Oligo:Antibody Conjugates—An Application Guide
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Transcript of Oligo:Antibody Conjugates—An Application Guide
© Innova Biosciences ltd. 2013. All rights reserved
Oligo:Ab conjugates an application guide
© Innova Biosciences ltd. 2013. All rights reserved
Ms. Alexander graduated with a B.S degree in Chemistry from the University of Iowa in 1997. Five days after her graduation she began working for IDT and has happily made oligos ever since. She oversees manufacturing operations for modified nucleic acids and GMP services.
Jessica Alexander, VP Specialty Manufacturing and Services Integrated DNA Technologies (Coralville, IA, USA)
Tom Speedy, Corporate Business Manager, Innova Biosciences (UK)
With an academic background in aquatic toxicology, Tom moved into the diagnostics industry as an R&D Scientist with a UK based forensic toxicology/drug testing company. He spent 7 years developing a number of successful products, including being part of the team that developed a saliva based drugs of abuse test for roadside and POC testing that won the Queens Award for Innovation. Tom now manages the corporate business for Innova Biosciences, working with diagnostics, pharma, biotech, R&D and contract research companies globally.
© Innova Biosciences ltd. 2013. All rights reserved
IDT Oligos + Thunder-Link® Conjugation Kit
© Innova Biosciences ltd. 2013. All rights reserved
Integrated DNA Technologies (IDT) is the world’s largest supplier of custom nucleic acids Key Operating Statistics >80,000 active customers >42,000 oligos ordered per day >2,800 orders shipped per day >95% orders entered over the web > 270,000 web site visits per month (117,000 unique visitors) > 85,000 phone calls per year > 23,000 web chats per year 29 global sales professionals >750 Employees
© Innova Biosciences ltd. 2013. All rights reserved
San Diego, CA • 1,654 square meters • ISO:9001:2008 certified • Next Day production for Western United States
Coralville, IA (Headquarters) • 14,300 square meters • Global Infrastructure, Production and support offices • ISO 9001:2008 certified • 3,100 square feet, ISO 13485:2003 and FDA-registered clean room
Skokie, IL •Administrative and Financial Office
Leuven, Belgium • 2,010 square meters • ISO 9001:2008 certified • Eurozone & Middle East Production and support
Singapore • 560 square meters • Asia Market Production and Support
IDT Facility Locations
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How to Order an Oligo
© Innova Biosciences ltd. 2013. All rights reserved
How to Design an Oligo
Standard sequence motifs to avoid • Minimize homopolymers (e.g. CCCCCCC) • 5’ Amino or Thiol modification is slightly
favored over 3’ positions • Avoid G-quadruplexes
© Innova Biosciences ltd. 2013. All rights reserved
5’ GCACTTCAGGCTCCTGGGCC 3’
How to Synthesize an Oligo
© Innova Biosciences ltd. 2013. All rights reserved
Electron microscope image
100–175 µm particle size
START HERE
5’ GCACTTCAGGCTCCTGGGCC 3’
How to Synthesize an Oligo
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #1
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #1
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #1
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #1
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #1
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
The synthesis cycle requires between 3 and 8 minutes.
Cycle #1
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #2
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5ʹ′ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #3
© Innova Biosciences ltd. 2013. All rights reserved
Synthesis Cycle
Coupling of next base
Oxidizing internucleotide
bond
Capping of coupling failures
Unmasking of growth protecting
group
5ʹ′ GCACTTCAGGCTCCTGGGCC 3ʹ′
How to Synthesize an Oligo
Cycle #19
A 20nt oligo can be synthesized in
one hour.
