How does an antibody bind to an ELISA plate?

An antibody binds to an ELISA plate in one of four ways. In passive adsorption it adheres to the polystyrene through ionic and hydrophobic interactions, in a random orientation. In affinity capture, a Protein A/G surface holds the antibody by its Fc, or a streptavidin surface holds it by a biotin tag, keeping the F(ab)2 arms free. Last but not least, through an antibody-antibody bond with a coated secondary antibody, which allows the antibody to bind via its Fc and thus leaves its F(ab)2 free to react with the antigen.

How much antibody do you need to coat an ELISA plate?

For passive adsorption on a High Binding surface, coat purified antibody at roughly 3–5 µg/mL in 100 µL per well, which deposits on the order of 400 ng/cm². Oriented surfaces differ: streptavidin and secondary-antibody capture work at 1–1.5 µg/mL, while Protein A/G is coated at ≥4 µg/mL. Run a checkerboard titration to find the optimum for your specific antibody, since coating above the surface capacity can raise background.

How long can you coat an ELISA plate?

Coating is usually done overnight at 4 °C, or for a shorter incubation at room temperature, depending on the antibody. Coated and blocked plates can then be dried and stored sealed with desiccant for later use. Optimal incubation time and storage stability are antibody-dependent and should be verified for each assay.

Can you overwash an ELISA plate?

Washing removes unbound and loosely bound antibody, but overly harsh or excessive washing can strip passively adsorbed antibody from the surface and raise well-to-well variability. Affinity-captured tolerates stringent washing better than passively adsorbed ones. Keep the number of cycles and the wash volume consistent across the plate and between runs.

Should you use a High Binding or an oriented surface for a capture antibody?

Use High Binding when a standard sandwich or competitive assay performs well and speed matters. Switch to an oriented surface (Streptavidin or Protein A/G or antibody-antibody) when functional activity is low because passive adsorption buries the F(ab)2 arms, or when you want tighter reproducibility from a defined orientation.

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Applications & Techniques

How to Coat an Antibody on a 96-Well ELISA Microplate

How to Coat an Antigen on a 96-Well ELISA Microplate

Microplate Surface Selection Guide: How to Choose the Right Surface for ELISA, CLIA, and FIA Assays

Choose the Right Microplate for Your Assay: A Practical Guide

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White 96-Well Microplates for Luminescence and CLIA

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Exploring diagnostic innovations with Biomat 96-Well ELISA plates

Black 96-Well Microplates for Fluorescence Assays

Medium and High Binding Well Plates

Biotin Binding Surfaces

Biomat has developed coated plates for Immunotoxicity ELISA tests

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ELISA Plates for Coronavirus COVID-19

Mouse Monoclonal Antibody Isotyping: Comprehensive ELISA Solutions for Accurate Immunoglobulin Characterization

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