01 0.04 0.05 0.93 0.83 -0.01 0.23 0.10 0.91 0.81 0.07 0.77 0.00 0.16 0.42 0.00 

0.82 ± 0.11 0.90 ± 0.13 23 1.25 0.83 0.98 0.87 0.67 0.75 0.91 0.88 0.04 0.68 0.82 0.04 0.04 0.46 1.00 0.81 0.35 0.02 0.73 0.97 0.91 0.66 0.05 0.51 0.19 0.35 0.02 

0.80 ± 0.12 0.56 ± 0.08 24 1.19 0.82 0.96 0.80 0.67 0.18 0.80 0.77 0.82 0.62 0.73 0.00 -0.01 0.10 0.89 0.70 0.02 0.33 0.20 0.84 0.74 0.16 0.74 0.07 0.13 0.38 0.00 

0.83 ± 0.12 0.27 ± 0.03 25 1.23 0.92 1.00 0.86 0.71 0.75 0.86 0.83 0.82 0.66 0.77 0.06 0.05 0.47 0.95 0.81 0.40 0.88 0.75 0.90 0.88 0.68 0.82 0.50 0.08 0.42 0.05 

0.81 ± 0.13 0.52 ± 0.09 26 0.23 0.81 0.03 0.84 0.68 0.73 0.05 0.04 0.68 0.63 0.04 0.02 0.02 0.49 0.96 0.77 0.35 0.88 0.65 0.05 0.03 0.62 0.65 0.43 0.16 0.05 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 1.28 0.95 1.03 0.86 0.72 0.74 0.89 0.85 0.81 0.69 0.81 0.05 0.04 0.51 0.95 0.77 0.40 0.86 0.74 0.88 0.81 0.61 0.71 0.46 0.18 0.39 0.03 



No to Low 

mAb Binding 

VISHAL KAMAT 


Response (

Data Analysis – Move mAb-1 Non-Binders to the Bottom 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 


Confidential 

mAb 

# 


1 2 3 4 5 6 7 8 9 10 11 14 15 16 17 18 19 20 21 22 23 24 26 25 12 13 

0.84 ± 0.12 1.20 ± 0.17 1 0.11 0.87 0.03 0.90 0.36 0.69 0.04 0.03 0.77 0.02 0.02 0.47 0.97 0.85 0.29 0.86 0.67 0.08 0.04 0.66 0.80 0.41 0.10 0.15 0.02 0.01 0.08 

0.88 ± 0.14 0.84 ± 0.12 2 1.24 0.02 1.06 0.87 0.70 0.78 0.07 0.08 0.73 0.07 0.08 0.53 0.96 0.86 0.39 0.93 0.07 0.13 0.10 0.71 0.81 0.54 0.44 0.19 0.03 0.06 0.04 

0.82 ± 0.11 0.88 ± 0.14 3 0.19 0.88 0.06 0.89 0.64 0.72 0.04 0.03 0.76 -0.03 0.04 0.48 0.93 0.81 0.34 0.83 0.71 0.06 0.02 0.63 0.78 0.41 0.07 0.16 0.00 0.05 0.05 

0.84 ± 0.11 0.78 ± 0.11 4 1.26 0.90 1.03 0.03 0.66 0.75 0.90 0.84 0.76 0.68 0.78 0.48 0.89 0.77 0.36 0.81 0.70 0.96 0.86 0.64 0.76 0.46 0.46 0.15 0.03 0.04 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 1.05 0.83 1.01 0.81 0.02 0.72 0.84 0.79 0.71 0.62 0.78 0.42 0.92 0.79 0.31 0.82 0.69 0.89 0.85 0.61 0.70 0.41 0.38 0.14 -0.01 0.00 -0.01 

0.83 ± 0.13 0.72 ± 0.11 6 1.23 0.85 1.05 0.87 0.70 0.04 0.85 0.81 0.78 0.63 0.79 0.05 0.95 0.82 0.02 0.22 0.08 0.91 0.78 0.05 0.76 0.03 0.39 0.19 0.02 0.04 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.22 0.02 0.02 0.89 0.69 0.76 0.02 0.02 0.81 0.01 0.01 0.49 0.98 0.84 0.36 0.82 0.72 0.02 0.02 0.62 0.77 0.39 0.05 0.17 0.01 0.01 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.24 0.02 0.07 0.90 0.70 0.77 0.04 0.03 0.88 0.05 0.06 0.50 1.02 0.87 0.37 0.96 0.74 0.07 0.04 0.70 0.83 0.45 0.10 0.23 0.04 0.04 0.07 

0.84 ± 0.12 0.89 ± 0.12 9 1.23 0.76 1.07 0.89 0.67 0.73 0.90 0.91 0.04 0.68 0.84 0.49 0.94 0.86 0.34 0.02 0.74 1.01 0.91 0.67 0.04 0.54 0.31 0.18 0.05 0.02 0.03 

0.82 ± 0.12 0.78 ± 0.10 10 0.33 0.06 0.13 0.83 0.68 0.79 0.12 0.10 0.84 0.03 0.10 0.51 0.97 0.84 0.41 0.90 0.08 0.13 0.11 0.68 0.84 0.46 0.44 0.18 0.05 0.03 0.02 

0.84 ± 0.12 0.89 ± 0.13 11 0.21 0.01 0.04 0.85 0.66 0.75 0.01 0.03 0.72 0.01 0.03 0.49 0.95 0.85 0.33 0.88 0.73 0.01 0.02 0.66 0.81 0.46 0.05 0.19 0.04 0.04 0.02 

0.83 ± 0.12 0.71 ± 0.10 14 1.28 0.89 1.04 0.88 0.72 0.09 0.87 0.85 0.83 0.67 0.81 0.04 0.92 0.83 -0.02 0.35 0.11 0.89 0.79 0.07 0.73 0.00 0.38 0.12 0.01 0.00 -0.02 

0.85 ± 0.13 0.78 ± 0.12 15 1.26 0.91 0.96 0.84 0.68 0.76 0.81 0.83 0.82 0.63 0.77 0.49 0.02 0.77 0.39 0.88 0.76 0.97 0.86 0.66 0.78 0.45 0.42 0.20 0.06 0.06 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 1.34 0.92 1.00 0.86 0.69 0.77 0.89 0.85 0.78 0.67 0.76 0.51 0.94 0.03 0.39 0.90 0.73 0.95 0.87 0.68 0.79 0.48 0.43 0.17 0.03 0.03 0.04 

0.82 ± 0.12 0.45 ± 0.06 17 1.27 0.87 0.92 0.78 0.66 0.29 0.79 0.80 0.80 0.64 0.68 0.18 0.88 0.77 0.07 0.44 0.31 0.87 0.79 0.26 0.76 0.13 0.37 0.15 0.04 0.00 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 1.22 0.82 0.97 0.82 0.64 0.15 0.85 0.84 0.06 0.59 0.78 0.11 0.94 0.78 0.02 0.01 0.69 0.94 0.88 0.06 0.07 0.02 0.40 0.16 0.03 0.02 -0.01 

0.80 ± 0.12 0.74 ± 0.11 19 1.23 0.11 1.02 0.80 0.64 0.06 0.81 0.78 0.75 0.09 0.78 0.03 0.85 0.77 0.04 0.83 0.02 0.91 0.77 0.06 0.73 0.02 0.38 0.16 0.05 0.01 0.03 

0.82 ± 0.13 0.88 ± 0.15 20 0.24 0.04 0.05 0.89 0.68 0.73 0.04 0.05 0.78 0.05 0.06 0.46 0.99 0.83 0.36 0.88 0.74 0.04 0.05 0.63 0.77 0.42 0.07 0.21 0.06 0.04 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.22 0.01 0.03 0.86 0.64 0.70 0.02 0.01 0.80 0.01 0.01 0.44 0.98 0.73 0.31 0.91 0.71 0.01 0.03 0.57 0.77 0.38 0.04 0.15 0.02 0.01 0.01 

0.86 ± 0.13 0.75 ± 0.11 22 1.25 0.86 1.00 0.84 0.69 0.07 0.82 0.81 0.80 0.66 0.75 0.05 0.93 0.83 -0.01 0.23 0.10 0.91 0.81 0.07 0.77 0.00 0.42 0.16 0.01 0.04 0.00 

0.82 ± 0.11 0.90 ± 0.13 23 1.25 0.83 0.98 0.87 0.67 0.75 0.91 0.88 0.04 0.68 0.82 0.46 1.00 0.81 0.35 0.02 0.73 0.97 0.91 0.66 0.05 0.51 0.35 0.19 0.04 0.04 0.02 

0.80 ± 0.12 0.56 ± 0.08 24 1.19 0.82 0.96 0.80 0.67 0.18 0.80 0.77 0.82 0.62 0.73 0.10 0.89 0.70 0.02 0.33 0.20 0.84 0.74 0.16 0.74 0.07 0.38 0.13 0.00 -0.01 0.00 

0.81 ± 0.13 0.52 ± 0.09 26 0.23 0.81 0.03 0.84 0.68 0.73 0.05 0.04 0.68 0.63 0.04 0.49 0.96 0.77 0.35 0.88 0.65 0.05 0.03 0.62 0.65 0.43 0.05 0.16 0.02 0.02 0.02 

0.83 ± 0.12 0.27 ± 0.03 25 1.23 0.92 1.00 0.86 0.71 0.75 0.86 0.83 0.82 0.66 0.77 0.47 0.95 0.81 0.40 0.88 0.75 0.90 0.88 0.68 0.82 0.50 0.42 0.08 0.06 0.05 0.05 

0.82 ± 0.13 0.02 ± 0.01 12 1.26 0.91 1.02 0.76 0.70 0.75 0.88 0.85 0.80 0.70 0.79 0.53 0.95 0.80 0.40 0.86 0.74 0.89 0.79 0.65 0.72 0.44 0.40 0.17 0.04 0.04 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 1.24 0.93 1.07 0.86 0.75 0.77 0.89 0.85 0.77 0.69 0.80 0.53 0.94 0.82 0.41 0.88 0.75 0.93 0.76 0.64 0.76 0.41 0.40 0.17 0.04 0.02 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 1.28 0.95 1.03 0.86 0.72 0.74 0.89 0.85 0.81 0.69 0.81 0.51 0.95 0.77 0.40 0.86 0.74 0.88 0.81 0.61 0.71 0.46 0.39 0.18 0.05 0.04 0.03 

No to Low 

mAb Binding 

VISHAL KAMAT 


Response (

Amount of 

Antigen 

Capture (nm) 


Confidential 

Data Analysis – Simultaneously Move Rows & Columns 

50 ug/mL 

mAb1 

Bound (nm) 

mAb# 20 & 21 were moved horizontally & vertically so that self-self competition is unchanged 

mAb 

# 


20 21 1 2 3 4 5 6 7 8 9 10 11 14 15 16 17 18 19 22 23 24 26 25 12 13 

0.82 ± 0.13 0.88 ± 0.15 20 0.04 0.05 0.24 0.04 0.05 0.89 0.68 0.73 0.04 0.05 0.78 0.05 0.06 0.46 0.99 0.83 0.36 0.88 0.74 0.63 0.77 0.42 0.07 0.21 0.06 0.04 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.01 0.03 0.22 0.01 0.03 0.86 0.64 0.70 0.02 0.01 0.80 0.01 0.01 0.44 0.98 0.73 0.31 0.91 0.71 0.57 0.77 0.38 0.04 0.15 0.02 0.01 0.01 

0.84 ± 0.12 1.20 ± 0.17 1 0.08 0.04 0.11 0.87 0.03 0.90 0.36 0.69 0.04 0.03 0.77 0.02 0.02 0.47 0.97 0.85 0.29 0.86 0.67 0.66 0.80 0.41 0.10 0.15 0.02 0.01 0.08 

0.88 ± 0.14 0.84 ± 0.12 2 0.13 0.10 1.24 0.02 1.06 0.87 0.70 0.78 0.07 0.08 0.73 0.07 0.08 0.53 0.96 0.86 0.39 0.93 0.07 0.71 0.81 0.54 0.44 0.19 0.03 0.06 0.04 

0.82 ± 0.11 0.88 ± 0.14 3 0.06 0.02 0.19 0.88 0.06 0.89 0.64 0.72 0.04 0.03 0.76 -0.03 0.04 0.48 0.93 0.81 0.34 0.83 0.71 0.63 0.78 0.41 0.07 0.16 0.00 0.05 0.05 

0.84 ± 0.11 0.78 ± 0.11 4 0.96 0.86 1.26 0.90 1.03 0.03 0.66 0.75 0.90 0.84 0.76 0.68 0.78 0.48 0.89 0.77 0.36 0.81 0.70 0.64 0.76 0.46 0.46 0.15 0.03 0.04 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 0.89 0.85 1.05 0.83 1.01 0.81 0.02 0.72 0.84 0.79 0.71 0.62 0.78 0.42 0.92 0.79 0.31 0.82 0.69 0.61 0.70 0.41 0.38 0.14 -0.01 0.00 -0.01 

