A good signal detection is necessary for the reproducibility of research findings when a researcher performs protein immunoassays. One certain way to obtain good immunoassay results is by selecting a secondary antibody that matches your primary antibody.

But what are primary antibodies and secondary antibodies? How do they help in signal detection? In this article, we provide you with a brief introduction about the two types of antibodies commonly used for signal detection in immunoassays, the differences between these antibodies, and some valuable tips on how to choose a primary and secondary antibody.

In this article

What is a protein immunoassay?

What is an antibody?

What are primary antibodies and secondary antibodies?

What is the main difference between a primary antibody and a secondary antibody?

How are primary and secondary antibodies produced?

How do primary and secondary antibodies work in immunoassays?

How do secondary antibodies produce a visual signal?

How do secondary antibodies amplify a signal?

How to choose primary and secondary antibodies

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What is a protein immunoassay?

Immunoassays are methods that use antibodies to detect proteins in your experimental sample, measure their concentration, and study protein functions or localizations in a cell or tissue sample.

While here are several detection techniques that heavily reply on antibody detection, we will highlight two examples, enzyme-linked immunosorbent assays (ELISA) and Western blotting.

1.Enzyme-linked immunosorbent assay (ELISA)

In an ELISA, an antibody, known as a primary antibody, attaches to a target protein, which is immobilized on a solid surface, such as a well in a plate. A secondary antibody binds to this antibody-target protein complex, aiding in the detection of the target protein. This type of assays is useful for detecting a small amount of specific proteins.

2.Western blotting (WB)

Another method to detect a target protein is Western blotting. Similar to ELISA, the target protein is immobilized on a solid surface, a membrane in this case. The primary antibody detects the target protein and the addition of a secondary antibody detects the target protein.

What is an antibody?

An antibody is a protein component of immune responses against invaders, such as viruses or bacteria. After detecting foreign substances on the invaders, known as antigens, the immune responses generate an antibody or an immunoglobulin against these antigens to neutralize them.

An antibody consists of four polypeptides with two identical light chains and two identical heavy chains.

Heavy chain, light chain, antibody

The fragment antigen-binding, or the Fab fragment, is a region of the light chain binding to an antigen. Whereas the fragment crystallizable region, or the Fc fragment, is the tail region of the antibody structure attaching to cell surface.

Fab region, Fc region, antibody

There are some classes of immunoglobulins based on their heavy chain regions (Alberts et al., 2012, Janeway Jr et al., 2001):

Immunoglobulin G (IgG)

Immunoglobulin G, or IgG, has four chains with two identical 50 kDa γ heavy chains and 25 kDa κ or λ light chains. IgG is the most abundant antibody in human serum. Based on their abundance in the serum, there are four subclasses in IgG: IgG1, IgG2, IgG3, and IgG4.

Immunoglobulin D (IgD)

Immunoglobulin D, or IgD, has δ heavy chains. IgD is present on the surface of immature naive B cells. B cells are a type of immune cells.

Immunoglobulin E (IgE)

Immunoglobulin E, or IgE, has ε heavy chains. IgE is associated with allergic reaction.

Immunoglobulin A (IgA)

Immunoglobulin A, or IgA, has λ heavy chains. IgA is an antibody present in secretions, such as saliva, tears, milk, and respiratory and intestinal secretions.

Immunoglobulin M (IgM)

Immunoglobulin M, or IgM, has µ heavy chains. IgM is the first antibody produced in developing B cells after infection.

Immunoglobulin classes, Ig classes

What are primary antibodies and secondary antibodies?

The two types of antibodies typically used in many immunoassays are primary and secondary antibodies.

Primary antibodies: antibodies that bind specifically to an antigen.

Secondary antibodies: antibodies that bind to primary antibodies.

Primary antibodies, Secondary antibodies

Both primary and secondary antibodies can be either monoclonal or polyclonal antibodies. To learn more about the differences between monoclonal and polyclonal antibodies, find our article below:

An Overview of Polyclonal and Monoclonal Antibodies: Their Differences and How to Choose

What is the main difference between a primary antibody and a secondary antibody?