© Innova Biosciences ltd. 2013. All rights reserved
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Full-
Leng
th P
rodu
ct (%
)
Oligo Length (nt)
Coupling Efficiency vs. Full-Length Product
99.25% - IDT Standard Oligos
98.5% - Industry Standard
© Innova Biosciences ltd. 2013. All rights reserved
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Full-
Leng
th P
rodu
ct (%
)
Oligo Length (nt)
Coupling Efficiency vs. Full-Length Product
99.25% - IDT Standard Oligos
98.5% - Industry Standard
© Innova Biosciences ltd. 2013. All rights reserved
5ʹ′ ATCTGACTATGACTGACTGACTGACAGACTACTGACTGACTATCTGACTGA TCTGCAACACGCAAGGTCGGCGAT-Amino 3ʹ′
98.4% Target Sequence 62.7% Target Sequence
Before HPLC Purification After HPLC Purification
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Amino and Thiol Modifiers
5ʹ′ Amino Modifier 5ʹ′ Thiol Modifier
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Amino and Thiol Modifiers – Molecular Dust Mops (Strong Nucleophiles)
5ʹ′ Amino Modifier 5ʹ′ Thiol Modifier
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5ʹ′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3ʹ′
70.8% Target Sequence
Before HPLC Purification
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5ʹ′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3ʹ′
70.8% Target Sequence
Before HPLC Purification
© Innova Biosciences ltd. 2013. All rights reserved
5ʹ′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3ʹ′
70.8% Target Sequence
Before HPLC Purification Target Sequence
Blocked Amino Modifier
8.8% Blocked Amino Modifier
© Innova Biosciences ltd. 2013. All rights reserved
Mass(Da) Intensity Delta Mass %Relative %Total Identity9394.2 9.27E+04 0 100 88.74 Target Mass9421.8 9.58E+03 27.6 10.32 9.16 Blocked Amino9447.5 2.19E+03 53.3 2.36 2.1 Blocked Amino
5ʹ′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3ʹ′ (MW=9394.1Da)
Electrospray Ionization Mass Spec (ESI-MS) – “Molecular Ruler”
Target Sequence
Blocked Amino Modifier
© Innova Biosciences ltd. 2013. All rights reserved
5ʹ′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3ʹ′
94.4% Target Sequence 70.8% Target Sequence
Before HPLC Purification After HPLC Purification
© Innova Biosciences ltd. 2013. All rights reserved
IDT Oligos + Thunder-Link® Conjugation Kit
© Innova Biosciences ltd. 2013. All rights reserved
1. Introduction
2. Traditional Immunoassays
3. Improving on colorimetric assays
4. Conjugating oligonucleotides to antibodies
5. Applications using oligonucleotide:antibody conjugates
© Innova Biosciences ltd. 2013. All rights reserved
Introduction
Oligonucleotide conjugation systems (‘Proactive’)
Founded, Cambridge UK 2002
First Lightning-Link® conjugation kit launched
2004 Assay services (pharmaceuticals)
Custom antibody labelling
2009
2006
2012
2005
2010 Nanoparticle R&D projects initiated
InnovaCoat® Gold kit launched; ISO9001 achieved
2013 Thunder-Link® kit launched
© Innova Biosciences ltd. 2013. All rights reserved
Introduction 4 main product categories:
• Lightning-Link®: enables labeling of biomolecules, with just 30 seconds hands-on time, to a wide range of enzymes, fluorescent dyes, biotin & streptavidin
• InnovaCoat®: range of gold nanoparticle conjugation kits including different particle sizes and surface chemistries
• Thunder-Link®: facilitates conjugation of oligonucleotides to antibodies, proteins or gold nanoparticles
• Phosphate detection: non-radioactive colorimetric assays with unparalleled stability
© Innova Biosciences ltd. 2013. All rights reserved
Traditional Immunoassays
1. Antibody specific for target antigen
DIRECT
SANDWICH
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Traditional Immunoassays
1. Antibody specific for target antigen 2. Suitable visualisation method – e.g. enzyme, fluorescent dye, nanoparticle
DIRECT
SANDWICH
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Traditional Immunoassays
ELISA Lateral Flow
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Traditional Immunoassays
ELISA
Detection Limits µg to pg
Amount of starting material required
µl
• Can be used to detect a variety of protein and hapten targets
• Linear signal and endpoint detection
• Established technology
• Tolerates a variety of sample matrices and contaminants
© Innova Biosciences ltd. 2013. All rights reserved
Improving on colorimetric detection
ELISA
Oligo-linked immunosorbent assay
Detection Limits µg to pg pg to fg Amount of starting material required
µl pl
Combines the specificity of antibodies with the amplification power of PCR
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Oligo-linked immunosorbent assay
Detection Limits pg to fg
Amount of starting material required
pl
• Combines the specificity of antibodies and the benefits of immunoassay technology, with the amplification power of qPCR
• Exponential signal and real time detection
• High sensitivity requires high quality reagents
• Tolerates a variety of sample matrices and contaminants
Improving on colorimetric detection
© Innova Biosciences ltd. 2013. All rights reserved
Conjugating oligonucleotides to antibodies
The quality and sensitivity of the assay is directly related to the quality of the reagents, therefore:
• Oligonucleotide based assays require high quality oligonucleotides
• The conjugate must NOT contain fragments of oligonucleotide
• The conjugate must NOT contain unbound oligonucleotide Traditional techniques for conjugating antibodies to oligonucleotides are costly, time consuming and require expert knowledge Several methods have been developed although they tend to be very complex, unreliable and often result in the cross linking
© Innova Biosciences ltd. 2013. All rights reserved
Conjugating oligonucleotides to antibodies
Furthermore: • The inclusion of positive controls enables the end user to confirm that the conjugation chemistry is working correctly.