0.83 ± 0.13 0.72 ± 0.11 6 0.91 0.78 1.23 0.85 1.05 0.87 0.70 0.04 0.85 0.81 0.78 0.63 0.79 0.05 0.95 0.82 0.02 0.22 0.08 0.05 0.76 0.03 0.39 0.19 0.02 0.04 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.02 0.02 0.22 0.02 0.02 0.89 0.69 0.76 0.02 0.02 0.81 0.01 0.01 0.49 0.98 0.84 0.36 0.82 0.72 0.62 0.77 0.39 0.05 0.17 0.01 0.01 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.07 0.04 0.24 0.02 0.07 0.90 0.70 0.77 0.04 0.03 0.88 0.05 0.06 0.50 1.02 0.87 0.37 0.96 0.74 0.70 0.83 0.45 0.10 0.23 0.04 0.04 0.07 

0.84 ± 0.12 0.89 ± 0.12 9 1.01 0.91 1.23 0.76 1.07 0.89 0.67 0.73 0.90 0.91 0.04 0.68 0.84 0.49 0.94 0.86 0.34 0.02 0.74 0.67 0.04 0.54 0.31 0.18 0.05 0.02 0.03 

0.82 ± 0.12 0.78 ± 0.10 10 0.13 0.11 0.33 0.06 0.13 0.83 0.68 0.79 0.12 0.10 0.84 0.03 0.10 0.51 0.97 0.84 0.41 0.90 0.08 0.68 0.84 0.46 0.44 0.18 0.05 0.03 0.02 

0.84 ± 0.12 0.89 ± 0.13 11 0.01 0.02 0.21 0.01 0.04 0.85 0.66 0.75 0.01 0.03 0.72 0.01 0.03 0.49 0.95 0.85 0.33 0.88 0.73 0.66 0.81 0.46 0.05 0.19 0.04 0.04 0.02 

0.83 ± 0.12 0.71 ± 0.10 14 0.89 0.79 1.28 0.89 1.04 0.88 0.72 0.09 0.87 0.85 0.83 0.67 0.81 0.04 0.92 0.83 -0.02 0.35 0.11 0.07 0.73 0.00 0.38 0.12 0.01 0.00 -0.02 

0.85 ± 0.13 0.78 ± 0.12 15 0.97 0.86 1.26 0.91 0.96 0.84 0.68 0.76 0.81 0.83 0.82 0.63 0.77 0.49 0.02 0.77 0.39 0.88 0.76 0.66 0.78 0.45 0.42 0.20 0.06 0.06 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 0.95 0.87 1.34 0.92 1.00 0.86 0.69 0.77 0.89 0.85 0.78 0.67 0.76 0.51 0.94 0.03 0.39 0.90 0.73 0.68 0.79 0.48 0.43 0.17 0.03 0.03 0.04 

0.82 ± 0.12 0.45 ± 0.06 17 0.87 0.79 1.27 0.87 0.92 0.78 0.66 0.29 0.79 0.80 0.80 0.64 0.68 0.18 0.88 0.77 0.07 0.44 0.31 0.26 0.76 0.13 0.37 0.15 0.04 0.00 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 0.94 0.88 1.22 0.82 0.97 0.82 0.64 0.15 0.85 0.84 0.06 0.59 0.78 0.11 0.94 0.78 0.02 0.01 0.69 0.06 0.07 0.02 0.40 0.16 0.03 0.02 -0.01 

0.80 ± 0.12 0.74 ± 0.11 19 0.91 0.77 1.23 0.11 1.02 0.80 0.64 0.06 0.81 0.78 0.75 0.09 0.78 0.03 0.85 0.77 0.04 0.83 0.02 0.06 0.73 0.02 0.38 0.16 0.05 0.01 0.03 

0.86 ± 0.13 0.75 ± 0.11 22 0.91 0.81 1.25 0.86 1.00 0.84 0.69 0.07 0.82 0.81 0.80 0.66 0.75 0.05 0.93 0.83 -0.01 0.23 0.10 0.07 0.77 0.00 0.42 0.16 0.01 0.04 0.00 

0.82 ± 0.11 0.90 ± 0.13 23 0.97 0.91 1.25 0.83 0.98 0.87 0.67 0.75 0.91 0.88 0.04 0.68 0.82 0.46 1.00 0.81 0.35 0.02 0.73 0.66 0.05 0.51 0.35 0.19 0.04 0.04 0.02 

0.80 ± 0.12 0.56 ± 0.08 24 0.84 0.74 1.19 0.82 0.96 0.80 0.67 0.18 0.80 0.77 0.82 0.62 0.73 0.10 0.89 0.70 0.02 0.33 0.20 0.16 0.74 0.07 0.38 0.13 0.00 -0.01 0.00 

0.81 ± 0.13 0.52 ± 0.09 26 0.05 0.03 0.23 0.81 0.03 0.84 0.68 0.73 0.05 0.04 0.68 0.63 0.04 0.49 0.96 0.77 0.35 0.88 0.65 0.62 0.65 0.43 0.05 0.16 0.02 0.02 0.02 

0.83 ± 0.12 0.27 ± 0.03 25 0.90 0.88 1.23 0.92 1.00 0.86 0.71 0.75 0.86 0.83 0.82 0.66 0.77 0.47 0.95 0.81 0.40 0.88 0.75 0.68 0.82 0.50 0.42 0.08 0.06 0.05 0.05 

0.82 ± 0.13 0.02 ± 0.01 12 0.89 0.79 1.26 0.91 1.02 0.76 0.70 0.75 0.88 0.85 0.80 0.70 0.79 0.53 0.95 0.80 0.40 0.86 0.74 0.65 0.72 0.44 0.40 0.17 0.04 0.04 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 0.93 0.76 1.24 0.93 1.07 0.86 0.75 0.77 0.89 0.85 0.77 0.69 0.80 0.53 0.94 0.82 0.41 0.88 0.75 0.64 0.76 0.41 0.40 0.17 0.04 0.02 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 0.88 0.81 1.28 0.95 1.03 0.86 0.72 0.74 0.89 0.85 0.81 0.69 0.81 0.51 0.95 0.77 0.40 0.86 0.74 0.61 0.71 0.46 0.39 0.18 0.05 0.04 0.03 

No to Low 

mAb Binding 

VISHAL KAMAT 


Response (

Data Analysis – Bin mAbs Based on Binding Response 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 


Confidential 

mAb 

# 


20 21 11 7 8 3 1 26 10 2 19 24 14 17 22 6 18 23 9 4 5 15 16 25 12 13 

0.82 ± 0.13 0.88 ± 0.15 20 0.04 0.05 0.06 0.04 0.05 0.05 0.24 0.07 0.05 0.04 0.74 0.42 0.46 0.36 0.63 0.73 0.88 0.77 0.78 0.89 0.68 0.99 0.83 0.21 0.06 0.04 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.01 0.03 0.01 0.02 0.01 0.03 0.22 0.04 0.01 0.01 0.71 0.38 0.44 0.31 0.57 0.70 0.91 0.77 0.80 0.86 0.64 0.98 0.73 0.15 0.02 0.01 0.01 

0.84 ± 0.12 0.89 ± 0.13 11 0.01 0.02 0.03 0.01 0.03 0.04 0.21 0.05 0.01 0.01 0.73 0.46 0.49 0.33 0.66 0.75 0.88 0.81 0.72 0.85 0.66 0.95 0.85 0.19 0.04 0.04 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.02 0.02 0.01 0.02 0.02 0.02 0.22 0.05 0.01 0.02 0.72 0.39 0.49 0.36 0.62 0.76 0.82 0.77 0.81 0.89 0.69 0.98 0.84 0.17 0.01 0.01 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.07 0.04 0.06 0.04 0.03 0.07 0.24 0.10 0.05 0.02 0.74 0.45 0.50 0.37 0.70 0.77 0.96 0.83 0.88 0.90 0.70 1.02 0.87 0.23 0.04 0.04 0.07 

0.82 ± 0.11 0.88 ± 0.14 3 0.06 0.02 0.04 0.04 0.03 0.06 0.19 0.07 -0.03 0.88 0.71 0.41 0.48 0.34 0.63 0.72 0.83 0.78 0.76 0.89 0.64 0.93 0.81 0.16 0.00 0.05 0.05 

0.84 ± 0.12 1.20 ± 0.17 1 0.08 0.04 0.02 0.04 0.03 0.03 0.11 0.10 0.02 0.87 0.67 0.41 0.47 0.29 0.66 0.69 0.86 0.80 0.77 0.90 0.36 0.97 0.85 0.15 0.02 0.01 0.08 

0.81 ± 0.13 0.52 ± 0.09 26 0.05 0.03 0.04 0.05 0.04 0.03 0.23 0.05 0.63 0.81 0.65 0.43 0.49 0.35 0.62 0.73 0.88 0.65 0.68 0.84 0.68 0.96 0.77 0.16 0.02 0.02 0.02 

0.82 ± 0.12 0.78 ± 0.10 10 0.13 0.11 0.10 0.12 0.10 0.13 0.33 0.44 0.03 0.06 0.08 0.46 0.51 0.41 0.68 0.79 0.90 0.84 0.84 0.83 0.68 0.97 0.84 0.18 0.05 0.03 0.02 

0.88 ± 0.14 0.84 ± 0.12 2 0.13 0.10 0.08 0.07 0.08 1.06 1.24 0.44 0.07 0.02 0.07 0.54 0.53 0.39 0.71 0.78 0.93 0.81 0.73 0.87 0.70 0.96 0.86 0.19 0.03 0.06 0.04 

0.80 ± 0.12 0.74 ± 0.11 19 0.91 0.77 0.78 0.81 0.78 1.02 1.23 0.38 0.09 0.11 0.02 0.02 0.03 0.04 0.06 0.06 0.83 0.73 0.75 0.80 0.64 0.85 0.77 0.16 0.05 0.01 0.03 

0.80 ± 0.12 0.56 ± 0.08 24 0.84 0.74 0.73 0.80 0.77 0.96 1.19 0.38 0.62 0.82 0.20 0.07 0.10 0.02 0.16 0.18 0.33 0.74 0.82 0.80 0.67 0.89 0.70 0.13 0.00 -0.01 0.00 

0.83 ± 0.12 0.71 ± 0.10 14 0.89 0.79 0.81 0.87 0.85 1.04 1.28 0.38 0.67 0.89 0.11 0.00 0.04 -0.02 0.07 0.09 0.35 0.73 0.83 0.88 0.72 0.92 0.83 0.12 0.01 0.00 -0.02 

0.82 ± 0.12 0.45 ± 0.06 17 0.87 0.79 0.68 0.79 0.80 0.92 1.27 0.37 0.64 0.87 0.31 0.13 0.18 0.07 0.26 0.29 0.44 0.76 0.80 0.78 0.66 0.88 0.77 0.15 0.04 0.00 0.02 

0.86 ± 0.13 0.75 ± 0.11 22 0.91 0.81 0.75 0.82 0.81 1.00 1.25 0.42 0.66 0.86 0.10 0.00 0.05 -0.01 0.07 0.07 0.23 0.77 0.80 0.84 0.69 0.93 0.83 0.16 0.01 0.04 0.00 

0.83 ± 0.13 0.72 ± 0.11 6 0.91 0.78 0.79 0.85 0.81 1.05 1.23 0.39 0.63 0.85 0.08 0.03 0.05 0.02 0.05 0.04 0.22 0.76 0.78 0.87 0.70 0.95 0.82 0.19 0.02 0.04 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 0.94 0.88 0.78 0.85 0.84 0.97 1.22 0.40 0.59 0.82 0.69 0.02 0.11 0.02 0.06 0.15 0.01 0.07 0.06 0.82 0.64 0.94 0.78 0.16 0.03 0.02 -0.01 

0.82 ± 0.11 0.90 ± 0.13 23 0.97 0.91 0.82 0.91 0.88 0.98 1.25 0.35 0.68 0.83 0.73 0.51 0.46 0.35 0.66 0.75 0.02 0.05 0.04 0.87 0.67 1.00 0.81 0.19 0.04 0.04 0.02 

0.84 ± 0.12 0.89 ± 0.12 9 1.01 0.91 0.84 0.90 0.91 1.07 1.23 0.31 0.68 0.76 0.74 0.54 0.49 0.34 0.67 0.73 0.02 0.04 0.04 0.89 0.67 0.94 0.86 0.18 0.05 0.02 0.03 

