5 Maximizing immunoassay performance with smart conjugate design

© Innova Biosciences ltd. 2013. All rights reserved© Innova Biosciences ltd. 2013. All rights reserved
Quality – Consistency - Expertise

Maximising immunoassay performance
with smart conjugate design

Dr Nick Gee
CEO/CSO of Innova Biosciences
Inventor of Lightning-Link®
Biochemist with over 20 years experience

IgG antibody structure

Each antibody has unique properties, including affinity
and selectivity.
A poor antibody will never make a good conjugate.
Smart conjugate design is about modifying antibodies
without any losing affinity.

Feature pKa
(variable*)
Forms Reactive
form
Lysine 10.8 -NH2/-NH3
+ -NH2
Cysteine 8.2 -SH/-S- -S-
Glu/Asp ~4.0 COOH/COO- -COO-
Carbohydrate n/a C(OH)-C(OH) -CHO
Possible points of label attachment in antibodies

+
R
NHS-Ester
R
Amide bond
(positive charge lost)
NHS (by-product)
R = e.g. fluorescent dyes,
biotin, maleimide,
hydrazide
Standard lysine modification technique to introduce labels or new
functional groups
protein protein

Too few
labels
What is the correct number of labels for an antibody?
0 2 4 6 8 10
0
20
40
60
80
100
No of labels attached
%Max.Performance
Altered folding (altered charge)
Steric hindrance
Reduced affinity
(Dye quenching)
Any conclusion is valid only for that particular antibody, label and assay

Conjugate optimisation and ranking of six PE conjugates.
Innova plate assay - Fc capture
1
2
3
4
5
6M
ouse
IgGH
um
an
IgG
0
10000
20000
30000
40000
50000
1:100 dilution
1:100 no antigen
1:400 dilution
1:400 no antigen
Fluorescenceunits
Best (client test)
What is ‘best’ is entirely application dependent – must screen
several conjugates in the intended application

Alkaline
phosphatase
HRP~10nm
Polymer
Labels vary over three orders of magnitude in size
Phycoerythrin

Lightning-Link®
One-step conjugation process
30 seconds hands-on time
No separation steps
No losses of antibody
Reproducible
Scalable up or down
Technology can be used to
produce conjugate libraries for
screening purposes
e.g. 10 conjugates with just 100ug
of antibody
50 labels in kit format:
HRP, fluorescent
proteins, dyes,
streptavidin…

PE
AP
HRP
Gold nanoparticle
40nm
Polymer
Nanoparticles

Latex
300nm
Particles

Antibody -
friendly
InnovaCoat
surface
Problems with surfaces:
Gold nanoparticle
40nm
Latex
300nm
Antibodies have not evolved to stick on metal or polystyrene!
Denaturation at surfaces occurs, especially with small fragments

A B C
A - InnovaCoat GOLD 10 OD
B – Washed
C – Naked gold 10 OD
IgG-spotted lateral flow strip,
blocked with BSA

Performance enhancement by oriented coupling
InnovaCoat
surface
InnovaCoat GOLD-
maleimide
Used with Fab’ fragments, or expressed antibody fragments
with a terminal cysteine; or any other thiolated substance.

Oriented coupling can lead to higher sensitivity
pepsin
reduce
blocking

0 10 20 30 40 50
0
100
200
300
400
500
600
700
800
Analyte conc (ng/ml)
Signal
Oriented (cys)
‘Random’ (lys)
Lateral flow data with InnovaCoat GOLD coupled to Fab’ or Fab

Periodate activation of antibodies for oriented labelling
periodate
aldehyde groups
Hydrazide-labels

Conjugate design tips:
• Screen multiple conjugates with a range of label densities in the
intended application.
• Position large labels away from the antibody binding domains
using site-specific modification techniques.
• Do not adsorb antibodies directly onto metallic/hydrophobic
surfaces if it can be avoided. Couple to a protective surface layer.
• For oriented covalent coupling on nanoparticles, use terminal
thiols on fragments or add appropriate functional groups to the Fc

One-step
labelling kits
Lightning-Link
InnovaCoat GOLD
conjugation kits
Conjugate
optimisation Oriented
coupling
Fc-specific
attachment
of labels
Innova bioconjugation toolkit and services
0
10000
20000
30000
40000
50000
Dye polymers
Antibody
fragmentation

© Innova Biosciences ltd. 2013. All rights reserved
Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

© 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

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