Non Specific Binding of Antibodies in Immunoassays

© Innova Biosciences ltd. 2012. All rights reserved© Innova Biosciences ltd. 2012. All rights reserved
Quality – Consistency - Expertise

© Innova Biosciences ltd. 2012. All rights reserved
Welcome to our third webinar
How to Overcome all your Problems with
Secondary Antibodies
Dr Andy Lane

Dr Andy Lane
• Immunoassay fundamentals
• Properties of secondary antibodies
• Direct labelling of primary antibodies
Dr Lane has recently joined Innova Biosciences,
where he is well positioned to utilise his antibody
conjugation and flow cytometry experience in
combination with Innova’s ground-breaking rapid
conjugation technology.

Immunoassay fundamentals
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

In many cases these reagents are not
available
DIRECT
SANDWICH
X
X

A common way to deal with this is to use a
“secondary antibody” in an “indirect” assay

Properties of secondary antibodies
Anti-species immunoglobulin reagents
Single reagent may be used in many assays
Binding of multiple antibodies may increase signal

BUT……….
Level of non-specific binding in the assay is increased, especially in assays
where immunoglobulins from different species are present
Multi-parameter assays are very difficult to run
Increase in sensitivity may not be realised
Assay times are increased due to the additional washes and incubation steps

Increase in non-specific binding

Increase in non-specific binding
especially in systems where immunoglobulins from different species are present

Multi-parameter assays are
very difficult to run

Species cross-reactivity may be reduced by the use of
antibodies that have been adsorbed against species
immunoglobulin.
However, as the most cross-reactive antibodies are those with
highest affinity, this results in a reduction in antibody affinity
and therefore performance of the antibody.
Cross-reactivity may also be due to other factors such as
binding to Fc receptors and general low-level non-specific
interactions, which increase simply with the amount of
antibody present and is not saturable.
Secondary antibody cross-reactivity

BUT……….
Level of non-specific binding in the assay is increased, especially in assays
where immunoglobulins from different species are present
Multi-parameter assays are very difficult to run
Increase in sensitivity may not be realised
Assay times are increased due to the additional washes and incubation
steps

All of these problems with
secondary antibodies can be
overcome by the use of directly
conjugated primary antibodies

No non-specific binding

No non-specific binding
even in systems where immunoglobulins from different species are present

Multi-parameter assays are
straightforward to run

The major problem with using directly
conjugated antibodies in assays is their
lack of availability, and also the difficulty
of conjugating antibodies yourself by
traditional methods.

Features of
Lightning-Link®
• Lightning-Link ® - the world’s easiest antibody labeling kits
• Simple, one step process
• Only 30 seconds hands-on
• Reproducible
• Scalable µg to mg
• 100% recovery
Just add primary antibody !
22

23

Lightning-Link®

Lightning-Link® Rapid
Lightning-Link® is a registered trademark of Innova Biosciences
DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

Conjugation considerations
Antibody doesn’t meet
these criteria?
You need to know some things about your antibody.
Lightning-Link conjugations are really simple
but you need antibody in the right format to work effectively.
Commercially available antibodies come in many forms,
and you may need to check with the supplier about some details.
Concentration – 1mg/ml or higher is preferred
Purity – ensure other proteins have been removed,
and also make sure they haven’t been put back again afterwards!
Buffer formulation – most common formulations are suitable,
but ensure that amines such as glycine are truly absent,
as well as thiols such as DTT or mercaptoethanol. Tris is OK up to 20mM
Use a purification kit to purify, concentrate
and/or change the buffer of your antibody

Using your new conjugates
• Use exactly as normal in terms of staining technique
• Titrate – possibly extensively!
• Storage – at 40C in concentrated form is always best.
• A preservative (e.g. 0.05% w/v sodium azide) may be useful, and if stored diluted a
carrier protein would be advised (e.g. 1% w/v BSA)
• Some conjugates may be safely frozen, but others should not be. Never freeze
RPE, APC or their tandem forms!
• Keep conjugates away from light – tandem dyes are especially sensitive
28

Directly Labeled Antibodies Indirectly Labeled Antibodies
primary antibody Goat Anti-NQO1 Goat Anti-NQO1
target quinone reductase 1 quinone reductase 1
sample lysate human kidney human kidney
primary antibody working concentration 0.00425 µg/ml 0.1 µg/ml
secondary antibody used no (direct conjugation) yes
exposure time (min) 10 10
primary antibody source Everest Biotech, Cat no: EB05370
western blot analysis
Western blotting data comparing conjugated and unconjugated antibodies
(direct and indirect detection)

conjugated unconjugated
primary antibody Goat Anti-GFAP Goat Anti-GFAP
target GFAP GFAP
sample lysate mouse brain mouse brain
primary antibody working concentration 0.185 µg/ml 0.5 µg/ml
secondary antibody used no (direct conjugation) yes
exposure time (min) 3 3
primary antibody source Everest Biotech, Cat no: EB07478
western blot analysis
Western blotting data comparing conjugated and unconjugated antibodies
(direct and indirect detection)

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.
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

Non Specific Binding of Antibodies in Immunoassays

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