Mouse skin 4 days after inoculation

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Antibody Generation in Proteomics
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Proteomics Challenge

• 35,000 human genes
• gt1million human proteins?

• Ligands are needed to create quantitative assays

3
Proteomics-Scale Antibody Production
GCGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTAATTAAGCGCTAGCTGCTAGCTGACTGCA
AGCACGCAAAGGTTTCTGGGATGATTTCAGCCAGACCTCTGGTGACAAGG
GCCGCGTGGAGCAGTGACACCAGCAAAAGATGGCACGCGAACCAGCCAAA
AGCTAGCTAGCATCGATAGCATCAGCATCAGCATCAGCACATTATATCAG
CGATAGACTAGTAGCATGAGTTATGAGCGCGCGCATTAGCATATTAGCGA
TAGCGCGGCGCATTATATAGCATCATCGACTAGTCGATCGATAGTAGATG
ACTACTAGCTAGATGCAGCATTATAGCATTATATATGCATGAGGCGCGGG
CCGATACGATATTATAGCTCTATCACACAACGTTGTAAACGTATATACGG
CAGTCAGAGATCAGCGCGCGCTATT
Genome Sequence
4
Ligands
scFv
Antibodies
Peptides
RNA
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Ligand Selection Technology
Phage
Animals
Microbes
Ribosome
mRNA
Protein
Beads
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Problem
GCGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTACTTAAGCGCTAGCTGCTAGCTGACTGCA
AGCACGTAAAGGTTTCTGGGTATTTAGCGACGAGCGTGCTAGTGTTCATT
Most ligand selection methods require at least
?g quantities of soluble purified target protein
Obtaining the target protein is a major
bottleneck
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Polyclonal Antibodies
Protein immunization
Johnston et al. (1992) Nature 356 p152-154
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DNA Delivery Methods
Needle delivers DNA outside cells and is
subsequently taken up
Gene gun delivers DNA directly into cells
Requires 1mg/shot
Requires 100?g/shot
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Helios Gene Gun
12 shot cartridge
350 psi optimal for mice
Helium
Gold particles carrying DNA are propelled into
the mouse tissues
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Gene Gun Cross-Section
Bullet holder
He
Gun nozzle
Bullet
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Gene Gun Bullets

