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Primary Antibody vs. Secondary Antibody, How to Choose?

 Antibodies (or immunoglobulins) are Y-shaped glycoproteins released by the immune system to detect and bind to "foreign" substances, known as "antigens", triggering a cascade of actions to destroy these foreign invaders. Scientists would develop different antibodies for research use, which basically can be classified into primary and secondary antibodies. However, how to choose the right type of antibodies for different immunoassays in experiment procedures, such as western blotting (WB), enzyme-linked immunosorbent assays (ELISA), and immunoprecipitation (IP), is one of the most common questions for green hands in the laboratory.

 

Primary Antibody vs. Secondary Antibody

 

Antibodies could be categorized into two types based on the binding capability and experimental applications—primary antibodies and secondary antibodies.

 

The main difference between these two types of antibodies is that primary antibody binds specifically to an antigen, like a protein or a biomolecule of interest, while a secondary antibody bind to the primary antibody that has attached with a target antigen. Compared to secondary antibodies, primary antibodies are produced from different host species like animal species of rats, mice, goats, and rabbits, though they could both be produced in form of monoclonal (mAbs) or polyclonal antibodies (pAbs).

 

Selection of Antibodies

 

In laboratory research and clinical trials, choosing right antibody reagents is essential for correct results, which often means more accurate and reproducible study results and less waste on valuable samples and funds. The selection of antibodies should be based on these factors:

 

l Applications

 

Primary antibodies are usually used in immunoassays to bind to the target antigen, including WB and cell staining, but a secondary antibody is not necessarily used. For instance, direct ELISA involves direct labeling, requiring the primary antibody to be directly labeled with an enzyme for enzymatic reaction and signal detection, in which secondary antibodies are not needed. The primary antibody is also applied in indirect ELISA but the enzyme is labeled on the secondary antibody and the secondary antibody bind to the primary antibody. Though with a relatively complex protocol and cross-reactivity risk, secondary antibodies are more sensitive and flexible in labeling and detection, which results in their usage in the colorimetric, chemiluminescent, and fluorescent detection of primary antibodies. Applications of secondary antibodies include flow cytometry, western blotting, and cell imaging, while primary antibodies are used in the detection of biomarkers for diabetes, cancer, Alzheimer's, and Parkinson's disease.

 

l Source of Antibodies

 

The species that the primary antibody is raised from is another factor researchers should take into consideration. The host species of primary antibodies should be different from the species of the sample, in case that the secondary antibody cross-react with endogenous immunoglobulins in the sample. For example, research on mouse protein should use a primary antibody that is not produced by mouse, such as a primary antibody raised in rabbit, and the followed secondary antibody should be against the host species of the primary antibody, here using anti-rabbit IgG secondary antibody.

 

l Clonality

 

As antibodies could be mAbs and pAbs, clonality is an additional characteristic to consider when researchers are choosing primary and secondary antibodies for research. MAbs only target one epitope of an antigen as they are produced from a single B-cell clone while pAbs that are produced from multiple B-cell clones of an animal can recognize multiple epitopes of an antigen.

 

Choosing monoclonal or polyclonal antibodies, which both have advantages and disadvantages, depends on experiment requirements. Monoclonal antibodies allow precision targeting due to high specificity and high reproducibility, but the higher expense and less possibility of working across different species are negative factors that can't be ignored. On the contrary, polyclonal antibodies are featured with higher affinity and the ability to detect and tolerate variations in the antigen and are widely available at a lower cost, but the risk of cross-reactivity is increased along with the presence of multiple epitopes.

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