0.84 ± 0.11 0.78 ± 0.11 4 0.96 0.86 0.78 0.90 0.84 1.03 1.26 0.46 0.68 0.90 0.70 0.46 0.48 0.36 0.64 0.75 0.81 0.76 0.76 0.03 0.66 0.89 0.77 0.15 0.03 0.04 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 0.89 0.85 0.78 0.84 0.79 1.01 1.05 0.38 0.62 0.83 0.69 0.41 0.42 0.31 0.61 0.72 0.82 0.70 0.71 0.81 0.02 0.92 0.79 0.14 -0.01 0.00 -0.01 

0.85 ± 0.13 0.78 ± 0.12 15 0.97 0.86 0.77 0.81 0.83 0.96 1.26 0.42 0.63 0.91 0.76 0.45 0.49 0.39 0.66 0.76 0.88 0.78 0.82 0.84 0.68 0.02 0.77 0.20 0.06 0.06 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 0.95 0.87 0.76 0.89 0.85 1.00 1.34 0.43 0.67 0.92 0.73 0.48 0.51 0.39 0.68 0.77 0.90 0.79 0.78 0.86 0.69 0.94 0.03 0.17 0.03 0.03 0.04 

0.83 ± 0.12 0.27 ± 0.03 25 0.90 0.88 0.77 0.86 0.83 1.00 1.23 0.42 0.66 0.92 0.75 0.50 0.47 0.40 0.68 0.75 0.88 0.82 0.82 0.86 0.71 0.95 0.81 0.08 0.06 0.05 0.05 

0.82 ± 0.13 0.02 ± 0.01 12 0.89 0.79 0.79 0.88 0.85 1.02 1.26 0.40 0.70 0.91 0.74 0.44 0.53 0.40 0.65 0.75 0.86 0.72 0.80 0.76 0.70 0.95 0.80 0.17 0.04 0.04 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 0.93 0.76 0.80 0.89 0.85 1.07 1.24 0.40 0.69 0.93 0.75 0.41 0.53 0.41 0.64 0.77 0.88 0.76 0.77 0.86 0.75 0.94 0.82 0.17 0.04 0.02 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 0.88 0.81 0.81 0.89 0.85 1.03 1.28 0.39 0.69 0.95 0.74 0.46 0.51 0.40 0.61 0.74 0.86 0.71 0.81 0.86 0.72 0.95 0.77 0.18 0.05 0.04 0.03 

No to Low 

mAb Binding 

VISHAL KAMAT 


Response (


Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 


Confidential 

mAb 

# 


20 21 11 7 8 3 1 26 10 2 19 24 14 17 22 6 18 23 9 4 5 15 16 25 12 13 

0.82 ± 0.13 0.88 ± 0.15 20 0.04 0.05 0.06 0.04 0.05 0.05 0.24 0.07 0.05 0.04 0.74 0.42 0.46 0.36 0.63 0.73 0.88 0.77 0.78 0.89 0.68 0.99 0.83 0.21 0.06 0.04 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.01 0.03 0.01 0.02 0.01 0.03 0.22 0.04 0.01 0.01 0.71 0.38 0.44 0.31 0.57 0.70 0.91 0.77 0.80 0.86 0.64 0.98 0.73 0.15 0.02 0.01 0.01 

0.84 ± 0.12 0.89 ± 0.13 11 0.01 0.02 0.03 0.01 0.03 0.04 0.21 0.05 0.01 0.01 0.73 0.46 0.49 0.33 0.66 0.75 0.88 0.81 0.72 0.85 0.66 0.95 0.85 0.19 0.04 0.04 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.02 0.02 0.01 0.02 0.02 0.02 0.22 0.05 0.01 0.02 0.72 0.39 0.49 0.36 0.62 0.76 0.82 0.77 0.81 0.89 0.69 0.98 0.84 0.17 0.01 0.01 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.07 0.04 0.06 0.04 0.03 0.07 0.24 0.10 0.05 0.02 0.74 0.45 0.50 0.37 0.70 0.77 0.96 0.83 0.88 0.90 0.70 1.02 0.87 0.23 0.04 0.04 0.07 

0.82 ± 0.11 0.88 ± 0.14 3 0.06 0.02 0.04 0.04 0.03 0.06 0.19 0.07 -0.03 0.88 0.71 0.41 0.48 0.34 0.63 0.72 0.83 0.78 0.76 0.89 0.64 0.93 0.81 0.16 0.00 0.05 0.05 

0.84 ± 0.12 1.20 ± 0.17 1 0.08 0.04 0.02 0.04 0.03 0.03 0.11 0.10 0.02 0.87 0.67 0.41 0.47 0.29 0.66 0.69 0.86 0.80 0.77 0.90 0.36 0.97 0.85 0.15 0.02 0.01 0.08 

0.81 ± 0.13 0.52 ± 0.09 26 0.05 0.03 0.04 0.05 0.04 0.03 0.23 0.05 0.63 0.81 0.65 0.43 0.49 0.35 0.62 0.73 0.88 0.65 0.68 0.84 0.68 0.96 0.77 0.16 0.02 0.02 0.02 

0.82 ± 0.12 0.78 ± 0.10 10 0.13 0.11 0.10 0.12 0.10 0.13 0.33 0.44 0.03 0.06 0.08 0.46 0.51 0.41 0.68 0.79 0.90 0.84 0.84 0.83 0.68 0.97 0.84 0.18 0.05 0.03 0.02 

0.88 ± 0.14 0.84 ± 0.12 2 0.13 0.10 0.08 0.07 0.08 1.06 1.24 0.44 0.07 0.02 0.07 0.54 0.53 0.39 0.71 0.78 0.93 0.81 0.73 0.87 0.70 0.96 0.86 0.19 0.03 0.06 0.04 

0.80 ± 0.12 0.74 ± 0.11 19 0.91 0.77 0.78 0.81 0.78 1.02 1.23 0.38 0.09 0.11 0.02 0.02 0.03 0.04 0.06 0.06 0.83 0.73 0.75 0.80 0.64 0.85 0.77 0.16 0.05 0.01 0.03 

0.80 ± 0.12 0.56 ± 0.08 24 0.84 0.74 0.73 0.80 0.77 0.96 1.19 0.38 0.62 0.82 0.20 0.07 0.10 0.02 0.16 0.18 0.33 0.74 0.82 0.80 0.67 0.89 0.70 0.13 0.00 -0.01 0.00 

0.83 ± 0.12 0.71 ± 0.10 14 0.89 0.79 0.81 0.87 0.85 1.04 1.28 0.38 0.67 0.89 0.11 0.00 0.04 -0.02 0.07 0.09 0.35 0.73 0.83 0.88 0.72 0.92 0.83 0.12 0.01 0.00 -0.02 

0.82 ± 0.12 0.45 ± 0.06 17 0.87 0.79 0.68 0.79 0.80 0.92 1.27 0.37 0.64 0.87 0.31 0.13 0.18 0.07 0.26 0.29 0.44 0.76 0.80 0.78 0.66 0.88 0.77 0.15 0.04 0.00 0.02 

0.86 ± 0.13 0.75 ± 0.11 22 0.91 0.81 0.75 0.82 0.81 1.00 1.25 0.42 0.66 0.86 0.10 0.00 0.05 -0.01 0.07 0.07 0.23 0.77 0.80 0.84 0.69 0.93 0.83 0.16 0.01 0.04 0.00 

0.83 ± 0.13 0.72 ± 0.11 6 0.91 0.78 0.79 0.85 0.81 1.05 1.23 0.39 0.63 0.85 0.08 0.03 0.05 0.02 0.05 0.04 0.22 0.76 0.78 0.87 0.70 0.95 0.82 0.19 0.02 0.04 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 0.94 0.88 0.78 0.85 0.84 0.97 1.22 0.40 0.59 0.82 0.69 0.02 0.11 0.02 0.06 0.15 0.01 0.07 0.06 0.82 0.64 0.94 0.78 0.16 0.03 0.02 -0.01 

0.82 ± 0.11 0.90 ± 0.13 23 0.97 0.91 0.82 0.91 0.88 0.98 1.25 0.35 0.68 0.83 0.73 0.51 0.46 0.35 0.66 0.75 0.02 0.05 0.04 0.87 0.67 1.00 0.81 0.19 0.04 0.04 0.02 

0.84 ± 0.12 0.89 ± 0.12 9 1.01 0.91 0.84 0.90 0.91 1.07 1.23 0.31 0.68 0.76 0.74 0.54 0.49 0.34 0.67 0.73 0.02 0.04 0.04 0.89 0.67 0.94 0.86 0.18 0.05 0.02 0.03 

0.84 ± 0.11 0.78 ± 0.11 4 0.96 0.86 0.78 0.90 0.84 1.03 1.26 0.46 0.68 0.90 0.70 0.46 0.48 0.36 0.64 0.75 0.81 0.76 0.76 0.03 0.66 0.89 0.77 0.15 0.03 0.04 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 0.89 0.85 0.78 0.84 0.79 1.01 1.05 0.38 0.62 0.83 0.69 0.41 0.42 0.31 0.61 0.72 0.82 0.70 0.71 0.81 0.02 0.92 0.79 0.14 -0.01 0.00 -0.01 

0.85 ± 0.13 0.78 ± 0.12 15 0.97 0.86 0.77 0.81 0.83 0.96 1.26 0.42 0.63 0.91 0.76 0.45 0.49 0.39 0.66 0.76 0.88 0.78 0.82 0.84 0.68 0.02 0.77 0.20 0.06 0.06 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 0.95 0.87 0.76 0.89 0.85 1.00 1.34 0.43 0.67 0.92 0.73 0.48 0.51 0.39 0.68 0.77 0.90 0.79 0.78 0.86 0.69 0.94 0.03 0.17 0.03 0.03 0.04 

0.83 ± 0.12 0.27 ± 0.03 25 0.90 0.88 0.77 0.86 0.83 1.00 1.23 0.42 0.66 0.92 0.75 0.50 0.47 0.40 0.68 0.75 0.88 0.82 0.82 0.86 0.71 0.95 0.81 0.08 0.06 0.05 0.05 

0.82 ± 0.13 0.02 ± 0.01 12 0.89 0.79 0.79 0.88 0.85 1.02 1.26 0.40 0.70 0.91 0.74 0.44 0.53 0.40 0.65 0.75 0.86 0.72 0.80 0.76 0.70 0.95 0.80 0.17 0.04 0.04 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 0.93 0.76 0.80 0.89 0.85 1.07 1.24 0.40 0.69 0.93 0.75 0.41 0.53 0.41 0.64 0.77 0.88 0.76 0.77 0.86 0.75 0.94 0.82 0.17 0.04 0.02 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 0.88 0.81 0.81 0.89 0.85 1.03 1.28 0.39 0.69 0.95 0.74 0.46 0.51 0.40 0.61 0.74 0.86 0.71 0.81 0.86 0.72 0.95 0.77 0.18 0.05 0.04 0.03 

No to Low 

mAb Binding 

VISHAL KAMAT 


Response (


Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 


Confidential 

mAb 

# 


20 21 11 7 8 3 1 26 10 2 19 24 14 17 22 6 18 23 9 4 5 15 16 25 12 13 

0.82 ± 0.13 0.88 ± 0.15 20 0.04 0.05 0.06 0.04 0.05 0.05 0.24 0.07 0.05 0.04 0.74 0.42 0.46 0.36 0.63 0.73 0.88 0.77 0.78 0.89 0.68 0.99 0.83 0.21 0.06 0.04 0.05 

0.82 ± 0.13 0.86 ± 0.14 21 0.01 0.03 0.01 0.02 0.01 0.03 0.22 0.04 0.01 0.01 0.71 0.38 0.44 0.31 0.57 0.70 0.91 0.77 0.80 0.86 0.64 0.98 0.73 0.15 0.02 0.01 0.01 

0.84 ± 0.12 0.89 ± 0.13 11 0.01 0.02 0.03 0.01 0.03 0.04 0.21 0.05 0.01 0.01 0.73 0.46 0.49 0.33 0.66 0.75 0.88 0.81 0.72 0.85 0.66 0.95 0.85 0.19 0.04 0.04 0.02 

0.83 ± 0.13 0.86 ± 0.14 7 0.02 0.02 0.01 0.02 0.02 0.02 0.22 0.05 0.01 0.02 0.72 0.39 0.49 0.36 0.62 0.76 0.82 0.77 0.81 0.89 0.69 0.98 0.84 0.17 0.01 0.01 0.02 

0.87 ± 0.13 0.91 ± 0.13 8 0.07 0.04 0.06 0.04 0.03 0.07 0.24 0.10 0.05 0.02 0.74 0.45 0.50 0.37 0.70 0.77 0.96 0.83 0.88 0.90 0.70 1.02 0.87 0.23 0.04 0.04 0.07 

0.82 ± 0.11 0.88 ± 0.14 3 0.06 0.02 0.04 0.04 0.03 0.06 0.19 0.07 -0.03 0.88 0.71 0.41 0.48 0.34 0.63 0.72 0.83 0.78 0.76 0.89 0.64 0.93 0.81 0.16 0.00 0.05 0.05 

