Using ELISA to Validate Antibody Affinity

Scientists employ antibodies as reagents in a broad range of applications, including research, diagnostics, and clinical tests. Antibodies vary greatly in how they interact with targets, and the quality of the antibodies significantly impacts the success of the experiment. Selecting the right antibodies for a specific application is critical for ensuring they result in the proper binding to the target of interest, perform consistently across different tests, and produce accurate, reproducible data.

What Is Antibody Validation?

Antibody validation consists of proving the following:

• Specificity – an antibody’s ability to differentiate between various antigens
• Affinity – the strength of the bond created between the epitope of the target antigen and the paratope of the antibody of interest
• Reproducibility – the ability of an antibody to yield consistent results when used in different lots, at different times, and/or by different researchers

Antibody validation should also include proving specificity of an antibody within the exact application for which it will be used. This validation can be performed for various immunoassays including ELISA, western blot, immunofluorescence, immunocytochemistry, immunohistochemistry, immunoprecipitation, chromatin immunoprecipitation, and protein or peptide arrays. If an antibody is intended to be used for a specific assay format, it must be tested in that context.

Many factors can impact an antibody’s performance. Improper storage conditions during transport can impact performance. Researchers must also be aware of the product specifications, including the application for which it has been validated, the appropriate protocols for using the antibody, and the recommended dilutions.

How Do You Validate Antibody Affinity?

The three main methods for validating antibody affinity are:

• Microscale thermophoresis (MST)
• Surface plasmon resonance (SPR)
• ELISA

MST is a biophysical method that detects antibody affinity in various concentrations by determining the specific movements of molecules in the substrate using a microscopic temperature gradient. The results of this method vary based on factors like molecular size and charge.

SPR is an optical method of affinity detection that involves immobilizing one molecule in a metal film and leaving the other molecule free floating. The combination of these molecules affects the refractive index of the film, so exposing the film to polarized light results in changes in the light’s extinction angle, which can be viewed through an optical detector.

ELISA is the most popular and effective method for validating antibody affinity due to its high degree of efficacy and precision. ELISA kits do not require a large concentration of antigens and antibodies and take less time to perform. In this method, a researcher incubates a fixed concentration of an antibody with an antigen in a solution until the mixture reaches a steady state. This allows equal amounts of binding and dissociation events to occur each second. Then, the amount of the unbound antibody is measured using indirect ELISA.

Monoclonal vs. Polyclonal Antibodies in Affinity Validation

It is important to note that validating an antibody’s affinity depends on the type of antibody that is used. Polyclonal antibody preparations contain heterogeneous mixtures of different antibodies from a pool of immunized animal samples and show varying affinities for multiple epitopes. Because only a portion of the antibodies in this pool will bind to the target of interest, and the antibodies vary among different animals and different batches, these antibodies do not produce uniform results.

When using polyclonal antibodies, researchers cannot guarantee that samples will have consistent, reproducible binding capabilities. They must perform affinity purification to strengthen target-specific antibodies within samples and remove serum proteins. This does not fully eliminate non-specific antibodies and can still result in cross-reactivity issues.

In contrast to polyclonal antibodies created through animal immunization, monoclonal antibodies are generated via hybridoma technology, meaning they are all identical clones and selective for a single epitope on an antigen. These preparations are homogeneous and only include the antibody of interest, meaning researchers are not required to perform the additional step of affinity purification to obtain consistent, reproducible results. Monoclonal antibodies demonstrate uniform performance with high accuracy across different batches and they can be produced in large quantities with the same binding capabilities. Therefore, they are often the preferred antibody for affinity validation, especially in long-term projects.

Sources:

https://www.labome.com/method/Antibody-Validation.html

https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/antibody-standard-validation

https://www.biomatik.com/blog/antibody-validation-how-to-confirm-the-specificity-of-an-antibody/