Transcript Poster - Surface pressure and temperature profile

US/Ireland Emerging Technologies,
Univ. of Massachusetts, Lowell, Oct 19-20th., 2009.
'Developing Immunoassays for
Bioprocess Analysis and Diagnostics'
Richard O’Kennedy,
Professor of Biological Sciences/Vice-President,
School of Biotechnology, Biomedical Diagnostics
Institute and National Centre for Sensors Research,
Dublin City University, Dublin 9, Ireland.
SUMMARY:
'Developing Immunoassays for Bioprocess
Analysis and Diagnostics'
• Background
• Antibodies: ideal reagents for assay development
• Key Characteristics: specificity, sensitivity,
structural format, stability and immobilization.
• Generation
• Genetic engineering facilitates optimisation
• Potential applications
Core Expertise
Display and Selection:
Phage, yeast and ribosomal
display of proteins
Antibody Production:
Monoclonal, polyclonal and
recombinant (human, chicken,
mouse and rabbit)
Fermentation:
Large scale protein expression
Liposomes:
Antibody labelling and
dye/contrast agent
encapsulation
Mutagenesis:
Random and site-specific
mutagenesis for protein
improvement
Biorecognition molecule:
Antibodies, fragments, peptides,
protein scaffolds and DNA
Lateral Flow Assays:
Point of care tests for
environmental and clinical
applications
Biosensor Assays:
Incorporation of biorecognition
elements into biosensor
platforms
Cloning:
Cloning, expression and
purification of antigens and
biomakers
Automated Screening:
Custom written software for high
throughput screening
Protein Kinetics:
Determination of interaction rate
constants and thermodynamic
profiles
Research Interests
Marine Toxins
Sialic Acids
Prostate Cancer
Markers
Cardiac Markers
Food
Drugs of Abuse
Aflatoxins
Contaminants:
Multiple Myeloma
They didn’t mention this in my contract
Recombinant antibodies
F(ab')2
CH2
Fab
IgG
scFv
Dimeric
scFv
Dimeric
bifunctional
scFv
Antibody Types
Characteristics
Polyclonal
Monoclonal
Recombinant
Ease of production
++++
+++
++
Low cost
++++
+++
++
Stability*
+++
++
++
Availability
++++
+++
+ ()
Ease of immobilization
++++
++++
++++++
Sensitivity*
++++
++++
+++++
-
-
+++++
Capacity for improvement*
Avian Antibodies: IgY
• Phylogenetically distant from humans
• Single primer sets
• Stable and long half-life
• Do not react with RF, HAMA or surface Fc receptors
Picture obtained from www.beckman.com
Antibody Production, Characterication and Applications
Derivatization of Target
Target
Target-Carrier Conjugate Design
and Synthesis
Linker
Immunize Mouse
Kinetic and
Thermodynamic
Characterisation
RU
Time
Polyclonal antibodies
Monoclonal antibodies
Development of
Target-specific tests using
a range of antibody labels
Response (RU)
Recombinant antibodies
Immunize Rabbit
Biosensor-based
Immunoassay
Target Conc.
Pimp My Antibody
Nature 446, 964-966 (26 April 2007)
Antibodies for Use – Key Issues
Issue
Strategy for Optimisation
Sensitivity
High-throughput screen / improve
Specificity
Increase / broaden
Stability
Genetic modification
Immobilisation
Chemical- multiple chemistries
Orientation
Insert tags e.g. biotin
Labelling
Chemical/Biological
Size
Smaller facilitates denser packing
Multi-use
Stabilise / immobilise antigen
Non-specific binding
Use blockers / adequate controls
PRODUCTION AND CHARACTERISATION OF MURINE
SINGLE CHAIN FV ANTIBODIES
Genetic source of antibody fragments
VH
Extract RNA
- Non-immunised/Immunised Mouse
Reverse Transcription
- Hybridomas
VL
SOE-PCR anneals the VH and
VL regions together
SfiI digest of SOE product
F1
Chloramphenicol
Ligation of amplified DNA into plasmid
Transform into E.coli
Phage Display scFv
antibodies
Phage displaying scFv antibody fragment
pIII phage coat protein
Recombinant scFv antibody fragment
Vector containing VH and VL
DNA sequences
Diagram of filamentous phage expressing scFv on the surface of the
phage as a fusion with the pIII phage coat protein. Light and heavy
chain genes are present in the vector contained within the phage and
the phage is ready for infection into E.coli.
1. Phage antibody
library
8. Prepare phage
particles
7. Amplify bacteria
2. Incubate with antigen
on immunotube
Phage
selection
cycle
6. Re-infect selected
phages into E. coli
3. Wash and remove nonbinders
4. Elute specific
phage
5. Analyse eluted
phage by phage-ELISA
Robotic Screening
A100 – rapid kinetic characterisation
• High throughput hardware and software
• High quality, high content data
• Parallel analysis array format system
A100 Screening approach
Complex Stability of 95 clones
Stability early
%left =
Stability late
Stability late
Stability early
X 100
Listeria monocytogenes
• Gram-positive bacterium, motile
• Ubiquitous in the environment
• ‘Listeriosis’ - manifested as
• food poisoning (‘Influenza-like’)
• spontaneous abortion (2nd/3rd trimester)
• meningitis/encephalitis
• >20% mortality rate
Food recalls
• In 2002, 27.4 million pounds of turkey produce
were recalled due to suspected contamination with
L. monocytogenes
•largest meat recall in the history of the United States
•resulted in several fatalities and a subsequent
nationwide class-action lawsuit
Limits allowed: 1 micro-organism per 50 g
Virulence proteins
Internalin B (InlB)
~65 kDa
p60 (60kDa),
Hpt
(cell growth/division)
Listeria
monocytogenes
Actin Tail Filaments (ActA)
90 kDa
(Intracellular motility)
Internalin A (InlA)
80 kDa
P66 (kDa)
Aminopeptidase
Listeriolysin (60 kDa),
PI-PLC,
PC-PLC
(escape from vacuole)
Antibodies
Protein
Antibodies produced
Polyclonal
Monoclonal
scFv
InlA/rInlA
-