0.84 ± 0.12 1.20 ± 0.17 1 0.08 0.04 0.02 0.04 0.03 0.03 0.11 0.10 0.02 0.87 0.67 0.41 0.47 0.29 0.66 0.69 0.86 0.80 0.77 0.90 0.36 0.97 0.85 0.15 0.02 0.01 0.08 

0.81 ± 0.13 0.52 ± 0.09 26 0.05 0.03 0.04 0.05 0.04 0.03 0.23 0.05 0.63 0.81 0.65 0.43 0.49 0.35 0.62 0.73 0.88 0.65 0.68 0.84 0.68 0.96 0.77 0.16 0.02 0.02 0.02 

0.82 ± 0.12 0.78 ± 0.10 10 0.13 0.11 0.10 0.12 0.10 0.13 0.33 0.44 0.03 0.06 0.08 0.46 0.51 0.41 0.68 0.79 0.90 0.84 0.84 0.83 0.68 0.97 0.84 0.18 0.05 0.03 0.02 

0.88 ± 0.14 0.84 ± 0.12 2 0.13 0.10 0.08 0.07 0.08 1.06 1.24 0.44 0.07 0.02 0.07 0.54 0.53 0.39 0.71 0.78 0.93 0.81 0.73 0.87 0.70 0.96 0.86 0.19 0.03 0.06 0.04 

0.80 ± 0.12 0.74 ± 0.11 19 0.91 0.77 0.78 0.81 0.78 1.02 1.23 0.38 0.09 0.11 0.02 0.02 0.03 0.04 0.06 0.06 0.83 0.73 0.75 0.80 0.64 0.85 0.77 0.16 0.05 0.01 0.03 

0.80 ± 0.12 0.56 ± 0.08 24 0.84 0.74 0.73 0.80 0.77 0.96 1.19 0.38 0.62 0.82 0.20 0.07 0.10 0.02 0.16 0.18 0.33 0.74 0.82 0.80 0.67 0.89 0.70 0.13 0.00 -0.01 0.00 

0.83 ± 0.12 0.71 ± 0.10 14 0.89 0.79 0.81 0.87 0.85 1.04 1.28 0.38 0.67 0.89 0.11 0.00 0.04 -0.02 0.07 0.09 0.35 0.73 0.83 0.88 0.72 0.92 0.83 0.12 0.01 0.00 -0.02 

0.82 ± 0.12 0.45 ± 0.06 17 0.87 0.79 0.68 0.79 0.80 0.92 1.27 0.37 0.64 0.87 0.31 0.13 0.18 0.07 0.26 0.29 0.44 0.76 0.80 0.78 0.66 0.88 0.77 0.15 0.04 0.00 0.02 

0.86 ± 0.13 0.75 ± 0.11 22 0.91 0.81 0.75 0.82 0.81 1.00 1.25 0.42 0.66 0.86 0.10 0.00 0.05 -0.01 0.07 0.07 0.23 0.77 0.80 0.84 0.69 0.93 0.83 0.16 0.01 0.04 0.00 

0.83 ± 0.13 0.72 ± 0.11 6 0.91 0.78 0.79 0.85 0.81 1.05 1.23 0.39 0.63 0.85 0.08 0.03 0.05 0.02 0.05 0.04 0.22 0.76 0.78 0.87 0.70 0.95 0.82 0.19 0.02 0.04 0.02 

0.83 ± 0.12 0.82 ± 0.11 18 0.94 0.88 0.78 0.85 0.84 0.97 1.22 0.40 0.59 0.82 0.69 0.02 0.11 0.02 0.06 0.15 0.01 0.07 0.06 0.82 0.64 0.94 0.78 0.16 0.03 0.02 -0.01 

0.82 ± 0.11 0.90 ± 0.13 23 0.97 0.91 0.82 0.91 0.88 0.98 1.25 0.35 0.68 0.83 0.73 0.51 0.46 0.35 0.66 0.75 0.02 0.05 0.04 0.87 0.67 1.00 0.81 0.19 0.04 0.04 0.02 

0.84 ± 0.12 0.89 ± 0.12 9 1.01 0.91 0.84 0.90 0.91 1.07 1.23 0.31 0.68 0.76 0.74 0.54 0.49 0.34 0.67 0.73 0.02 0.04 0.04 0.89 0.67 0.94 0.86 0.18 0.05 0.02 0.03 

0.84 ± 0.11 0.78 ± 0.11 4 0.96 0.86 0.78 0.90 0.84 1.03 1.26 0.46 0.68 0.90 0.70 0.46 0.48 0.36 0.64 0.75 0.81 0.76 0.76 0.03 0.66 0.89 0.77 0.15 0.03 0.04 0.04 

0.82 ± 0.13 0.72 ± 0.10 5 0.89 0.85 0.78 0.84 0.79 1.01 1.05 0.38 0.62 0.83 0.69 0.41 0.42 0.31 0.61 0.72 0.82 0.70 0.71 0.81 0.02 0.92 0.79 0.14 -0.01 0.00 -0.01 

0.85 ± 0.13 0.78 ± 0.12 15 0.97 0.86 0.77 0.81 0.83 0.96 1.26 0.42 0.63 0.91 0.76 0.45 0.49 0.39 0.66 0.76 0.88 0.78 0.82 0.84 0.68 0.02 0.77 0.20 0.06 0.06 0.04 

0.88 ± 0.12 0.69 ± 0.10 16 0.95 0.87 0.76 0.89 0.85 1.00 1.34 0.43 0.67 0.92 0.73 0.48 0.51 0.39 0.68 0.77 0.90 0.79 0.78 0.86 0.69 0.94 0.03 0.17 0.03 0.03 0.04 

0.83 ± 0.12 0.27 ± 0.03 25 0.90 0.88 0.77 0.86 0.83 1.00 1.23 0.42 0.66 0.92 0.75 0.50 0.47 0.40 0.68 0.75 0.88 0.82 0.82 0.86 0.71 0.95 0.81 0.08 0.06 0.05 0.05 

0.82 ± 0.13 0.02 ± 0.01 12 0.89 0.79 0.79 0.88 0.85 1.02 1.26 0.40 0.70 0.91 0.74 0.44 0.53 0.40 0.65 0.75 0.86 0.72 0.80 0.76 0.70 0.95 0.80 0.17 0.04 0.04 0.02 

0.82 ± 0.13 0.03 ± 0.01 13 0.93 0.76 0.80 0.89 0.85 1.07 1.24 0.40 0.69 0.93 0.75 0.41 0.53 0.41 0.64 0.77 0.88 0.76 0.77 0.86 0.75 0.94 0.82 0.17 0.04 0.02 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 0.88 0.81 0.81 0.89 0.85 1.03 1.28 0.39 0.69 0.95 0.74 0.46 0.51 0.40 0.61 0.74 0.86 0.71 0.81 0.86 0.72 0.95 0.77 0.18 0.05 0.04 0.03 

No to Low 

mAb Binding 

VISHAL KAMAT 

Competing mAbs 

27 

(-) 

Negative Control

Data Analysis – Re-order the mAb# for Simplistic Representation 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 


Confidential 

mAb 

# 


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.82 ± 0.13 0.88 ± 0.15 1 0.04 0.05 0.06 0.04 0.05 0.05 0.24 0.07 0.05 0.04 0.74 0.42 0.46 0.36 0.63 0.73 0.88 0.77 0.78 0.89 0.68 0.99 0.83 0.21 0.06 0.04 0.05 

0.82 ± 0.13 0.86 ± 0.14 2 0.01 0.03 0.01 0.02 0.01 0.03 0.22 0.04 0.01 0.01 0.71 0.38 0.44 0.31 0.57 0.70 0.91 0.77 0.80 0.86 0.64 0.98 0.73 0.15 0.02 0.01 0.01 

0.84 ± 0.12 0.89 ± 0.13 3 0.01 0.02 0.03 0.01 0.03 0.04 0.21 0.05 0.01 0.01 0.73 0.46 0.49 0.33 0.66 0.75 0.88 0.81 0.72 0.85 0.66 0.95 0.85 0.19 0.04 0.04 0.02 

0.83 ± 0.13 0.86 ± 0.14 4 0.02 0.02 0.01 0.02 0.02 0.02 0.22 0.05 0.01 0.02 0.72 0.39 0.49 0.36 0.62 0.76 0.82 0.77 0.81 0.89 0.69 0.98 0.84 0.17 0.01 0.01 0.02 

0.87 ± 0.13 0.91 ± 0.13 5 0.07 0.04 0.06 0.04 0.03 0.07 0.24 0.10 0.05 0.02 0.74 0.45 0.50 0.37 0.70 0.77 0.96 0.83 0.88 0.90 0.70 1.02 0.87 0.23 0.04 0.04 0.07 

0.82 ± 0.11 0.88 ± 0.14 6 0.06 0.02 0.04 0.04 0.03 0.06 0.19 0.07 -0.03 0.88 0.71 0.41 0.48 0.34 0.63 0.72 0.83 0.78 0.76 0.89 0.64 0.93 0.81 0.16 0.00 0.05 0.05 

0.84 ± 0.12 1.20 ± 0.17 7 0.08 0.04 0.02 0.04 0.03 0.03 0.11 0.10 0.02 0.87 0.67 0.41 0.47 0.29 0.66 0.69 0.86 0.80 0.77 0.90 0.36 0.97 0.85 0.15 0.02 0.01 0.08 

0.81 ± 0.13 0.52 ± 0.09 8 0.05 0.03 0.04 0.05 0.04 0.03 0.23 0.05 0.63 0.81 0.65 0.43 0.49 0.35 0.62 0.73 0.88 0.65 0.68 0.84 0.68 0.96 0.77 0.16 0.02 0.02 0.02 

0.82 ± 0.12 0.78 ± 0.10 9 0.13 0.11 0.10 0.12 0.10 0.13 0.33 0.44 0.03 0.06 0.08 0.46 0.51 0.41 0.68 0.79 0.90 0.84 0.84 0.83 0.68 0.97 0.84 0.18 0.05 0.03 0.02 

0.88 ± 0.14 0.84 ± 0.12 10 0.13 0.10 0.08 0.07 0.08 1.06 1.24 0.44 0.07 0.02 0.07 0.54 0.53 0.39 0.71 0.78 0.93 0.81 0.73 0.87 0.70 0.96 0.86 0.19 0.03 0.06 0.04 

0.80 ± 0.12 0.74 ± 0.11 11 0.91 0.77 0.78 0.81 0.78 1.02 1.23 0.38 0.09 0.11 0.02 0.02 0.03 0.04 0.06 0.06 0.83 0.73 0.75 0.80 0.64 0.85 0.77 0.16 0.05 0.01 0.03 

0.80 ± 0.12 0.56 ± 0.08 12 0.84 0.74 0.73 0.80 0.77 0.96 1.19 0.38 0.62 0.82 0.20 0.07 0.10 0.02 0.16 0.18 0.33 0.74 0.82 0.80 0.67 0.89 0.70 0.13 0.00 -0.01 0.00 

0.83 ± 0.12 0.71 ± 0.10 13 0.89 0.79 0.81 0.87 0.85 1.04 1.28 0.38 0.67 0.89 0.11 0.00 0.04 -0.02 0.07 0.09 0.35 0.73 0.83 0.88 0.72 0.92 0.83 0.12 0.01 0.00 -0.02 

0.82 ± 0.12 0.45 ± 0.06 14 0.87 0.79 0.68 0.79 0.80 0.92 1.27 0.37 0.64 0.87 0.31 0.13 0.18 0.07 0.26 0.29 0.44 0.76 0.80 0.78 0.66 0.88 0.77 0.15 0.04 0.00 0.02 

0.86 ± 0.13 0.75 ± 0.11 15 0.91 0.81 0.75 0.82 0.81 1.00 1.25 0.42 0.66 0.86 0.10 0.00 0.05 -0.01 0.07 0.07 0.23 0.77 0.80 0.84 0.69 0.93 0.83 0.16 0.01 0.04 0.00 

0.83 ± 0.13 0.72 ± 0.11 16 0.91 0.78 0.79 0.85 0.81 1.05 1.23 0.39 0.63 0.85 0.08 0.03 0.05 0.02 0.05 0.04 0.22 0.76 0.78 0.87 0.70 0.95 0.82 0.19 0.02 0.04 0.02 

0.83 ± 0.12 0.82 ± 0.11 17 0.94 0.88 0.78 0.85 0.84 0.97 1.22 0.40 0.59 0.82 0.69 0.02 0.11 0.02 0.06 0.15 0.01 0.07 0.06 0.82 0.64 0.94 0.78 0.16 0.03 0.02 -0.01 

