E. coli - ForteBio

Detecting Common Food-borne
Pathogens Using KPL BacTrace ®
Antibodies and the Octet RED96
Dr. Brian Bullard
R&D Scientist, Kirkegaard & Perry Labs
Gaithersburg, MD

Who KPL Is
• Founded 1979, KPL developed the first affinity-purified polyclonal antibodies
• KPL offers >800 catalog products
• Offer kits and applications for protein detection and labeling
– ELISA, Western blot, Immunohistology
• Polyclonal antibodies
– Secondary antibodies & conjugates
– Primary antibodies against bacteria

• Model System
Outline
• Initial Studies with Intact Bacteria
• Improving Sensitivity
• Improving Signal
• Complex Matrices
• Future Directions

E. coli O157:H7
• Gram-negative bacterium
• Food-borne pathogen
– Causative agent of the Jack In The Box outbreak in
the early 90’s
• Easy to grow
• Anti-E. coli O157:H7 is well studied

ELISA Data Demonstrating the Specificity of
Polyclonal Anti-E. coli O157
OD (405 nm)
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Heat Fixed
Chemically Fixed
E. coli O157:H7 KPL
E. coli O157:H7 ATCC
E. coli O1:H7
E. coli K12
E. coli ATCC
E. coli O125a
E. coli O128a,128b:H
E. coli O111:H8
E. coli O124:NM
E. coli O26:K60
E. coli O29:NM
Citrobacter freundii
Edwardsiella tarda
Enterobacter cloacae
Hafnia alvei
Klebsiella pneumoniae
Providencia Stuartii
Salmonella typhimurium
Serratia marcescens
Shigella flexneri
Shigella sonnei
Staphylococcus aureus
Yersinia ruckeri

Why is KPL Using an Octet to Detect
Bacteria?
• Improved speed and sensitivity
• Compatibility with complex matrices
• Improved in-process testing of
antibodies/antisera
• Improved understanding of customers’ needs
when working with Bio-Layer Interferometry
(BLI)

Whole Bacteria: Culture Manipulation
Grow E. coli
Plate dilutions to
determine cfu/ml
Scrape colonies off
the plate and
suspend in buffer
10-fold Serial Dilution
Assay on Octet
RED96

Protein G
Biosensors
Whole Bacteria: Detection
Regenerate and
Wash (x3) Antibody Load
(300 sec.)
Protein G Biosensor
Block w/ Goat
Serum (300 sec.)
Baseline
(180 sec.)
Capture Bacteria
(300 sec.)

l (nm)
0.45
0.25
0
The Octet Can Detect Intact Bacteria
E. coli O157 detected with anti-E. coli O157 Ab
8.5 x 10 8 cfu/ml
8.5 x 10 7 cfu/ml
0 60 120 180 240 290
Since bacteria Time are (seconds) so massive, why is
the limit of detection so poor?

Why Aren’t Bound Bacteria Inducing More
Signal?
• Bacteria are massive compared to the
antibodies on the surface on the tip
– ~950 bacteria should be able to bind the tip under
perfect conditions
• Bacteria aren’t all that dense
– Mostly water, which light can penetrate easily
– E. coli are Gram-negative and have a thin cell well
• Bacteria are odd shaped
– Comparing a cube to a football
– Leads to inefficient loading at the tip surface

Simulating Very Dense Particles
Goat-
Anti-
Rabbit
Baseline
(30 sec.)
x56
Rabbit-
Anti-
Goat
Protein G Biosensor
Baseline
(30 sec.)

l (nm)
200
150
100
50
Results from Antibody Layering
Raw Sensor Data
0
0 2000 4000 6000 8000 10,000 12,000 14,000 16,000
Time (Seconds)

l (nm)
Subtracted
Data
0.60
0.40
0.20
0
200
150
100
50
0
Differences in Y-Axes
Raw Sensor Data
Time

Improving Detection Limit
Intact Bacteria Antibody Layering
Antibody
(10 nm)
Biosensor (1900 mm 2 )

Improving Detection Limit
Intact Bacteria Disrupted Bacteria
Antibody
(10 nm)
Biosensor (1900 mm 2 )

Disrupted Cells: Culture Manipulation
Grow E. coli
Assay on Octet
RED96
Scrape colonies of the
plate and suspend in
buffer
Pellet for 2 min.
Bath Sonicate for 30 min.
in EDTA + 1% Triton-X
10-fold Serial Dilution
Plate dilutions to
determine cfu/ml

Protein G
Biosensors
Disrupted Cells: Detection
Regenerate and
Wash (x3)
Protein G Biosensor
Antibody Load
(300 sec.)
Block w/ Goat
Serum (300 sec.)
Baseline
(180 sec.)
Capture Bacteria
Particles (300 sec.)

l (nm)
Disrupting Cells Improves Detection Limit
0.45
0.25
5 x 10 8 cfu/ml
5 x 10 7 cfu/ml
5 x 10 6 cfu/ml
5 x10 5 cfu/ml
5 x 10
0
0 60 120 180 240 290
Time (seconds)
4 cfu/ml
Previous limit was 8.7 x 107 cfu/ml
Can we improve sensitivity even more?

