Binding and Amplification: How It Works

Antibodies are Y-shaped proteins produced by an animal’s immune system that are capable of binding to an antigen. Antigens may be nucleotides, carbohydrates, or other small molecules, but they are usually other proteins. When an antibody binds to a protein, it does not bind to the entire protein but to a specifically targeted segment known as an epitope, often five or six amino acids in length.

A typical full-length protein sequence consists of multiple epitopes that antibodies can bind to and many different types of antibodies can recognize a protein. Each antibody is composed of a constant region found in all antibodies produced by a certain animal species as well as a variable region specific to certain epitopes.

Binding Action

When the immune system of an organism encounters an antigen for the first time, macrophages and dendritic cells capture the antigen, break it down, and present it to the B cell lymphocytes. This initiates the process of somatic hypermutation. The B cell codes for a new antibody that contains a unique antigen-binding site (a paratope) located at the tip of the variable region. 

Each B cell produces one specific antibody against one unique epitope. The paratope is capable of binding to unique epitopes from the antigen. After the antibodies are encoded with high enough specificity, the B cells release antibodies into the organism’s bloodstream. These antibodies bind with the antigen and allow the immune system to remove it from the body. 

This binding action allows antibodies to remove numerous types of pathogens. For example, antibodies disable viruses directly through binding. Antibodies eliminate bacterial pathogens by binding to the surface proteins on the bacteria and signaling to the immune system that the bacteria must be destroyed. After the immune system removes the antigen, the B cells remain in the animal’s bloodstream and are primed to produce more antibodies if the antigen reappears. When developing a custom antibody to bind with a specific antigen, an animal’s immune system will capture the protein, break it down into individual epitopes, and present these epitopes to the B cells to initiate the development of antibodies. Researchers can then collect the antibodies in serum or isolate the B cells for specific binding to the epitope.

Detecting and Amplifying Antibody Binding

When studying antibody binding, researchers can visualize antibody binding through direct or indirect methods of detection. Direct detection uses primary antibodies conjugated to a label, while indirect detection uses a labeled secondary antibody developed against the host species with the primary antibody. Direct detection methods ensure the secondary antibody will not unintentionally bind to other nonspecific epitopes. However, it can lack the sensitivity necessary to detect antibodies with lower expression levels. Direct detection may also negatively impact the primary antibody’s affinity. 

Indirect detection methods involve higher sensitivity and generate more intensely amplified signals. It is possible to further amplify a signal by using avidin (a glycoprotein in egg whites) or streptavidin (protein purified from a Streptomyces avidinii bacterium) to bind biotin. However, both amplification methods require taking additional steps to avoid nonspecific binding.

Why Is Antibody Binding Important?

Antibodies are valuable research tools because they make it possible to detect a protein in an assay without detecting unrelated proteins. Antibodies’ binding capability also allows researchers to use them in a wide range of diagnostic and therapeutic applications, including pregnancy tests and cancer treatment. 

Because antibodies can bind exclusively to regions of interest, they can be used for the detection and quantification of proteins through Western Blot or an ELISA test kit. With techniques like immunohistochemistry and immunoprecipitation, a protein can be localized within a cell and its tissue or isolated from a mixture of multiple proteins, respectively. 


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