0.82 ± 0.11 0.90 ± 0.13 18 0.97 0.91 0.82 0.91 0.88 0.98 1.25 0.35 0.68 0.83 0.73 0.51 0.46 0.35 0.66 0.75 0.02 0.05 0.04 0.87 0.67 1.00 0.81 0.19 0.04 0.04 0.02 

0.84 ± 0.12 0.89 ± 0.12 19 1.01 0.91 0.84 0.90 0.91 1.07 1.23 0.31 0.68 0.76 0.74 0.54 0.49 0.34 0.67 0.73 0.02 0.04 0.04 0.89 0.67 0.94 0.86 0.18 0.05 0.02 0.03 

0.84 ± 0.11 0.78 ± 0.11 20 0.96 0.86 0.78 0.90 0.84 1.03 1.26 0.46 0.68 0.90 0.70 0.46 0.48 0.36 0.64 0.75 0.81 0.76 0.76 0.03 0.66 0.89 0.77 0.15 0.03 0.04 0.04 

0.82 ± 0.13 0.72 ± 0.10 21 0.89 0.85 0.78 0.84 0.79 1.01 1.05 0.38 0.62 0.83 0.69 0.41 0.42 0.31 0.61 0.72 0.82 0.70 0.71 0.81 0.02 0.92 0.79 0.14 -0.01 0.00 -0.01 

0.85 ± 0.13 0.78 ± 0.12 22 0.97 0.86 0.77 0.81 0.83 0.96 1.26 0.42 0.63 0.91 0.76 0.45 0.49 0.39 0.66 0.76 0.88 0.78 0.82 0.84 0.68 0.02 0.77 0.20 0.06 0.06 0.04 

0.88 ± 0.12 0.69 ± 0.10 23 0.95 0.87 0.76 0.89 0.85 1.00 1.34 0.43 0.67 0.92 0.73 0.48 0.51 0.39 0.68 0.77 0.90 0.79 0.78 0.86 0.69 0.94 0.03 0.17 0.03 0.03 0.04 

0.83 ± 0.12 0.27 ± 0.03 24 0.90 0.88 0.77 0.86 0.83 1.00 1.23 0.42 0.66 0.92 0.75 0.50 0.47 0.40 0.68 0.75 0.88 0.82 0.82 0.86 0.71 0.95 0.81 0.08 0.06 0.05 0.05 

0.82 ± 0.13 0.02 ± 0.01 25 0.89 0.79 0.79 0.88 0.85 1.02 1.26 0.40 0.70 0.91 0.74 0.44 0.53 0.40 0.65 0.75 0.86 0.72 0.80 0.76 0.70 0.95 0.80 0.17 0.04 0.04 0.02 

0.82 ± 0.13 0.03 ± 0.01 26 0.93 0.76 0.80 0.89 0.85 1.07 1.24 0.40 0.69 0.93 0.75 0.41 0.53 0.41 0.64 0.77 0.88 0.76 0.77 0.86 0.75 0.94 0.82 0.17 0.04 0.02 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 0.88 0.81 0.81 0.89 0.85 1.03 1.28 0.39 0.69 0.95 0.74 0.46 0.51 0.40 0.61 0.74 0.86 0.71 0.81 0.86 0.72 0.95 0.77 0.18 0.05 0.04 0.03 

No to Low 

mAb Binding 

VISHAL KAMAT 

Competing mAbs 

27 

(-) 

Negative Control

Data Analysis – Representation of Cross-Competition Data 

Amount of 

Antigen 

Capture (nm) 

50 ug/mL 

mAb1 

Bound (nm) 

mAb 

# 


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.82 ± 0.13 0.88 ± 0.15 1 0.04 0.05 0.06 0.04 0.05 0.05 0.24 0.07 0.05 0.04 0.74 0.42 0.46 0.36 0.63 0.73 0.88 0.77 0.78 0.89 0.68 0.99 0.83 0.21 0.06 0.04 0.05 

0.82 ± 0.13 0.86 ± 0.14 2 0.01 0.03 0.01 0.02 0.01 0.03 0.22 0.04 0.01 0.01 0.71 0.38 0.44 0.31 0.57 0.70 0.91 0.77 0.80 0.86 0.64 0.98 0.73 0.15 0.02 0.01 0.01 

0.84 ± 0.12 0.89 ± 0.13 3 0.01 0.02 0.03 0.01 0.03 0.04 0.21 0.05 0.01 0.01 0.73 0.46 0.49 0.33 0.66 0.75 0.88 0.81 0.72 0.85 0.66 0.95 0.85 0.19 0.04 0.04 0.02 

0.83 ± 0.13 0.86 ± 0.14 4 0.02 0.02 0.01 0.02 0.02 0.02 0.22 0.05 0.01 0.02 0.72 0.39 0.49 0.36 0.62 0.76 0.82 0.77 0.81 0.89 0.69 0.98 0.84 0.17 0.01 0.01 0.02 

0.87 ± 0.13 0.91 ± 0.13 5 0.07 0.04 0.06 0.04 0.03 0.07 0.24 0.10 0.05 0.02 0.74 0.45 0.50 0.37 0.70 0.77 0.96 0.83 0.88 0.90 0.70 1.02 0.87 0.23 0.04 0.04 0.07 

0.82 ± 0.11 0.88 ± 0.14 6 0.06 0.02 0.04 0.04 0.03 0.06 0.19 0.07 -0.03 0.88 0.71 0.41 0.48 0.34 0.63 0.72 0.83 0.78 0.76 0.89 0.64 0.93 0.81 0.16 0.00 0.05 0.05 

0.84 ± 0.12 1.20 ± 0.17 7 0.08 0.04 0.02 0.04 0.03 0.03 0.11 0.10 0.02 0.87 0.67 0.41 0.47 0.29 0.66 0.69 0.86 0.80 0.77 0.90 0.36 0.97 0.85 0.15 0.02 0.01 0.08 

0.81 ± 0.13 0.52 ± 0.09 8 0.05 0.03 0.04 0.05 0.04 0.03 0.23 0.05 0.63 0.81 0.65 0.43 0.49 0.35 0.62 0.73 0.88 0.65 0.68 0.84 0.68 0.96 0.77 0.16 0.02 0.02 0.02 

0.82 ± 0.12 0.78 ± 0.10 9 0.13 0.11 0.10 0.12 0.10 0.13 0.33 0.44 0.03 0.06 0.08 0.46 0.51 0.41 0.68 0.79 0.90 0.84 0.84 0.83 0.68 0.97 0.84 0.18 0.05 0.03 0.02 

0.88 ± 0.14 0.84 ± 0.12 10 0.13 0.10 0.08 0.07 0.08 1.06 1.24 0.44 0.07 0.02 0.07 0.54 0.53 0.39 0.71 0.78 0.93 0.81 0.73 0.87 0.70 0.96 0.86 0.19 0.03 0.06 0.04 

0.80 ± 0.12 0.74 ± 0.11 11 0.91 0.77 0.78 0.81 0.78 1.02 1.23 0.38 0.09 0.11 0.02 0.02 0.03 0.04 0.06 0.06 0.83 0.73 0.75 0.80 0.64 0.85 0.77 0.16 0.05 0.01 0.03 

0.80 ± 0.12 0.56 ± 0.08 12 0.84 0.74 0.73 0.80 0.77 0.96 1.19 0.38 0.62 0.82 0.20 0.07 0.10 0.02 0.16 0.18 0.33 0.74 0.82 0.80 0.67 0.89 0.70 0.13 0.00 -0.01 0.00 

0.83 ± 0.12 0.71 ± 0.10 13 0.89 0.79 0.81 0.87 0.85 1.04 1.28 0.38 0.67 0.89 0.11 0.00 0.04 -0.02 0.07 0.09 0.35 0.73 0.83 0.88 0.72 0.92 0.83 0.12 0.01 0.00 -0.02 

0.82 ± 0.12 0.45 ± 0.06 14 0.87 0.79 0.68 0.79 0.80 0.92 1.27 0.37 0.64 0.87 0.31 0.13 0.18 0.07 0.26 0.29 0.44 0.76 0.80 0.78 0.66 0.88 0.77 0.15 0.04 0.00 0.02 

0.86 ± 0.13 0.75 ± 0.11 15 0.91 0.81 0.75 0.82 0.81 1.00 1.25 0.42 0.66 0.86 0.10 0.00 0.05 -0.01 0.07 0.07 0.23 0.77 0.80 0.84 0.69 0.93 0.83 0.16 0.01 0.04 0.00 

0.83 ± 0.13 0.72 ± 0.11 16 0.91 0.78 0.79 0.85 0.81 1.05 1.23 0.39 0.63 0.85 0.08 0.03 0.05 0.02 0.05 0.04 0.22 0.76 0.78 0.87 0.70 0.95 0.82 0.19 0.02 0.04 0.02 

0.83 ± 0.12 0.82 ± 0.11 17 0.94 0.88 0.78 0.85 0.84 0.97 1.22 0.40 0.59 0.82 0.69 0.02 0.11 0.02 0.06 0.15 0.01 0.07 0.06 0.82 0.64 0.94 0.78 0.16 0.03 0.02 -0.01 

0.82 ± 0.11 0.90 ± 0.13 18 0.97 0.91 0.82 0.91 0.88 0.98 1.25 0.35 0.68 0.83 0.73 0.51 0.46 0.35 0.66 0.75 0.02 0.05 0.04 0.87 0.67 1.00 0.81 0.19 0.04 0.04 0.02 

0.84 ± 0.12 0.89 ± 0.12 19 1.01 0.91 0.84 0.90 0.91 1.07 1.23 0.31 0.68 0.76 0.74 0.54 0.49 0.34 0.67 0.73 0.02 0.04 0.04 0.89 0.67 0.94 0.86 0.18 0.05 0.02 0.03 

0.84 ± 0.11 0.78 ± 0.11 20 0.96 0.86 0.78 0.90 0.84 1.03 1.26 0.46 0.68 0.90 0.70 0.46 0.48 0.36 0.64 0.75 0.81 0.76 0.76 0.03 0.66 0.89 0.77 0.15 0.03 0.04 0.04 

0.82 ± 0.13 0.72 ± 0.10 21 0.89 0.85 0.78 0.84 0.79 1.01 1.05 0.38 0.62 0.83 0.69 0.41 0.42 0.31 0.61 0.72 0.82 0.70 0.71 0.81 0.02 0.92 0.79 0.14 -0.01 0.00 -0.01 

0.85 ± 0.13 0.78 ± 0.12 22 0.97 0.86 0.77 0.81 0.83 0.96 1.26 0.42 0.63 0.91 0.76 0.45 0.49 0.39 0.66 0.76 0.88 0.78 0.82 0.84 0.68 0.02 0.77 0.20 0.06 0.06 0.04 

0.88 ± 0.12 0.69 ± 0.10 23 0.95 0.87 0.76 0.89 0.85 1.00 1.34 0.43 0.67 0.92 0.73 0.48 0.51 0.39 0.68 0.77 0.90 0.79 0.78 0.86 0.69 0.94 0.03 0.17 0.03 0.03 0.04 

0.83 ± 0.12 0.27 ± 0.03 24 0.90 0.88 0.77 0.86 0.83 1.00 1.23 0.42 0.66 0.92 0.75 0.50 0.47 0.40 0.68 0.75 0.88 0.82 0.82 0.86 0.71 0.95 0.81 0.08 0.06 0.05 0.05 

0.82 ± 0.13 0.02 ± 0.01 25 0.89 0.79 0.79 0.88 0.85 1.02 1.26 0.40 0.70 0.91 0.74 0.44 0.53 0.40 0.65 0.75 0.86 0.72 0.80 0.76 0.70 0.95 0.80 0.17 0.04 0.04 0.02 

0.82 ± 0.13 0.03 ± 0.01 26 0.93 0.76 0.80 0.89 0.85 1.07 1.24 0.40 0.69 0.93 0.75 0.41 0.53 0.41 0.64 0.77 0.88 0.76 0.77 0.86 0.75 0.94 0.82 0.17 0.04 0.02 0.02 

0.81 ± 0.13 0.02 ± 0.01 27 (-) 0.88 0.81 0.81 0.89 0.85 1.03 1.28 0.39 0.69 0.95 0.74 0.46 0.51 0.40 0.61 0.74 0.86 0.71 0.81 0.86 0.72 0.95 0.77 0.18 0.05 0.04 0.03 

27 

(-) 

Confidential 

VISHAL KAMAT

Tutorial: Drawing Circles to Represent Cross-Competition Data 

~50kD Antigen – 18x18 

Amount of 

Antigen 

Captured ± Std 

Dev (nm) 

Amount of 

mAb-1 Bound 

± Std Dev (nm) 

mAb 

# 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 

19 

(-) 

0.21 ± 0.02 0.38 ± 0.03 1 -0.01 0.00 0.00 -0.01 0.00 0.00 0.23 0.30 0.19 0.24 0.22 0.20 0.20 0.36 0.36 0.41 0.26 0.00 0.00 