1mm Gold Particle
Nucleic acids
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DNA Dose
Dose is limited to about 2.5mg per shot
The limitation is due to the capacity of the
gold for binding DNA and the amount of gold that
can be delivered into tissue without causing
damage.
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DNA Dose
10ng
1000ng
1ng
100ng
Dose immunized into mice
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Bullet Preparation
Nitrogen gas regulator
Syringes to inject slurry into tubing
Ethanol to resuspend gold
Modified 8-channel bullet dryer
Vortex to create gold/ethanol slurry
2ft section of Tefzel tubing (enough for 50
bullets)
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Bullet Preparation
Guillotine slices tubing into 14mm pieces
Tefzel tubing is fed in here
Cut bullets collect here
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Bullet Storage
Parafilm
Stable for at least 6 months if kept in a dry
environment
Bullets
Tissue paper
Drierite
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Gene Gun Delivery
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Gene Gun Delivery
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Gene Gun Delivery
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Gene Gun Delivery
Ears are convenient sites to immunize
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Target cells for Immunization
High Throughput Antibody Production
Epidermis
DC
DC
DC
Dendritic cells are potent initiators of the
immune response
Large numbers are found in the skin
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Immune Response
DC
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Immunization Regime
Antibody levels
3 weeks
3 weeks
On average most mice produce high titer responses
after two immunizations
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Antibody Responses
100
80
Anti-AAT Equivalents (mg/ml)
60
40
20
0
1
2
3
4
5
Ave.
Individual Mice
Typical titer 20,000
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Advantages of Producing Antibodies by Genetic
Immunization
Antibodies recognize native antigen
No contaminants
Faster
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!
Warning!
Genetic immunization is a completely different
technology from protein-based methods. DNA cannot
simply be substituted for protein in protocols.
Special considerations
-Special care in the design of constructs to
ensure efficient expression
-Different kinds of adjuvants are required
27
Antigen Localization
DC
MHCII
B cell
MHCII
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Expression of Antigen
Nucleus
Golgi
E.R.
Folding/Solubility
Translation
Transcription
Processing
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Transcriptional Promoters
Immediate early enhancer/promoter from human
cytomegalovirus with an intron from b-globin
(hCMVieI) 1150bp
Synthetic enhancer/promoter designed from
transcription factor consensus binding sites and
assembled from oligonucleotides (SP72) 600bp
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Transcriptional Terminators
Rabbit beta globin terminator 500bp
RGT
Human growth hormone terminator 700bp
HGH
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Optimizing Translation
Translation can be enhanced by recoding genes
with codons that are decoded by high abundance
tRNAs
GCT GCC GCA GCG
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Codon Optimization Examples
Antigen
Control
Control
Antigen
Antigen
Control
Control
Antigen
Wild Type
Codon Optimized
Wild Type
Codon Optimized
Bacterial example
Mammalian example
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Codon Optimization
ATGTGGGCCGCCGACTCGGACAGGGAGGACCTGACCATGATGATCGACGA
CCTGGACCGCAGAGGCTTCAGATCCATCTACCGGCTGCTGGCCAGCCGCT
GCAAGCTGGCCGACGCGTTCCAGAATTGGGCGCGCTCCCGGCTCGCCCGC
TACATCATCCTGTCGGCCGCGATCGTCATCTACTACGTGCTCTCGTCGAT
CCTGTCCCTGTCGATCTCGATCTACCGGGCCTCCTCGATGTG
ATGTGCGCTGCTGATTCTGATAGGGAGGATCTTACTATGATGATAGATGA
TCTAGATCGTAGAGGATTTAGATCTATATACCGGCTATTGGCTAGTCGAT
GCAAGCTAGCTGATGCTTTTCAAAATTGGGCGCGCTCTCGGCTCGCTCGC
TATATTATTTTATCGGCTGCTATCGTCATCTACTATGTTCTCTCGTCGAT
ATTATCTCTATCTATTTCTATCTATCGGGCCTCTTCGATGTG
Assemble gene from oligonucleotides
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Gene Synthesis
Pool of Oligos
Assembly (PCR)
etc
Amplify (PCR)
PCR assembly method (Gene v164 p49 1995)
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DNA Builder Software
Designs oligos for gene synthesis
Available free at pga.swmed.edu
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Gene Synthesis Example
Assembly
PCR
Markers
Gene 3
Gene 1
Gene 2
Gene 3
Gene 1
Gene 2
1500-
1000-
500-
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Antigen Selection
Small regions (20-100aa) are selected to allow
high levels of expression and is a convenient
size for gene synthesis.
Surface regions are selected to ensure efficient
expression and that the resulting antibody will
recognize the native antigen.
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Antigenic Index
Select for immunization
Jameson Wolf algorithm combines
hydrophilicity, surface probability, flexibility
and secondary structure prediction scores.
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Passing Quality Control
Nucleus
Golgi
E.R.
Folding/Solubility
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COMP Fusions
Antigen
COMP
Secretion leader peptide
A fragment of cartilage oligomeric matrix protein
(COMP) is a small, well folded naturally secreted
protein and is highly soluble
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Post-translational modifications
Antigen
Glycosylation
NX
S
T
Furin proteases
R
RX R
K
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Immune Response Problems
Antigen uptake
B cell stimulation
B cell epitopes
DC
DC stimulation
T cell stimulation
Antigen uptake
T cell epitopes
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Enhancing Antigen Uptake
DC
B cell
COMP forms pentamers
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Genetic Adjuvants
GMCSF/Flt3L
Antibodies
GMCSF
None
Time (weeks)
GMCSF and Flt3L are DC growth factors
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T cell epitopes
Universal P2 and P30 epitopes from tetanus toxin
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Scaffold Antigenicity
T cell epitopes
Antigen
COMP
Tag
Leader
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Creating a HTP Antibody Production System
Combination of Technologies
Codon Optimization
IVT
LEEs
Scaffold
Adjuvants
High-Throughput Antibody Production System
Genetic Immunization
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HTP Antibody System
Oligonucleotides
Overexpression LEE
Immunization LEE
In vitro Expression
Immunoassay
Genetic Immunization
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Linear Expression Elements
Antigen
Control
Antigen
Promoter
Terminator
Terminator
Antigen
Promoter
Sykes Johnston (1999) Nat. Biotech. 17 p355-359
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Assembly of LEEs by Overlap PCR
Ag
Antigen
P
T
100aa regions
20aa regions
SP72 and RGT are used for LEEs since they are
smaller and assemble more efficiently
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Gel Analysis of Overlap PCR LEEs
Ag
Antigen
P
T
100aa regions
20aa regions
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In Vitro Transcription and Translation
RTS continuous-exchange cell-freetechnology
(CECF) boosts yields

• Continuous exchange boosts protein yields by
• Replenishing energy sources
• Removing inhibitory reaction by-products
• Extending translation period

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LEE-Based IVT
T7T
T7P
Markers
GST-----------TEV-Flag-----Gene-----SII
DNA
-DNA
GST Fusions
PDGFb
PDGFa
TNFb
TNNCI
TNFa
TGFb
15 mL Reactions 100ng/mL
Vegf
SOD
a-SII Probe
Western
Coomassie
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Sensitivity of Antibodies
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Detecting the Natural Antigen
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Specialty Antibodies
Ubiquitin
Peptide Antibodies
ELISA
Exon1
Exon3
IEERLGS VSGGELF
Poly K48
Poly K29
Poly K63
500ng
100ng
20ng
4ng
7
6
5
4
4
3
3
3
2
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Chickens Rabbits
Control
Antigen
Control
Antigen
Rabbit anti-ABCG5
Chicken anti-HDAC5
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Summary
ATGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTACTTAAGCGCTAGCTGCAGCGTGCTAGTG
TTCGCGGTAG
ATGTTAGGCTCCTAGGATGATCGATGATGCTTATATCGCGGCTGCTGATG
CTAGTCGATTATGCTCTCGCTACTTAAGCGCTAGCTGCAGCGTGCTAGTG
TTCGCGGTAG
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Acknowledgements
Stephen Johnston
Joey Nguyen
Tom Kodadek
Vickie Seward
Dave Fancy
Alice Shaw
Xiang Chen
Greg Urquhart
Jerry Lin
Eileen Ward
Rosemary Wilson
Steve Liu
Southwestern PGA- project 4