Finding the right antibody for your protein immunoassays can be challenging. An antibody, which works well for an assay, may fail to work in another assay. Therefore, this article will provide you with helpful information about the differences between two widely used antibodies: monoclonal and polyclonal antibodies. In addition, we include some considerations before you choose an antibody.
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What is an antibody?An antibody is a protein element of the immune system in the blood, which recognizes invaders, such as bacteria and viruses. An antigen is foreign substances inducing the activation of immune responses, including antibody production. Whereas, an epitope is a part of an antigen to which an antibody binds.
In protein research, there are two popular types of antibodies: monoclonal antibodies and polyclonal antibodies.
What are monoclonal and polyclonal antibodies?
- Monoclonal antibodies is an identical population of antibodies targeting a single epitope of the antigen.
- Polyclonal antibodies is a heterogeneous population of antibodies targeting many epitopes within the same antigen .
What is the difference between monoclonal antibodies and polyclonal antibodies?
The main differences between these two types of antibodies:
- The population of antibodies in the monoclonal antibodies is homogenous.
- They recognize a single epitope of the antigen.
- The production of monoclonal antibody involves animals and hybridoma cells (cells originated from a fusion of spleen cells and myeloma cells).
- Population of polyclonal antibodies are heterogeneous.
- They recognize multiple epitopes in the antigen.
- The production of polyclonal antibodies only requires animals.
How are monoclonal and polyclonal antibodies produced?
The production of monoclonal antibodies starts by injecting an animal with an antigen. After several weeks, the spleen cells are harvested and fused with myeloma cells. Spleen cells produce antibodies, but they are unable to grow in culture. On the contrary, myeloma cells growing in culture are unable to produce antibody. After the fusion of both cells, the hybrid cells, also known as hybridoma cells, are selected and cultured to produce identical monoclonal antibodies.
Polyclonal antibodies are produced by injecting a particular antigen into an animal. After several days, the animal receives a booster of antigen injection to produce large amounts of antibodies against the antigen. Then, blood serum containing polyclonal antibodies are collected or purified.
What are advantages and disadvantages of monoclonal and polyclonal antibodies?
- High specificity to a single epitope
- Low risk of cross reactivity
- Less prone to batch-to-batch variability
- Prone to slight changes of antigen
- More time to produce
- Less prone to slight changes of antigen
- High sensitivity and affinity against the target antigen
- Quick to produce
- High risk of cross reactivity
- Prone to batch-to-batch variability
What are monoclonal and polyclonal antibodies used for?
Monoclonal antibodies have high specificity, so they are useful for some research applications requiring antibodies specific to a single epitope. Due to this reason, producing therapeutic antibodies is one attractive approach for drug development (Lu et al., 2020).
Monoclonal antibodies are also useful for quantifying the levels of a particular target protein; for example, in ELISA or radioimmunoassay.
They can also be used for Western Blotting (WB), immunohistochemistry (IHC), and immunofluorescence assay (IF). However, depending on the epitope, monoclonal antibodies can also be ineffective for these methods. A change in the structure of protein caused by chemicals, pH, or temperature, can affect the efficiency of the monoclonal antibodies.
Monoclonal antibodies produced against native proteins may recognize a conformational epitope, or an epitope containing discontinuous amino acid residues (Liang, 1998). As a result, these monoclonal antibodies may fail to recognize the epitope on the denatured state of the target proteins.
Discontinuous residues are amino acids far apart in primary sequences, but close to each other in the folded structures. In this case, choose a monoclonal antibody that can recognize continuous residues, or linear epitopes. Otherwise, consider to use a polyclonal antibody.
Polyclonal antibodies are useful for many general research applications and diagnostic assays, because of their high affinity towards the target antigen and their tolerance to minor changes of the target protein,
Applications for polyclonal antibodies include one or more of these following methods: Western Blotting, ELISA, immunohistochemistry, immunofluorescence assay, and immunoprecipitation (IP).
If produced against a denatured antigen, polyclonal antibodies recognize epitopes of the target protein in the denatured state. Treatments during assays, such as formalin fixation during immunohistochemistry, can change the molecules of the tissue to form crosslinks and mask the epitopes (Dapson, 2009). Therefore, a polyclonal antibody, which works well on Western Blots, may fail to recognize a target protein in immunohistochemistry.
Polyclonal antibodies can cause cross-reactivity and high background noise. Therefore, when performing your assays, it is essential to include good controls, such as a purified recombinant protein in Western Blots, as a reference.
Before choosing, match the antibody with the assay and investigate the details about it, including the specificity of an antibody.
One way to validate the antibody specificity is by using knockout (KO) validation. A specific antibody validated with this method usually produces no signal on the Western Blots for the knockout cell line, but yields a good signal with the expected size for the wild-type cell line.
How to Choose an Antibody
- Check the information about the type of antibody and the validation.
- Match the antibody with your protein assays based on the information about the applications.
- Choose monoclonal antibody when an assay requires high specificity.
- Choose polyclonal antibody for an assay requiring high sensitivity and affinity.
- Consider the cost of the antibody and the amount you’ll need for your assay. As an example, it may become too costly to use an expensive antibody and run a particular assay, such as an ELISA.
- Check the sensitivity of the antibody by finding the information about how much dilution you need in your assay.
- Make sure the additives, such as sodium azide or BSA, are compatible with the reagents you’ll use in your assay.
- Test the antibody with a recombinant target protein before using it with important samples.
- Include a positive and negative control in your protein assay.
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