0.19 ± 0.06 0.18 ± 0.06 2 0.14 0.07 0.03 0.11 0.13 0.07 0.24 0.30 0.20 0.25 0.21 0.18 0.19 0.35 0.35 0.12 0.24 0.01 -0.01 

0.22 ± 0.02 0.40 ± 0.02 3 0.01 -0.01 0.02 -0.01 0.02 0.00 0.27 0.27 0.23 0.29 0.21 0.18 0.16 0.38 0.41 0.46 0.34 -0.01 0.00 

0.13 ± 0.01 0.30 ± 0.01 4 0.03 0.00 0.04 0.00 0.02 0.00 0.13 0.23 0.17 0.16 0.15 0.12 0.11 0.31 0.27 0.32 0.17 -0.03 -0.03 

0.13 ± 0.01 0.31 ± 0.01 5 0.00 0.00 0.02 0.00 0.00 0.00 0.16 0.26 0.21 0.20 0.18 0.16 0.14 0.35 0.31 0.36 0.20 0.00 -0.01 

0.21 ± 0.02 0.21 ± 0.01 6 0.14 0.08 0.17 0.12 0.14 0.07 0.27 0.32 0.23 0.28 0.23 0.21 0.21 0.35 0.34 0.36 0.26 0.02 0.00 

0.21 ± 0.02 0.31 ± 0.03 7 0.28 0.21 0.32 0.24 0.29 0.19 0.00 0.01 -0.02 0.00 0.20 0.19 0.19 0.35 0.33 0.38 0.23 0.01 -0.01 

0.20 ± 0.05 0.31 ± 0.03 8 0.29 0.21 0.32 0.25 0.28 0.19 0.00 0.00 -0.01 0.00 0.17 0.14 0.14 0.35 0.34 0.40 0.24 0.00 0.00 

0.14 ± 0.01 0.29 ± 0.01 9 0.27 0.14 0.30 0.24 0.28 0.19 -0.01 0.00 -0.02 -0.01 0.21 0.21 0.20 0.35 0.32 0.36 0.27 0.00 -0.01 

0.14 ± 0.01 0.27 ± 0.02 10 0.25 0.13 0.29 0.22 0.25 0.18 -0.01 0.00 -0.02 -0.01 0.19 0.21 0.18 0.34 0.30 0.35 0.20 0.00 -0.01 

0.14 ± 0.01 0.22 ± 0.01 11 0.28 0.14 0.29 0.23 0.26 0.19 0.15 0.16 0.19 0.20 0.00 0.00 0.00 0.34 0.31 0.36 0.22 0.00 0.00 

0.22 ± 0.02 0.23 ± 0.02 12 0.32 0.20 0.34 0.27 0.31 0.20 0.26 0.19 0.22 0.29 0.00 0.00 0.00 0.36 0.36 0.41 0.27 0.01 0.00 

0.21 ± 0.02 0.23 ± 0.02 13 0.31 0.20 0.33 0.26 0.30 0.19 0.26 0.19 0.21 0.27 0.00 0.01 0.00 0.35 0.35 0.40 0.25 0.01 0.00 

0.23 ± 0.02 0.43 ± 0.02 14 0.35 0.17 0.42 0.29 0.34 0.17 0.28 0.27 0.24 0.30 0.22 0.18 0.16 -0.01 0.00 0.49 0.25 -0.01 -0.01 

0.13 ± 0.01 0.38 ± 0.01 15 0.30 0.17 0.33 0.23 0.29 0.18 0.17 0.27 0.22 0.21 0.18 0.17 0.14 0.00 0.00 0.36 0.22 0.00 0.00 

0.14 ± 0.01 0.40 ± 0.01 16 0.30 0.09 0.31 0.22 0.26 0.10 0.18 0.28 0.21 0.21 0.18 0.16 0.15 0.35 0.33 0.02 0.24 -0.05 -0.04 

0.21 ± 0.02 0.33 ± 0.03 17 0.32 0.21 0.33 0.25 0.29 0.19 0.26 0.33 0.21 0.27 0.24 0.21 0.22 0.36 0.35 0.42 0.00 0.00 0.00 

0.14 ± 0.01 0.01 ± 0.01 18 0.29 0.15 0.31 0.25 0.27 0.18 0.19 0.27 0.22 0.22 0.19 0.19 0.17 0.33 0.32 0.35 0.23 0.01 0.00 

0.23 ± 0.02 -0.02 ± 0.01 19 (-) 0.32 0.15 0.39 0.27 0.30 0.16 0.27 0.27 0.23 0.28 0.20 0.18 0.16 0.33 0.35 0.42 0.24 -0.01 -0.02 

mAb-1, mAb-2, 

mAb-11, 

mAb-12, mAb-13 

mAb-16 

mAb-3, mAb-4, 

mAb-7, mAb-8, 

mAb-5, mAb-6 

mAb-9, mAb-10 

mAb-14, 

mAb-17 

mAb-15 

Confidential 

VISHAL KAMAT

Tutorial: Drawing Circles to Represent Cross-Competition Data 

~60kD Antigen – 14x14 

Amount of 

Antigen 

Captured ± 

Std Dev (nm) 

mAb1 Bound 

± Std Dev 

(nm) 

mAb 

# 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 

0.44 ± 0.02 0.51 ± 0.03 1 0.00 0.03 0.08 0.01 0.04 0.02 0.46 0.40 0.39 0.26 0.19 0.38 -0.01 0.00 

0.45 ± 0.02 0.57 ± 0.03 2 0.00 0.00 0.05 0.01 0.03 0.00 0.45 0.42 0.41 0.26 0.22 0.43 0.00 0.02 

0.44 ± 0.02 0.48 ± 0.04 3 0.04 0.09 0.03 0.00 0.03 0.02 0.42 0.37 0.37 0.25 0.15 0.34 -0.03 -0.03 

0.44 ± 0.03 0.45 ± 0.04 4 0.07 0.12 0.11 0.05 0.18 0.14 0.52 0.48 0.47 0.31 0.30 0.37 -0.05 -0.04 

0.47 ± 0.04 0.52 ± 0.03 5 0.04 0.07 0.03 0.03 0.02 0.00 0.01 0.38 0.40 0.27 0.20 0.39 0.00 0.01 

0.41 ± 0.08 0.47 ± 0.10 6 0.03 0.02 0.02 0.02 0.03 0.00 0.01 0.41 0.39 0.27 0.18 0.42 0.00 0.01 

0.45 ± 0.03 0.51 ± 0.04 7 0.40 0.45 0.44 0.54 0.11 0.06 0.03 0.02 0.05 0.02 -0.02 0.37 -0.02 -0.01 

0.43 ± 0.04 0.48 ± 0.04 8 0.45 0.45 0.42 0.55 0.57 0.47 0.08 0.06 0.09 0.07 0.04 0.42 0.04 0.04 

0.42 ± 0.05 0.48 ± 0.05 9 0.47 0.48 0.41 0.54 0.55 0.39 0.08 0.06 0.05 0.06 0.04 0.39 0.04 0.05 

0.42 ± 0.04 0.32 ± 0.03 10 0.41 0.41 0.39 0.51 0.51 0.36 0.18 0.14 0.15 0.08 0.05 0.39 0.02 0.02 

0.41 ± 0.03 0.24 ± 0.02 11 0.35 0.46 0.43 0.45 0.48 0.40 0.26 0.21 0.22 0.14 0.06 0.38 0.00 0.00 

0.42 ± 0.02 0.40 ± 0.03 12 0.40 0.39 0.36 0.39 0.48 0.42 0.42 0.41 0.35 0.22 0.19 0.02 0.00 0.01 

0.44 ± 0.03 -0.02 ± 0.01 13 0.48 0.47 0.45 0.49 0.48 0.40 0.48 0.46 0.43 0.34 0.30 0.41 0.00 0.00 

0.45 ± 0.03 -0.03 ± 0.01 14 0.50 0.47 0.47 0.41 0.52 0.42 0.48 0.45 0.45 0.36 0.28 0.40 0.00 0.00 

mAb-1, mAb-2, 

mAb-3, mAb-4 

mAb-5, 

mAb-6 

mAb-7 

mAb-8, mAb-9, 

mAb-10, mAb-11 

mAb-12 

Confidential 

VISHAL KAMAT

We Have Our MOMENTS!!!!!!!! 

Confidential 

VISHAL KAMAT

Challenges: Cross-Competition Data is Not Always Interpretable 

Uni-directional Competition 

Amount of 

Antigen 

Captured ± 

Std Dev (nm) 

Amount of 

mAb-1 

Bound ± Std 

Dev (nm) 

mAb 

# 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 

24 

(-) 

0.27 ± 0.05 0.38 ± 0.1 1 0.02 0.00 0.00 0.00 0.00 0.01 0.04 0.01 0.16 0.08 0.35 0.33 0.36 0.37 0.42 0.40 0.37 0.33 0.75 0.43 0.36 0.33 0.24 0.01 

0.27 ± 0.06 0.56 ± 0.13 2 0.02 -0.01 0.00 0.00 0.00 0.00 0.05 0.02 0.17 0.09 0.41 0.39 0.42 0.43 0.44 0.44 0.42 0.38 0.82 0.48 0.41 0.38 0.29 0.00 

0.27 ± 0.06 0.47 ± 0.11 3 0.02 0.01 0.01 0.00 0.01 0.01 0.05 0.02 0.17 0.22 0.40 0.38 0.42 0.42 0.44 0.43 0.41 0.37 0.81 0.48 0.41 0.38 0.28 0.01 

0.27 ± 0.06 0.35 ± 0.09 4 0.02 0.01 0.00 0.00 0.00 0.01 0.06 0.02 0.16 0.21 0.39 0.38 0.42 0.42 0.44 0.43 0.41 0.36 0.79 0.47 0.40 0.37 0.28 0.00 

0.29 ± 0.02 0.37 ± 0.02 5 0.01 0.00 0.00 0.00 0.00 0.01 0.05 0.02 0.16 0.11 0.39 0.38 0.41 0.42 0.43 0.43 0.41 0.36 0.77 0.47 0.40 0.37 0.27 0.00 

0.28 ± 0.02 0.41 ± 0.02 6 0.02 0.00 0.00 0.00 0.00 0.01 0.06 0.02 0.16 0.12 0.38 0.36 0.41 0.41 0.42 0.41 0.40 0.35 0.77 0.47 0.38 0.35 0.26 0.00 

0.27 ± 0.05 0.37 ± 0.09 7 0.00 -0.02 -0.01 -0.01 -0.01 -0.01 0.02 -0.01 0.13 0.06 0.04 0.03 0.36 0.37 0.39 0.40 0.36 0.24 0.78 0.41 0.32 0.37 0.25 -0.01 

0.29 ± 0.02 0.36 ± 0.02 8 0.01 -0.01 0.00 0.00 0.00 0.00 0.03 0.00 0.13 0.07 0.04 0.04 0.37 0.37 0.39 0.40 0.36 0.25 0.77 0.41 0.32 0.37 0.25 -0.01 

0.34 ± 0.01 0.41 ± 0.02 9 0.02 -0.01 -0.01 -0.01 0.00 0.00 0.05 0.00 0.09 0.43 0.00 -0.01 0.26 0.26 0.28 0.28 0.26 0.17 0.72 0.42 0.35 0.39 0.28 -0.01 

0.28 ± 0.02 0.76 ± 0.04 10 0.00 -0.02 0.08 0.08 -0.01 -0.01 0.01 0.02 0.40 0.01 0.04 0.03 0.37 0.38 0.40 0.40 0.37 0.26 0.76 0.41 0.33 0.37 0.25 -0.02 

0.30 ± 0.02 0.37 ± 0.02 11 0.46 0.42 0.39 0.41 0.43 0.44 0.48 0.44 0.33 0.47 -0.02 -0.02 0.06 0.00 0.01 0.01 0.00 0.01 0.49 0.38 0.34 0.36 0.30 0.02 

0.37 ± 0.01 0.47 ± 0.02 12 0.49 0.41 0.46 0.44 0.47 0.46 0.47 0.44 0.45 0.51 0.00 -0.01 0.04 0.00 -0.01 -0.01 -0.02 -0.01 0.52 0.14 0.38 0.33 0.24 0.01 

0.27 ± 0.06 0.41 ± 0.10 13 0.42 0.33 0.40 0.39 0.41 0.41 0.42 0.37 0.40 0.49 0.02 0.01 0.04 0.02 0.00 0.01 0.00 0.05 0.50 0.39 0.38 0.36 0.22 0.08 

0.28 ± 0.02 0.28 ± 0.02 14 0.42 0.38 0.36 0.38 0.39 0.41 0.47 0.41 0.29 0.46 -0.01 0.01 0.07 0.02 0.03 0.03 0.02 0.07 0.49 0.41 0.37 0.42 0.36 0.10 