How to Increase Sensitivity
• Disrupt bacterial cells more efficiently
– Add glass beads to sonication mixture
• Increase “density” of the bacteria
– Gold labeled antibody
– Precipitating substrate
• Metal Enhanced DAB
– Increased capture duration

Disrupted Cells: Culture Manipulation
Grow E. coli
Assay on Octet
RED96
Scrape colonies of the
plate and suspend in
buffer
Pellet for 2 min.
Bath Sonicate for 30 min.
in EDTA + 1% Triton-X
10-fold Serial Dilution
Plate dilutions to
determine cfu/ml

Disrupted Cells + Enzymatic Reaction:
Detection
Streptavidin
Biosensors
Biotin
Streptavidin
Biotin
Biosensor
Biotin Biotin Biotin
HRP HRP HRP HRP HRP
Antibody Load
(300 sec.)
Incubate with
Metal Enhanced
DAB
Block w/ Goat
Serum (600 sec.)
Baseline
(30 sec.)
Block w/ Goat
Serum (300 sec.)
Block w/ Goat
Serum (300 sec.)
Capture Bacteria
Particles (300 sec.)
HRP

l (nm)
Use of Metal Enhanced DAB Gains ~1 Log of
Sensitivity
30.00
15.00
5.5 x 10 5 cfu/ml
0
0 60 120 180 240 290
Time (seconds)
5.5 x10 4 cfu/ml
5.5 x 10 3 cfu/ml

What Else Can Be Done to Improve
Sensitivity?
• Factors to Consider:
– Capturing bacteria with polyclonal antibody
– Bacteria/particles are NOT uniform
• Longer bacterial incubation time would
increase particles captured on the tip surface
– Necessitates the use of sandwich assay due to
offline capture of bacteria

Disrupted Cells: Culture Manipulation
Grow E. coli
Assay on Octet
RED96
Scrape colonies of the
plate and suspend in
buffer
Pellet for 2 min.
Bath Sonicate for 30 min.
in EDTA + 1% Triton-X
10-fold Serial Dilution
Plate dilutions to
determine cfu/ml

Disrupted Cells + Enzymatic Reaction:
Detection
Streptavidin
Biosensors
Biotin
Streptavidin
Biotin
Biosensor
Biotin Biotin Biotin
HRP HRP HRP HRP HRP
Antibody Load
(300 sec.)
Incubate with
Metal Enhanced
DAB
Block w/ Goat
Serum (600 sec.)
Baseline
(30 sec.)
Block w/ Goat
Serum (300 sec.)
Block w/ Goat
Serum (300 sec.)
Off-line Capture of
Bacteria Particles
(22.5 hours)
HRP

Offline Capture of Bacteria in Combination with
Metal Enhanced DAB Yields Best Sensitivity
3 x 10 3 cfu/ml
3 x 10 2 cfu/ml
Now we can detect 30 bacteria/ml!
3 x 10 1 cfu/ml

Bacteria in Hamburger Extract: Culture
Manipulation
Grow E. coli
10-fold Serial Dilution in
Hamburger Extract
Wash Cells in PBS
10-fold Serial Dilution
in PBS
Assay on Octet
RED96
Plate Dilutions to
Determine cfu/ml

Bacteria in Hamburger Extract : Detection
Streptavidin
Biosensors
Biotin
Regenerate and
Wash (x3) Antibody Load
(300 sec.)
Streptavidin Biosensor
Biotin
Biotin
Biotin
Biotin
Block w/ Goat
Serum (300 sec.)
Baseline
(180 sec.)
Capture Bacteria
Suspended in Hamburger
Extract (300 sec.)

l (nm)
Bacteria in Hamburger Extract are Detected
as Well as in PBS
0.45
0.25
E. coli O157 detected with anti-E. coli O157 Ab
0
0 60 120 180 240 290
Time (seconds)
9.7 x 10 8 cfu/ml
9.7 x 10 7 cfu/ml

Summary
• The Octet RED96 can be used to detect
bacteria in complex matrices
• Bacterial disruption can improve the limit of
detection
• The use of metal enhanced DAB, which
precipitates on the biosensor, increases signal
and sensitivity

Octet vs. Traditional Methods
Traditional Methodology
• Assay requires several
hours
• Limit of sensitivity is about
10 4 bacteria
• Often require enrichment
• Bacterial suspension limited
to buffers (e.g. PBS)
Octet Methodology
• 8 samples can be assayed
in as little < 45 minutes
• Limit of sensitivity < 10 2
• Does not require
enrichment
• Compatible with complex
matrices

Future Studies
• Shortening the off-line incubation time
• Detecting other food-borne Gram-negative
bacteria
• Detection of Gram-positive bacteria
• Detecting bacteria with monoclonal antibodies
against toxins or surface exposed epitopes
• Quantification experiments

Acknowledgements
KPL R&D Team

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