Secondary antibody

Definition

A secondary antibody is an antibody that binds to a primary antibody to assist in detection, sorting, and purification processes. It is often conjugated with a marker, such as an enzyme or fluorescent dye, for visualization.

5 Must Know Facts For Your Next Test

1. Secondary antibodies are used in various immunoassays like ELISA and Western Blotting.
2. They are typically raised in a different species than the primary antibody to avoid cross-reactivity.
3. Conjugation of secondary antibodies with enzymes (e.g., HRP) allows for colorimetric detection.
4. Fluorescently-labeled secondary antibodies are used in Fluorescent Antibody Techniques.
5. Blocking steps are essential prior to applying secondary antibodies to prevent non-specific binding.

Review Questions

• What role does a secondary antibody play in an ELISA?
• Why are secondary antibodies usually raised in a different species than the primary antibody?
• How does the conjugation of secondary antibodies with enzymes facilitate detection?

Related terms

Primary Antibody: An antibody that specifically binds to an antigen of interest.

Enzyme-Linked Immunosorbent Assay (ELISA): A plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones.

Fluorophore: A fluorescent chemical compound that can re-emit light upon light excitation; often conjugated to antibodies for fluorescent labeling.