0.27 ± 0.05 0.36 ± 0.08 15 0.43 0.41 0.43 0.40 0.40 0.39 0.45 0.41 0.40 0.54 0.04 0.03 0.07 0.04 0.02 0.03 0.03 0.14 0.53 0.46 0.48 0.49 0.39 0.20 

0.34 ± 0.01 0.43 ± 0.02 16 0.42 0.40 0.43 0.39 0.39 0.39 0.44 0.41 0.38 0.53 0.03 0.03 0.06 0.03 0.03 0.02 0.02 0.14 0.48 0.40 0.51 0.50 0.41 0.19 

0.34 ± 0.01 0.43 ± 0.02 17 0.41 0.40 0.42 0.38 0.39 0.38 0.44 0.39 0.38 0.53 0.04 0.03 0.06 0.04 0.02 0.03 0.02 0.15 0.51 0.46 0.49 0.49 0.38 0.20 

0.28 ± 0.02 0.46 ± 0.03 18 0.52 0.44 0.48 0.47 0.49 0.48 0.49 0.46 0.48 0.53 0.01 -0.01 0.04 0.01 -0.01 0.00 -0.01 -0.02 0.55 0.40 0.36 0.32 0.18 0.00 

0.34 ± 0.01 0.43 ± 0.02 19 0.54 0.45 0.51 0.49 0.52 0.52 0.53 0.48 0.52 0.54 0.12 0.12 0.16 0.13 0.15 0.14 0.10 0.16 0.09 0.38 0.39 0.36 0.30 0.07 

0.28 ± 0.02 0.35 ± 0.02 20 0.43 0.39 0.36 0.39 0.40 0.41 0.46 0.41 0.37 0.46 0.04 0.01 0.40 0.37 0.38 0.35 0.35 0.27 0.63 0.07 0.38 0.48 0.31 0.10 

0.36 ± 0.01 0.46 ± 0.02 21 0.50 0.43 0.47 0.45 0.49 0.47 0.47 0.44 0.58 0.51 0.37 0.35 0.41 0.39 0.43 0.44 0.39 0.36 0.72 0.42 0.01 0.01 0.01 0.03 

0.35 ± 0.01 0.45 ± 0.02 22 0.47 0.39 0.43 0.41 0.44 0.44 0.43 0.40 0.53 0.48 0.31 0.31 0.38 0.34 0.41 0.39 0.36 0.33 0.68 0.40 0.02 0.02 0.31 0.02 

0.35 ± 0.01 0.45 ± 0.02 23 0.35 0.28 0.33 0.31 0.33 0.33 0.34 0.30 0.45 0.39 0.29 0.21 0.26 0.31 0.30 0.28 0.25 0.26 0.64 0.29 0.09 0.32 0.00 -0.05 

0.27 ± 0.06 -0.02 ± 0.01 24 (-) 0.36 0.31 0.3 0.32 0.32 0.34 0.4 0.35 0.37 0.42 0.04 0.03 0.37 0.33 0.35 0.35 0.32 0.27 0.66 0.38 0.35 0.41 0.31 0.10 

Confidential 

VISHAL KAMAT

Challenges: Cross-Competition Data is Not Always Interpretable 

Uni-directional Competition 

Amount of 

Antigen 

Captured ± Std 

Dev (nm) 

Amount of mAb-1 

Bound ± Std Dev 

(nm) 

mAb 

# 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 

0.40 ± 0.02 1.23 ± 0.04 1 0.02 0.05 0.16 0.06 0.12 0.12 0.13 -0.03 0.83 0.10 1.03 0.94 0.75 1.04 0.14 0.14 1.30 1.35 1.36 1.44 1.47 1.10 1.08 1.18 1.15 1.12 -0.02 

0.48 ± 0.03 0.73 ± 0.04 2 0.38 0.23 0.48 0.36 0.49 0.50 0.49 1.24 0.30 1.06 1.20 0.82 0.34 0.64 1.22 1.33 1.28 1.31 1.25 1.34 1.49 1.26 0.49 1.36 1.28 1.27 0.08 

0.43 ± 0.03 1.29 ± 0.03 3 0.04 -0.01 0.00 0.02 0.03 0.04 0.06 1.21 0.05 0.89 1.16 0.38 0.13 0.23 1.27 1.30 1.32 1.33 1.32 1.28 1.35 1.27 1.17 1.33 1.27 1.26 -0.01 

0.41 ± 0.03 1.13 ± 0.03 4 -0.04 -0.13 0.05 -0.11 0.01 0.01 0.01 1.19 0.86 1.01 0.75 0.33 0.12 0.91 1.04 1.05 0.75 0.80 0.74 0.71 0.77 1.03 1.01 1.14 0.99 0.98 -0.20 

0.40 ± 0.03 1.22 ± 0.03 5 0.01 0.01 0.05 -0.02 0.04 0.04 0.04 1.10 0.52 0.85 1.11 0.95 0.63 1.06 0.96 1.17 1.17 1.18 1.17 1.13 1.18 1.17 0.90 1.23 1.16 1.20 0.00 

0.39 ± 0.03 1.16 ± 0.04 6 0.01 0.01 0.07 -0.01 0.07 0.06 0.05 1.17 1.01 0.90 1.16 1.04 0.51 1.10 1.14 1.18 1.17 1.17 1.17 1.14 1.18 1.16 0.36 1.22 1.11 1.17 0.01 

0.38 ± 0.02 1.13 ± 0.04 7 0.01 0.05 0.08 0.00 0.08 0.07 0.03 1.16 0.43 0.91 1.11 0.92 0.53 1.12 1.12 1.17 1.19 1.19 1.17 1.14 1.19 1.18 1.10 1.25 1.12 1.17 0.03 

0.46 ± 0.02 1.30 ± 0.03 8 0.11 0.77 1.23 1.24 1.19 1.25 1.24 0.08 0.07 0.08 0.02 0.11 0.07 0.09 0.01 0.12 1.22 1.24 1.20 1.23 1.34 1.25 1.17 1.36 1.24 1.30 0.05 

0.39 ± 0.02 0.95 ± 0.03 9 0.73 -0.14 0.24 0.73 0.51 0.91 0.40 0.16 -0.15 -0.01 0.15 0.07 -0.21 0.15 0.18 0.30 0.87 0.90 0.91 0.96 1.00 0.52 0.84 0.73 0.70 0.92 -0.28 

0.37 ± 0.02 0.95 ± 0.04 10 0.16 0.65 1.01 0.96 0.90 1.06 1.04 0.12 0.05 0.08 0.10 0.11 0.04 0.13 0.09 0.15 1.07 1.12 1.10 1.18 1.24 1.06 0.85 1.13 1.06 1.11 0.03 

0.44 ± 0.02 1.35 ± 0.05 11 1.01 0.23 1.00 0.94 1.15 1.02 1.12 -0.05 -0.05 -0.04 -0.04 -0.04 -0.05 -0.04 -0.05 1.33 1.08 1.13 1.14 1.21 1.26 1.05 1.02 1.24 1.33 1.09 -0.05 

0.42 ± 0.02 1.16 ± 0.04 12 0.92 0.06 0.26 0.42 0.93 0.85 0.81 -0.03 -0.08 -0.04 -0.02 -0.04 -0.07 -0.02 -0.02 0.62 1.21 1.26 1.28 1.36 1.38 0.92 1.18 1.20 1.13 1.16 -0.06 

0.39 ± 0.02 0.97 ± 0.03 13 0.69 -0.10 0.30 0.20 0.64 0.54 0.54 0.20 -0.11 0.03 0.20 0.12 -0.19 0.21 0.22 1.00 0.66 0.69 0.70 0.75 0.79 0.74 0.79 0.87 0.70 0.90 -0.36 

0.38 ± 0.02 1.10 ± 0.04 14 0.89 0.04 0.15 0.84 0.97 0.96 1.03 0.01 -0.02 0.00 -0.01 -0.02 -0.02 0.00 -0.01 1.14 1.02 1.07 1.07 1.18 1.20 0.98 1.02 1.04 1.09 1.11 -0.02 

0.45 ± 0.03 1.42 ± 0.04 15 -0.03 0.65 1.34 1.14 1.12 1.23 1.26 -0.05 -0.04 -0.03 -0.04 -0.04 -0.04 -0.03 -0.04 1.06 1.32 1.34 1.34 1.30 1.37 1.31 1.25 1.31 1.34 1.24 -0.05 

0.38 ± 0.03 1.17 ± 0.04 16 0.07 0.62 1.06 0.85 0.97 1.03 1.03 -0.02 0.09 0.05 1.10 0.58 0.94 1.08 0.76 0.09 1.23 1.22 1.25 1.23 1.26 1.16 1.08 1.18 1.15 1.12 0.05 

0.45 ± 0.02 1.50 ± 0.03 17 0.90 0.26 0.75 0.45 0.65 0.60 0.74 1.11 0.81 0.87 0.97 1.06 0.52 1.10 1.03 1.28 0.01 0.01 0.01 0.01 0.02 0.15 1.21 0.14 1.22 1.40 -0.03 

0.44 ± 0.02 1.46 ± 0.03 18 0.89 0.28 0.79 0.49 0.68 0.63 0.77 1.12 0.78 0.86 0.96 1.04 0.52 1.09 1.03 1.26 0.00 0.01 0.01 0.01 0.02 0.16 1.20 0.15 1.21 1.38 -0.02 

0.44 ± 0.02 1.42 ± 0.03 19 0.86 0.26 0.71 0.39 0.64 0.60 0.69 1.03 0.75 0.80 0.90 1.03 0.49 1.07 0.91 1.23 0.01 0.02 0.01 0.01 0.03 0.19 1.18 0.17 1.18 1.37 -0.03 

0.43 ± 0.02 1.45 ± 0.03 20 0.81 0.21 0.61 0.32 0.56 0.51 0.61 0.96 0.70 0.76 0.84 0.98 0.46 1.03 0.84 1.20 0.00 0.00 -0.01 -0.01 0.01 0.18 1.14 0.16 1.16 1.34 -0.05 

0.42 ± 0.02 1.47 ± 0.04 21 0.82 0.26 0.66 0.35 0.61 0.56 0.66 1.03 0.71 0.80 0.86 0.98 0.48 1.04 0.90 1.20 0.00 0.00 -0.01 -0.01 0.01 0.22 1.14 0.18 1.12 1.37 -0.03 

0.39 ± 0.03 1.19 ± 0.03 22 1.05 0.74 1.18 0.95 1.10 1.16 1.15 1.21 0.54 1.05 1.09 1.00 0.73 1.07 1.22 1.23 0.03 0.03 0.04 0.03 0.03 0.05 0.87 0.07 0.00 1.33 0.00 

0.49 ± 0.03 1.35 ± 0.04 23 1.09 0.00 1.19 1.11 1.11 0.57 1.23 1.41 1.05 1.07 1.25 1.26 0.99 1.32 1.36 1.50 1.46 1.47 1.48 1.55 1.63 1.08 0.01 1.47 1.25 1.42 0.06 

0.36 ± 0.02 1.21 ± 0.06 24 0.83 0.48 0.99 0.85 0.96 0.93 0.99 0.91 0.51 0.69 0.84 0.82 0.60 0.84 0.85 0.97 0.02 0.01 0.02 0.02 0.04 0.06 0.92 0.07 1.03 1.15 -0.01 

0.43 ± 0.02 1.24 ± 0.03 25 0.90 0.56 1.05 0.79 1.11 1.07 1.06 1.08 0.59 0.93 0.92 0.87 0.61 1.00 0.97 1.22 1.21 1.21 1.21 1.28 1.34 0.16 0.96 1.15 -0.03 1.13 0.00 

0.46 ± 0.02 1.44 ± 0.06 26 0.74 0.37 0.93 0.87 0.97 0.95 0.98 1.00 0.92 0.78 1.11 0.82 0.81 1.01 0.99 1.17 1.23 1.28 1.23 1.36 1.55 1.11 0.98 1.32 1.16 0.06 0.04 

0.44 ± 0.02 -0.06 ± 0.01 27 (-) 1.04 0.75 1.07 0.91 1.19 1.20 1.18 1.10 0.78 1.00 1.01 0.89 0.63 1.05 1.06 1.23 1.23 1.25 1.22 1.31 1.45 1.25 1.14 1.28 1.17 1.27 0.11 

27 

(-) 

Confidential 

VISHAL KAMAT

Biacore 3000/T200 vs Biacore 4000 vs Octet QK384 

Factors 

Number of mAbs tested 

for cross-competition 

Biacore 3000 / 

Biacore T200 

Biacore 4000 Octet QK 384 

32 32 31 

Assay Format Classical Sandwich Format Classical Sandwich Format In Tandem Format 

Amount of sensors 

required 

Measurement 

Times 

8 Biacore sensor chips 

(4 mAbs per chip) 

Cost: ~$1500 

90sec Antigen loading, 

180sec mAb-2 binding 

2 Biacore sensor chips 

(16 mAbs per chip) 

Cost: ~$375 

90sec Antigen loading, 

180sec mAb-2 binding 

64 anti-His Octet Biosensors 

Cost: ~$440 

180sec Antigen Capture, 

300sec mAb-1 Binding, 

120sec mAb-2 Binding 

Experiment run time ~ 64 hours ~ 14 hours ~ 17 hours 

Actual run time 

(with chip changes & 

preparing plates) 

Amount of 

mAb-2 required 

Amount of 

Antigen required 

Other 

Considerations 

Confidential 

8 days 

(assuming 1 chip/day) 

VISHAL KAMAT 

2 days 

(assuming 1 chip/day) 

1 day 

(assuming 2 plates/day) 

~1.4 ml @ 50nM ~0.6 ml @ 50nM ~1.8ml @ 300nM 

~15ml @ 50nM ~22ml @ 50nM ~1.8ml @ 100nM 

• Need experienced users 

• Requires optimization of 

surface regeneration 

• Need experienced users 

• Requires optimization of 

surface regeneration 

• Minimal user training 

required 

• Easy experimental set-up

Questions 


Scientist, Therapeutic Proteins 

Office: 914-847-7771 

Email: vishal.kamat@regeneron.com 

Confidential 

VISHAL KAMAT



Thilo Riedl, Ph.D. 

