Non Specific Binding of Antibodies in Immunoassays

3. © Innova Biosciences ltd. 2012. All rights reserved 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.

5. © Innova Biosciences ltd. 2012. All rights reserved Immunoassay fundamentals 1. Antibody specific for target antigen 2. Suitable visualisation method – e.g. enzyme, fluorescent dye, nanoparticle DIRECT SANDWICH

8. © Innova Biosciences ltd. 2012. All rights reserved Properties of secondary antibodies Anti-species immunoglobulin reagents Single reagent may be used in many assays Binding of multiple antibodies may increase signal

9. © Innova Biosciences ltd. 2012. All rights reserved Properties of secondary antibodies 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

15. © Innova Biosciences ltd. 2012. All rights reserved 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

21. © Innova Biosciences ltd. 2012. All rights reserved 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.

22. © Innova Biosciences ltd. 2012. All rights reserved 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

25. © Innova Biosciences ltd. 2012. All rights reserved 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

26. © Innova Biosciences ltd. 2012. All rights reserved 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

27. © Innova Biosciences ltd. 2012. All rights reserved27

28. © Innova Biosciences ltd. 2012. All rights reserved 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

29. © Innova Biosciences ltd. 2012. All rights reserved 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)

30. © Innova Biosciences ltd. 2012. All rights reserved 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)

33. © Innova Biosciences ltd. 2012. 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

34. © Innova Biosciences ltd. 2012. 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

Non Specific Binding of Antibodies in Immunoassays

Non Specific Binding of Antibodies in Immunoassays

Recommended

More Related Content

What's hot

What's hot(20)

Viewers also liked

Viewers also liked(20)

Similar to Non Specific Binding of Antibodies in Immunoassays

Similar to Non Specific Binding of Antibodies in Immunoassays (20)

More from Expedeon

More from Expedeon (20)

Recently uploaded

Recently uploaded(20)

Non Specific Binding of Antibodies in Immunoassays