Oligo:Antibody 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) • 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


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 3ʹ′



Synthesis Cycle



bond



group



Cycle #1


Synthesis Cycle



bond



group



Cycle #1


Synthesis Cycle



bond



group



Cycle #1


Synthesis Cycle



bond



group



Cycle #1


Synthesis Cycle



bond



group



Cycle #1


Synthesis Cycle



bond



group



The synthesis cycle requires between 3 and 8 minutes.

Cycle #1


Synthesis Cycle



bond



group



Cycle #2


Synthesis Cycle



bond



group

5ʹ′ GCACTTCAGGCTCCTGGGCC 3ʹ′


Cycle #3


Synthesis Cycle



bond



group

5ʹ′ GCACTTCAGGCTCCTGGGCC 3ʹ′


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-

Leng

th P

rodu

ct (%

)

Oligo Length (nt)

Coupling Efficiency vs. Full-Length Product

99.25% - IDT Standard Oligos

98.5% - Industry Standard


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


5ʹ′ ATCTGACTATGACTGACTGACTGACAGACTACTGACTGACTATCTGACTGA TCTGCAACACGCAAGGTCGGCGAT-Amino 3ʹ′

98.4% Target Sequence 62.7% Target Sequence

Before HPLC Purification After HPLC Purification


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′

70.8% Target Sequence

Before HPLC Purification




Before HPLC Purification




Before HPLC Purification Target Sequence

Blocked Amino Modifier

8.8% Blocked Amino Modifier


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



94.4% Target Sequence 70.8% 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 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


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


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 D C E

Banding is more often seen with mAbs than pAbs


Conjugating oligonucleotides to antibodies 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 20130

25

50

75

100

Year

No

of o

ccur

renc

es

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.


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

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

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


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