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



BUT……….






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……….






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 !

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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

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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 [email protected]

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Innova Biosciences Ltd.

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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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use of antibodies

antibody present

crossreactive antibodies

multiple antibodies

present multiparameter

level of nonspecific