The main difference between primary and secondary antibody is that primary antibodies bind to a specific antigen whereas secondary antibodies bind to primary antibodies.
Primary and secondary antibodies are two types of antibodies important in different immunoassays, including ELISA, western blot, and immunostaining.
Key Areas Covered
1. What is a Primary Antibody
– Definition, Structure, Importance
2. What is a Secondary Antibody
– Definition, Structure, Importance
3. Similarities Between Primary and Secondary Antibody
– Outline of Common Features
4. Difference Between Primary and Secondary Antibody
– Comparison of Key Differences
Key Terms
Primary Antibody, Secondary Antibody
What is a Primary Antibody
A primary antibody is the first antibody in immunoassays such as ELISA for the detection of the presence of a specific protein in the sample. Generally, the primary antibody can be a monoclonal or polyclonal antibody with the origin of the mouse, rat, rabbit, goat, and other animal species as hosts. The most important feature of a primary antibody is that it specifically binds to an antigen of interest in a particular protein in the sample.
Figure 1: Primary and Secondary Antibodies
Furthermore, the specific detection of a particular protein in a sample is important in different immunoassays including ELISA, immunopurification, western blotting, immunohistochemistry, and flow cytometry. It is also important in the diagnosis of diseases, therapeutic drug monitoring, and clinical pharmacokinetic and bioequivalence studies in the discovery of drugs and pharmaceutical industries.
What is Secondary Antibody
The secondary antibody is the second antibody in immunoassays. It binds to the primary antibody present in the sample. Therefore, the main function of the secondary antibody is to detect the primary antibody that binds to a specific antigen in the sample. This means it involves indirect detection of the target. The other significant feature of the secondary antibody is the presence of a detectable tag in the antibody itself.
Figure 2: ELISA
Moreover, secondary antibodies are conjugated to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), fluorescent dyes, and other molecules for the detection of their binding to the primary antibodies. The detection of the binding of the primary antibody to the specific protein is important for the detection of the presence of a particular protein in the sample.
Similarities Between Primary and Secondary Antibody
- Primary and secondary antibodies are two types of antibodies used in ELISA, western blot, and immunostaining.
- They are important to detect a specific antigen in a sample.
Difference Between Primary and Secondary Antibody
Definition
Primary antibody refers to an immunoglobulin that specifically binds to a particular protein or another biomolecule of research interest for the purpose of purifying or detecting and measuring it. Secondary antibody refers to polyclonal or monoclonal antibodies that bind to primary antibodies or antibody fragments, such as the Fc or Fab regions.
Bind to
The primary antibody binds to a specific antigen in the sample while the secondary antibody binds to the primary antibody.
Binding
Moreover, the primary antibody binds to the antigen of a protein while the secondary antibody binds to the Fc or Fab region of the primary antibody.
Importance
The primary antibody detects the presence of a specific antigen in the sample. While, the secondary antibody detects the presence of the primary antibody in the sample.
Addition
The primary antibody is the first antibody added to the sample while the secondary antibody is the second antibody added to the sample.
Detection
Besides, the primary antibody does not have a detectable tag while the secondary antibody contains a detectable tag.
Conclusion
In brief, primary antibody and secondary antibody are two types of antibodies used in different immunoassays including ELISA and western blot. The primary antibody is the first antibody added to the sample. Generally, it binds to a specific antigen of a protein present in the sample. Therefore, primary antibody is important for the detection of the presence of a particular antigen in the sample. The secondary antibody, on the other hand, is the second antibody added to the sample. It binds to the primary antibody. Therefore, the binding of the secondary antibody to the primary antibody detects the presence of the primary antibody in the sample. Hence, the main difference between primary antibody and secondary antibody is the type of binding and importance.
References:
- “Primary Antibodies.” Thermo Fisher Scientific – US,
- “Introduction to Secondary Antibodies.” Thermo Fisher Scientific – US,
Image Courtesy:
- “Primary-Secondaryantibody” By David Munch – Own work (CC BY-SA 3.0) via Commons Wikimedia
- “ELISA TMB.” By Ajpolino – Own Work (CC0) via Commons Wikimedia
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