-
InlB/rInlB



p60/rp60


-
Whole
cells



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mAb2b3 cross - reactivity
• Direct capture ELISA ( Anti-Inl A antibody)
– mAb2b3 only bound to L. monocytogenes cells tested
15
13
11
9
7
5
3
1
Listeria monocytogenes determination
1.1
1
A/A0..
0.9
0.8
0.7
0.6
0.5
0.4
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
Cell Concentration (Cells/ml)
1.E+09
1.E+10
Detection of Listeria
Anti-InlA antibody-linked red light emitting quantum dots (605nm)
The development of rapid fluorescence-based immunoassays, using quantum dot-labelled antibodies for the detection of Listeria monocytogenes
cell surface proteins - Int. J. Biol. Macromol., 39(1-3), 127-134. Tully, E., Hearty, S., Leonard, P. and O’Kennedy, R. (2006).
Aflatoxin B1
• Potent naturally occurring carcinogen
•
Ability to induce specific mutations in
specific mammalian genes
•
Reported 1 million cases of liver cancer
per year due to aflatoxins
•
Linked to human hepatocellular
carcinoma
•
Listed as group 1 carcinogens by the
IARC
AFB1
Biacore assay development
Anti-aflatoxin scFv preincubated
with aflatoxin
Anti-aflatoxin scFv
Aflatoxin
Antibody and free aflatoxin
injected over aflatoxinimmobilised CM5 chip surface
Conversion of scFv to Fab format
•
Anti-AFB1 murine scFv was converted to a chimeric Fab format by the
addition of human constant regions using a number of overlap extension
polymerase chain reactions (PCRs)
•
Potential benefits of converting
an scFv to Fab format in terms of
stability, sensitivity and specificity
were evaluated
Fab
scFv
•
Influence of additional constant regions
and presence of the interchain disulphide
bonds in the Fab fragment was assessed
Improving Affinity
• in vitro (directed) evolution strategies
Heavy chain CDR3
QEXXXYSMD
NNK NNK NNK
Comparison of Antibodies:
Detection of aflatoxin B1
Antibody Fragment
ELISA Limit of
Detection
Biacore Limit of
detection
Monomeric scFv
12ppb
0.390ppb
Dimeric scFv
3ppb
0.19ppb
Bifunctional dimeric
scFv
3ppb
n/a
D11 Fab
4.2ppb
0.29ppb
G6 Fab (mutant)
1.3ppb
0.12ppb
Summary of Lecture
Aim: offer perspective on selection and use
of antibodies
• Specificity
• Sensitivity
• Sources
• Selection
• Screening
• Sensing
Six-s = success
Acknowledgements
• Lab group
• Biacore
• FIRM
• SFI
• Fusion
• EI