10 oligo ab conjugates an application guide

© Innova Biosciences ltd. 2013. All rights reserved
Oligo:Ab conjugates
an application guide

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.

IDT Oligos + Thunder-Link® Conjugation Kit

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

San Diego, CA
• 1,654 square meters
• ISO:9001:2008 certified
• Next Day production for
Western United States
Coralville, IA (Headquarters)
• 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
• ISO 9001:2008 certified
• Eurozone & Middle East Production and
support
Singapore
• 560 square meters
• Asia Market Production and
Support
IDT Facility Locations

How to Order an Oligo

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

5’ GCACTTCAGGCTCCTGGGCC 3’
How to Synthesize an Oligo

Electron microscope image
100–175 µm
particle size
START HERE
5’ GCACTTCAGGCTCCTGGGCC 3’

Synthesis
Cycle
Coupling of
next base
Oxidizing
internucleotide
bond
Capping of
coupling failures
Unmasking of growth
protecting
group
5’ GCACTTCAGGCTCCTGGGCC 3′

Synthesis
Cycle
Coupling of
next base
Oxidizing
internucleotide
bond
Capping of
coupling failures
Unmasking of growth
protecting
group
Cycle #1

Synthesis
Cycle
Coupling of
next base
Oxidizing
internucleotide
bond
Capping of
coupling failures
Unmasking of growth
protecting
group
The synthesis cycle
requires between
3 and 8 minutes.
Cycle #1

Synthesis
Cycle
Coupling of
next base
Oxidizing
internucleotide
bond
Capping of
coupling failures
Unmasking of growth
protecting
group
Cycle #2

Synthesis
Cycle
Coupling of
next base
Oxidizing
internucleotide
bond
Capping of
coupling failures
Unmasking of growth
protecting
group
5′ GCACTTCAGGCTCCTGGGCC 3′
Cycle #3

Synthesis
Cycle
Coupling of
next base
Oxidizing
internucleotide
bond
Capping of
coupling failures
Unmasking of growth
protecting
group
5′ GCACTTCAGGCTCCTGGGCC 3′
Cycle #19
A 20nt oligo can
be synthesized in
one hour.

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-LengthProduct(%)
Oligo Length (nt)
Coupling Efficiency vs. Full-Length Product
99.25% - IDT Standard Oligos
98.5% - Industry Standard

5′ ATCTGACTATGACTGACTGACTGACAGACTACTGACTGACTATCTGACTGA
TCTGCAACACGCAAGGTCGGCGAT-Amino 3′
98.4% Target Sequence62.7% Target Sequence
Before HPLC Purification After HPLC Purification

Amino and Thiol Modifiers
5′ Amino Modifier 5′ Thiol Modifier

Amino and Thiol Modifiers – Molecular Dust Mops
(Strong Nucleophiles)
5′ Amino Modifier 5′ Thiol Modifier

5′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3′
70.8% Target Sequence
Before HPLC Purification

70.8% Target Sequence
Before HPLC Purification
Target Sequence
Blocked Amino Modifier
8.8% Blocked Amino Modifier

Mass(Da) Intensity Delta Mass %Relative %Total Identity
9394.2 9.27E+04 0 100 88.74 Target Mass
9421.8 9.58E+03 27.6 10.32 9.16 Blocked Amino
9447.5 2.19E+03 53.3 2.36 2.1 Blocked Amino
5′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3′ (MW=9394.1Da)
Electrospray Ionization Mass Spec (ESI-MS) – “Molecular Ruler”
Target Sequence
Blocked Amino Modifier

94.4% Target Sequence70.8% Target Sequence
Before HPLC Purification After HPLC Purification

1. Introduction
2. Traditional Immunoassays
3. Improving on colorimetric assays
4. Conjugating oligonucleotides to antibodies
5. Applications using oligonucleotide:antibody conjugates

Introduction
Oligonucleotide conjugation systems (‘Proactive’)
Founded, Cambridge UK2002
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

Introduction4 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

Traditional Immunoassays
1. Antibody specific for target antigen
DIRECT
SANDWICH

1. Antibody specific for target antigen
2. Suitable visualisation method – e.g.
enzyme, fluorescent dye, nanoparticle
DIRECT
SANDWICH

ELISA Lateral Flow

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

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

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

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

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

Non-Reduced antibody
A B DC E
Banding is more often seen
with mAbs than pAbs

Protein DNA
H
L
L chain-oligo
conjugate
H chain-oligo
conjugate
Higher order
conjugates
Reducing gel

Applications using oligonucleotide:antibody conjugates
• ImmunoPCR assays
• Proximity Ligation Assays
• Proximity Extension Assays
• 3DNA Technology in Lateral Flow
• 3DNA Technology in Cellular Targeting

Applications using oligonucleotide:antibody conjugates
2003 2005 2007 2009 2011 2013
0
25
50
75
100
Year
Noofoccurrences
The number of publications discussing ImmunoPCR (Pubmed)

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.

ImmunoPCRA 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

Proximity Ligation Assays

Proximity Extension Assays

Conventional immunoassays: cross-reactivity due to unspecific binding of antibodies
limits the degree of multiplexing.

Proseek Multiplex: unique DNA oligo sequences report only matched DNA-pairs (e.g.
1A+1B). Cross-reactive events are not detected.

3DNA Technology in Lateral Flow
Figure 2: 3DNA® Dendrimer with multiple
targeting 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

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

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.
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

10 oligo ab conjugates an application guide

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