What is nonspecific antibody binding?

Non-specific antibody binding occurs when an antibody binds to a cell that does not have an epitope specifically for that antibody. There are several reasons for non-specific antibody binding. The most common cause is an excess of antibody.

How can we prevent non-specific antibodies binding?

Tissue incubation with heat-inactivated normal serum or bovine serum albumin (BSA) is a common procedure used to reduce non-specific hydrophobic binding. Selection of the type of normal serum is important to prevent interactions with the primary or secondary antibodies, or with the tissues/cells being stained.

How does non-specific binding work?

When an antibody binds to unintended proteins (with highly similar epitopes) we speak of non-specificity. Then the actual binding of the antibody may be specific, yet the antibody is deemed non-specific in relation to the intended target protein.

How do you prevent non-specific binding?

Strategies to reduce non-specific binding Common strategies include: Adjusting the pH of your buffer. Using protein blocking additives. Adding non-ionic surfactants.

What’s the difference between avidity and affinity in antibodies?

While affinity is the measure of the binding strength at a single binding site, avidity is a measure of the total binding strength. Antibodies have between two and ten binding sites. Antibodies with fewer binding sites tend to have high affinity and low avidity, while those with greater binding sites tend to have low affinity and high avidity.

How is the avidity of a polyclonal antibody determined?

Polyclonal antibodies are heterogeneous and will contain a mixture of antibodies of different affinities recognizing several epitopes – therefore only an average affinity can be determined. Avidity gives a measure of the overall strength of an antibody-antigen complex.

What can I do about non specific antibody binding?

Fc regions of many antibodies readily bind to the Fc-receptors at different levels. Most commercially available antibodies contain Fc segments. To eliminate this problem, one can use an Fc blocking reagent, which contains a recombinant protein derived from immunoglobulin that will bind to the Fc-receptors and minimize the non-specific binding.

How many binding sites does an antibody have?

Antibodies have between two and ten binding sites. Antibodies with fewer binding sites tend to have high affinity and low avidity, while those with greater binding sites tend to have low affinity and high avidity.