Senior Scientist 

Assay & Bio-Analytical Science 

Genmab

Rapid characterization of antibody antigen 

binding by label-free cross-competition 

binning and hybrid library mapping 

Thilo Riedl, PhD 

Sr.Scientist Assay & BioAnalytical Science 

December 13, 2012

Genmab 

is a leading international biotechnology company 

developing Fully Human Antibody Therapeutics 

Founded in 1999 

 

 

 

Headquarters in Copenhagen, Denmark 

R&D facilities in Utrecht, The Netherlands 

UltiMAb® transgenic mouse technology platform 

Various product formats including 

Genmab NL 

Research, Drug Discovery & Development 

110 employees, 4000m 2 lab and office space 

 

 

ADCs, DuoBody® format, and UniBody® format 

Marketed product in collaboration with GSK: Arzerra® 

Genmab partners include 

Novartis, Janssen Biotech, GSK, Lundbeck, Roche, 

Seattle Genetics,…

Approved therapeutic antibodies 

52

Current and future antibody product innovation 

Future product formats will be developed from 

building blocks with diverse characteristics and functions 

53

Conventional antibody screening cascade 

Immunisations 

Library generation 

10,000s 

Primary screening 

100s 

Primary Screening 

170,000 assay points per run - 1536w format 

Secondary Screening 

Secondary screening 

10s 

In vitro screening 

10 

In vivo screening 

Cell based functional 

Later Stage 

Panel Diversity 

Characterization 

Biochemical 

1 

54

State-of-the-Art antibody discovery 

• Obtain antibody libraries with large diversity 

• Binding assays 

• CDR Sequences 

• Region mapping 

Early Stage 

Panel Diversity 

(Pre)Characterization 

• Affinity 

(I) 

• Functional in vitro Characterization 

• Assays on selected cell lines and models 

(II) 

(IV) 

(III) 

• In vivo characterization 

• Tumor models in mice 

55

Panel diversity & characterization 

Octet BLI contributions 

• Binding region assessment 

• Cross-competition binning 

• Hybrid library mapping 

• Species cross reactivity 

• Affinity 

• Functional assays 

(e.g. Ligand binding inhibition assays) 

56

Octet RED384 integration to Tecan Freedom EVO 

Off-line 

Biosensor handling 

Integrated 

Data Managment 

ActivityBase XE 

Enabling HTS BLI 

57

BLI cross-competition binning 

58

BLI cross-competition designs 

Sandwich 

Premix 

In Tandem 

Y 

AG 

Y 

AG 

AG 

+ Affinity information 

- k off AB 1 limiting 

Y 

Y 

AB 1 AB 2 

- AB 2 in excess 

- K D AB 2 limiting 

- AG-AB 2 premixes 

(no re-use) 

+ Avidity (/Affinity) information 

- Putative epitope masking 

- k off AB 1 limiting 

- AB 1 in saturation 

- Buffer control mandatory 

59

BLI cross-competition - Sandwich format 

NO cross-competition 

Y 

AG 

Y 

AG 

Y 

DISTINCT 

cross-competition bins 

cross-competition 

Y 

Y 

AG 

+ 

Y 

ONE 

cross-competition bin 

• Amine Reactive biosensors allow covalent antibody coupling 

• Kinetic data on antigen binding retrieved from buffer control 

60

HTS cross-competition process design 

• Automation by docking Octet 384RED to Tecan Freedom EVO 

• Coupled or Uncoupled Bi-modular process 

• Module A: Bulk production of Antibody Biosensor Arrays 

• Module B: Automated Cross-Competition Matrix 

61

Quality Control of Antibody Biosensor Arrays 

• AmineReactive biosensors preferred format 

• Generic pH value for coupling 

• Storage of Antibody Biosensor Arrays 

• Automation on Tecan Freedom EVO 

Antigen binding capacity 

Antigen binding kinetics 

(Average of 16 biosensors) 

(Average of 16 biosensors) 

62

Regeneration of Antibody Biosensor Arrays 

Eight antibody A biosensors (bulk AR coupling) 

Acidic regeneration each cross-competition round 

• Stable assay quality over regeneration cycles 

• Regeneration allows efficient HTS cross-competition binning 

63

HTS BLI versus ELISA cross-competition binning 

HTS BLI ELISA 

Antibody pairs analyzed (excluding Controls) 528 397 

Non-bidirectional cross-competition 

27 / 5.1% 

(14 / 3.5%)* 

18 / 4.5% 

Non-bidirectional cross-competition overlap 2 / 0.5%* 

*: 397 antibody pairs analyzed by ELISA 

64

Cross-competition binning: HTS BLI versus ELISA 

 

Comparable data sets obtained 

 

Real-Time Quality Control (antigen binding) 

 

Additional kinetic information 

 

Reduced hands-on time and material consumption 

 

A matrix analysis of 34x34 antibodies (1,156 reactions) can be 

accomplished in 6h run time in an automated setting 

65

BLI hybrid library mapping 

66


Principle: Loss of binding shuffle assay 

Ortholog and/or paralog shuffling 

Tox species cross-reactivity 

Plus factor: Maintenance of antigen structural integrity 

Example: 8x linear, duo species shuffle (human - mouse): 

67

BLI hybrid library mapping design 

• Reducing the risk of epitope masking: 

Terminal tagged antigens 

• Streptavidin biosensors preferred: 

High K D Biotin-Streptavidin 

Optional regeneration of antigen coated biosensors 

• Introducing BAP technology: 

Need of antigen purification eliminated 

Strep 

AG-Bio 

human 

shuffle species 

hybrid constructs 

tox species 

others 

68

Combining Technologies: 

Biotin Acceptor Peptide (BAP)-tag 

• Biotin, Vitamin B 7 (H) 

• BAP-tag: G L N D I F E A Q K I E W H E 

• Co-Expression Biotin ligase: BirA (E. Coli) 

• Site-specific in vivo biotinylation 

• Use of crude supernatants in combination with Streptavidin biosensors 

69

BLI hybrid library mapping process 

Repeated cycles 

Regeneration 

Regeneration 

Buffer 

• Generate hybrid library 

biosensores (up to 16) 

• Analyse samples sequentially 

• Regeneration optional and 

antigen dependent 

• Real time QC 

• Automated runs 

Octet sensorgram: 

Association of Antibody X to 16 hybrid biosensors 

70

BLI hybrid library mapping example 

• 265 antibodies tested in 4688 dips (16x hybrid library) 

• 43 samples excluded by QC (low or aberrant binding) 

• 152 samples yielded high quality data (binding > 0.20 nm) 

• 22h unattented run time with 2% Biosensor drop-off 

Benchmark Negative Control 01B03 01C03 

X reactive D1 D3 D4/11 

D5/6 D7 D8 D9 

D9/10 D10 D11 others 

71

Alternative HTS hybrid library mapping format 

• Biotinylated hybrid library proteins 

immobilized on streptavidin beads 

• HTS homogeneous binding assay: 

1536 well format 

Fluorescence scanning 

• Up to 16x hybrid library (16 quadrants) 

Shuffle 1 

Shuffle 4 

Shuffle 2 

Shuffle 5 

Shuffle 3 

Shuffle 6 

Human 

Mouse 

72

Bead based HTS versus BLI hybrid library mapping 

265 antibodies case study with 16x hybrid library: 

87% result match 

HTS hybrid library mapping 


• Fluorescence based assay 

+ Tens of thousands samples 

+ ~ 0.02 US$/ assay point 

+ ~ 10,000 samples/ 24h run time 

- No re-use of samples 

- No real time QC control 

• Label-free method 

- Hundreds of samples 

- ~ 0.25 US$/ assay point 

- 300 samples/ 24h run time 

+ Re-use of samples 

+ Real time QC control 

* Sensor integrity 

* Binding rates 

+ Additional kinetic information 

73

Added value of 

binding region assesment 

to antibody library generation 

74

Correlating degrees of diversity 

Hybrid library mapping 

CDR3HC sequence analysis 

# Antibodies # Antibodies 

# Antibodies 

0 10 20 30 0 40 10 50 20 60 30 70 40 80 50 60 70 80 

# different CDR3HC # different sequences CDR3HC sequences 

# distinct CDR3HC sequences 

0 10 0 20 10 30 20 30 

D9mm/D10mm 

D10mm 

D11mm 

D9mm 

D4mm/ D11mm 

D5mm/ D6mm 

D7mm 

D8mm 

Mouse X reactive 

D1mm 

D2mm 

D3mm 

D9mm/D10mm 

D10mm 

D11mm 

D9mm 

D4mm/ D11mm 

D5mm/ D6mm 

D7mm 

D8mm 


D1mm 

D2mm 

D3mm 

D9mm/D10mm 

D10mm 

D11mm 

D4mm/ D11mm 

D5mm/ D6mm 

D7mm 

D9mm 

D8mm 


D1mm 

D2mm 

D3mm 

D9mm/D10mm 

D10mm 

D11mm 

D4mm/ D11mm 

D5mm/ D6mm 

D7mm 

D9mm 

D8mm 


D1mm 

D2mm 

D3mm 

Zoo hybrid Zoo mapping hybrid mapping 

CDR3HC CDR3HC sequence sequence analysis analysis 

75

Internalization [RFU] 

Correlating cross-competition bins with function 

Antibody internalization assay 

BLI cross-competition 

2.010 5 

1.510 5 

1.010 5 

5.010 4 

0 

Group 1 

Group 2 

Group 3 

Group 4 

Cross-competition bins might correlate 

with specific biological functions 

76

BLI cross-competition binning 

versus 


77


compared to BLI cross-competition binning 

+ Binning to defined target regions 

+ Applicable also to small antigens 

+ Tox species cross-reactivity can be easily integrated 

+ Higher Throughput capability 

- Limitated by cross-reactivity with shuffle species 

- Generation of shuffle constructs required 

- Possible limitation to avidity data (AB format dependent) 

- Antigen biosensor versus Antibody biosensor regeneration 

• Both methods show results comparable to classical approaches 

• Combination: Cross-competition with mapped antibodies 

78

Summary 

79

Summary 

• Important role of binding region assessment in antibody panel 

generation and characterization 

• Octet BLI technology offers various formats for assessing antibody 

binding regions and kinetics 

• BLI cross-competition binning and BLI hybrid library mapping 

provide comparable binning results to classical methods 

• BLI approaches provide additional kinetic data and quality control 

• Integration of the 16-channel Octet 384RED to automated liquid 

handling significantly increases throughput and opportunities of the 

Octet platform 

80

Acknowledgements 

Ferdi van der Horst 

Arnout Gerritsen 

Dennis Verzijl 

Rob de Jong 

Bart de Goeij 

Cell & Molecular Sciences 

Protein Separations 

Biochemical Analysis 

81



Label-Free Assays for High-Throughput Monoclonal 

Antibody Characterization 

Q&A



Your Moderator 

Tamlyn Oliver 

Managing Editor 

Genetic Engineering & Biotechnology News



Sriram Kumaraswamy, Ph.D. 

Director, Applications 

Fortebio - A Division of Pall Life Sciences




Scientist 

Therapeutic Proteins 

Regeneron Pharmaceuticals



Thilo Riedl, Ph.D. 

Senior Scientist 

Assay & Bio-Analytical Science 

Genmab



Thank You For Attending 

Label-Free Assays for High-Throughput Monoclonal 

Antibody Characterization 

Broadcast Date: Thursday, December 13, 2012 

Time: 2 pm ET, 11 am PT 

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