• Technology is unidirectional and thus only allows formation of antibody-oligonucleotide complexes and never antibody-antibody or oligonucleotide-oligonucleotide.
To overcome these issues, the Thunder-Link® conjugation system has been designed to facilitate the generation of antibody-oligonucleotide conjugates
© Innova Biosciences ltd. 2013. All rights reserved
Conjugating oligonucleotides to antibodies
© Innova Biosciences ltd. 2013. All rights reserved
Conjugating oligonucleotides to antibodies
Non-Reduced antibody
A B D C E
Banding is more often seen with mAbs than pAbs
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Conjugating oligonucleotides to antibodies Protein DNA
H
L
L chain-oligo conjugate
H chain-oligo conjugate
Higher order conjugates
Reducing gel
© Innova Biosciences ltd. 2013. All rights reserved
© Innova Biosciences ltd. 2013. All rights reserved
Applications using oligonucleotide:antibody conjugates
• ImmunoPCR assays
• Proximity Ligation Assays
• Proximity Extension Assays
• 3DNA Technology in Lateral Flow
• 3DNA Technology in Cellular Targeting
© Innova Biosciences ltd. 2013. All rights reserved
© Innova Biosciences ltd. 2013. All rights reserved
Applications using oligonucleotide:antibody conjugates
2003 2005 2007 2009 2011 20130
25
50
75
100
Year
No
of o
ccur
renc
es
The number of publications discussing ImmunoPCR (Pubmed)
© Innova Biosciences ltd. 2013. All rights reserved
ImmunoPCR
Sano, T., Smith, C. and Cantor, C., 1992. Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA Conjugates. Published by the American Association for the Advancement of Science.
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ImmunoPCR A New Tool for Paleomicrobiology: The Plague Paradigm
Malou N, Tran T-N-N, Nappez C, Signoli M, et al. (2012) Immuno-PCR - A New Tool for Paleomicrobiology: The Plague Paradigm. PLoS ONE 7(2): e31744. doi:10.1371/journal.pone.0031744 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031744
• Degradation of DNA limits ability to detect ancient infections
• ImmunoPCR against Yersinia pestis can detect protein conc. 70 times lower than standard ELISA (0.74ng compared to 50ng cutoff)
• 34 teeth from mass graves of plague victims and 10 negative teeth were tested
• ELISA detected 3 of 34 +ves (1 false +ve) • PCR detected 10 of 34 +ves • iPCR detected 14 of 34 +ves
© Innova Biosciences ltd. 2013. All rights reserved
Proximity Ligation Assays
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Proximity Extension Assays
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Proximity Extension Assays
© Innova Biosciences ltd. 2013. All rights reserved
Proximity Extension Assays
Conventional immunoassays: cross-reactivity due to unspecific binding of antibodies limits the degree of multiplexing.
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Proximity Extension Assays
Proseek Multiplex: unique DNA oligo sequences report only matched DNA-pairs (e.g. 1A+1B). Cross-reactive events are not detected.
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3DNA Technology in Lateral Flow
Figure 2: 3DNA® Dendrimer with multipletargeting antibodies and hundreds of labels
Highly biotinylated 3DNA is customized with specific detection antibody-oligo conjugates. Materials are dried in a Lateral flow Point-of-Care Test.
3DNA
© Innova Biosciences ltd. 2013. All rights reserved
3DNA Assay Multiple 40nm streptavidin-gold
nanoparticles bind to ~1000 biotin labels on 3DNA with
detection antibody conjugates
Reference Assay 40nm streptavidin-gold
nanoparticles bind to biotinylated detection antibody
SA
SA SA SA
SA
SA
SA
SA
SA SA SA
SA
SA
SA
3DNA Technology in Lateral Flow
© Innova Biosciences ltd. 2013. All rights reserved
Fluorescent 3DNA customized with transferrin receptor antibody-oligonucleotide conjugates used for HepG2 cell staining
3DNA Technology in Cellular Staining
Cy3 3DNA (no targeting; no cell staining)
Cy3 3DNA hybridized with transferrin receptor antibody-oligo conjugate (lovely cell staining)
© Innova Biosciences ltd. 2013. All rights reserved
IDT Oligos + Thunder-Link® Conjugation Kit
© Innova Biosciences ltd. 2013. All rights reserved
Innova Biosciences Ltd. Babraham Research Campus,
Cambridge, UK, CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries