Oligo:Antibody Conjugates—An Application Guide

Oligo:Ab conjugates
an application guide

© Innova Biosciences ltd. 2013. All rights reserved

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.
Jessica Alexander, VP Specialty Manufacturing and Services
Integrated DNA Technologies (Coralville, IA, USA)

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.


IDT Oligos + Thunder-Link® Conjugation Kit


Integrated DNA Technologies (IDT) is the world’s largest supplier of custom
nucleic acids

Key Operating Statistics
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>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


IDT Facility Locations

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
• ISO 9001:2008 certified
• Eurozone & Middle East Production and

support

San Diego, CA
• ISO:9001:2008 certified
• Next Day production for
Western United States

Singapore
• 560 square meters
• Asia Market Production and
Support


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


How to Synthesize an Oligo

5’ GCACTTCAGGCTCCTGGGCC 3’



5’ GCACTTCAGGCTCCTGGGCC 3’
START HERE

100–175 µm
particle size
Electron microscope image



5’ GCACTTCAGGCTCCTGGGCC 3ʹ′

Unmasking of growth
protecting
group

Coupling of
next base

Synthesis
Cycle
Capping of
coupling failures

Oxidizing
internucleotide
bond



Unmasking of growth
protecting
group

Coupling of
next base

Synthesis
Cycle

Cycle #1

Capping of
coupling failures

Oxidizing
internucleotide
bond



Unmasking of growth
protecting
group

The synthesis cycle
requires between
3 and 8 minutes.

Coupling of
next base

Synthesis
Cycle

Cycle #1

Capping of
coupling failures

Oxidizing
internucleotide
bond



Unmasking of growth
protecting
group

Coupling of
next base

Synthesis
Cycle

Cycle #2

Capping of
coupling failures

Oxidizing
internucleotide
bond


5ʹ′ GCACTTCAGGCTCCTGGGCC 3ʹ′

Unmasking of growth
protecting
group

Coupling of
next base

Synthesis
Cycle

Cycle #3

Capping of
coupling failures

Oxidizing
internucleotide
bond


5ʹ′ GCACTTCAGGCTCCTGGGCC 3ʹ′

Unmasking of growth
protecting
group

A 20nt oligo can
be synthesized in
one hour.

Coupling of
next base

Synthesis
Cycle

Cycle #19

Capping of
coupling failures

Oxidizing
internucleotide
bond

Coupling Efficiency vs. Full-Length Product
100%
90%

Full-Length Product (%)

80%
70%

60%
50%

99.25% - IDT Standard Oligos

40%

98.5% - Industry Standard

30%
20%
10%
0%
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Oligo Length (nt)


5ʹ′ ATCTGACTATGACTGACTGACTGACAGACTACTGACTGACTATCTGACTGA
TCTGCAACACGCAAGGTCGGCGAT-Amino 3ʹ′
Before HPLC Purification

After HPLC Purification

62.7% Target Sequence


Amino and Thiol Modifiers

5ʹ′ Amino Modifier

5ʹ′ Thiol Modifier


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

5ʹ′ Amino Modifier

5ʹ′ Thiol Modifier


5ʹ′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3ʹ′



Target Sequence

Blocked Amino Modifier

8.8% Blocked Amino Modifier

5ʹ′ Amino – GTGGATCTGCGCACTTCAGGCTCCTGGGCA 3ʹ′ (MW=9394.1Da)
Electrospray Ionization Mass Spec (ESI-MS) – “Molecular Ruler”
Target Sequence

Mass(Da)
9394.2
9421.8
9447.5

Intensity Delta Mass %Relative
9.27E+04
0
100
9.58E+03
27.6
10.32
2.19E+03
53.3
2.36

%Total
88.74
9.16
2.1

Identity
Target Mass
Blocked Amino
Blocked Amino

Blocked Amino Modifier



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
2002

Founded, Cambridge UK

2004

Assay services (pharmaceuticals)

2005

Custom antibody labelling

2006

First Lightning-Link® conjugation kit launched

2009

Oligonucleotide conjugation systems (‘Proactive’)

2010

Nanoparticle R&D projects initiated

2012

InnovaCoat® Gold kit launched; ISO9001 achieved

2013

Thunder-Link® kit launched

4 main product categories:

Introduction

• 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

SANDWICH
DIRECT


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

SANDWICH

DIRECT


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
Detection Limits
Amount of starting
material required

Oligo-linked immunosorbent
assay

µg to pg

pg to fg

µl

pl

Combines the specificity of antibodies with the amplification power of PCR

Improving on colorimetric detection
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

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

To overcome these issues, the Thunder-Link® conjugation
system has been designed to facilitate the generation of
antibody-oligonucleotide conjugates
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.

A

B

C

D

E

Non-Reduced antibody

Banding is more often seen
with mAbs than pAbs


Protein

DNA

Higher order
conjugates
H chain-oligo
conjugate
H

L

L chain-oligo
conjugate

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
The number of publications discussing ImmunoPCR (Pubmed)

No of occurrences

100

75
50

25
0
2003

2005

2007

2009

2011

2013

Year

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.

A New Tool for Paleomicrobiology:
The Plague Paradigm

ImmunoPCR

• 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

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

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
Highly biotinylated 3DNA is customized
with specific detection antibody-oligo
conjugates. Materials are dried in a
Lateral flow Point-of-Care Test.

3DNA
Figure 2: 3DNA® Dendrimer with multiple
targeting antibodies and hundreds of labels


3DNA Technology in Lateral Flow
3DNA Assay

Reference Assay
40nm streptavidin-gold
nanoparticles bind to biotinylated
detection antibody

SA

SA
SA

SA

SA

Multiple 40nm streptavidin-gold
nanoparticles bind to ~1000 biotin
labels on 3DNA with
detection antibody conjugates
SA
SA

SA
SA

SA
SA

SA
SA

SA


3DNA Technology in Cellular Staining
Fluorescent 3DNA customized with transferrin receptor antibodyoligonucleotide conjugates used for HepG2 cell 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


Oligo:Antibody Conjugates—An Application Guide

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