The difference between a primary antibody and a secondary antibody primarily lies on the binding to an antigen.

A primary antibody binds directly to a particular antigen. In this case, it would be the target protein in your experiment. A secondary antibody binds to the primary antibody instead of binding to the target antigen.

During antibody production, the target antigen and host species used to produce the primary antibodies are different from those used to make the secondary antibodies.

How are primary and secondary antibodies produced?

The production of primary anibodies requires one host species, such as a rabbit, mouse, goat, or chicken. In the first production step, the host will be immunized against an antigen to produce a primary antibody.

The production of secondary antibodies starts from the injection of a primary antibody from one particular host species into a completely different host species. For example, if the antibody generated is from a mouse, the secondary antibodies can be produced in a rabbit, a goat or a chicken.

Secondary antibody production

Secondary antibodies, which are produced against the whole antibody or IgG, recognize all IgG fragments. They recognize both the light and heavy chains. They are also relatively inexpensive and useful for many general immunoassays.

However, they can bind to the Fc regions of both primary antibodies and Fc receptors on cells. To increase the specificity and reduce cross-reactivity of a secondary antibody in your assays, you can use a particular antibody generated against Fc fragments, Fab fragments, or other fragments.

For example, secondary antibodies against Fab regions can be useful in Western blotting after immunoprecipitations. This type of antibody only recognizes Fab fragments, so they are much more specific than the ones raised against the whole IgG. As a result, using antibodies specific to Fab fragments avoids the detection of unwanted heavy chains with the size of 50 kDa on the blots, which prevents the visualization of the target protein with a similar size.

For detecting a target protein, an attachment of signal-producing molecules to either the primary or the secondary antibody is essential.

How do primary and secondary antibodies work in immunoassays?

You can visualize the binding of antigens to primary antibodies either by using a direct or indirect immunoassay.

In the direct immunoassay, a primary antibody attaches to a signal molecule. When a primary antibody binds to an antigen, this antigen-antibody interaction activates a signal molecule, allowing for detection.

In indirect immunoassays, secondary antibodies help with detection because these antibodies are the ones linking to a signal molecule. The way this method works: a secondary antibody recognizes a primary antibody binding to an antigen, then the formation of this complex activates the signal molecules.

Direct Immunoassay and indirect immunoassay

How do secondary antibodies produce a visual signal?

When using secondary antibodies, you’ll be performing the indirect immune assay. Here, secondary antibodies bind to antigen-bound primary antibodies. This interaction causes the signal molecules attached to secondary antibodies to emit a signal, or convert undetectable substrates into a visual signal on Western blots (Yu et al., 2015).

Below are some examples of signal molecules:


A method you can use for indirect detection is by using secondary antibodies labeled with radioisotopes, such as Iodine-125. This radioisotope emits gamma rays with low-energy photons, which are detectable by using X-ray film.


In Western blotting, secondary antibodies conjugated to an enzyme, such as horseradish peroxide, are commonly used. After the addition of the substrate for horseradish peroxide, the enzyme cleaves the colorless substrate and produces a visible colored signal on the blot.


Secondary antibodies can be labeled with a fluorescent dye, such as fluorescein isothiocyanate (FITC), R-phycoerythrin, rhodamine, and Texas red. The binding of antibodies excites the fluor at a specific wavelength and it will emit light at another wavelength.

Radioisotope, Enzymatic signal molecule, fluorochrome

How do secondary antibodies amplify a signal?

In the indirect immunoassay, more than one secondary antibody with signal molecule can bind to the primary antibody molecule. In this case, the use of secondary antibodies that bind to a signal molecule may result in a signal amplification (Yu et al., 2015). Hence, the indirect detection is much more sensitive than the direct immunoassay.

How to choose primary and secondary antibodies

  • Learn the details of the primary antibody. For example: the validation, the host species in which the antibody was produced, the compatibility with your immunoassays, the sensitivity, the cost, and the specificity to your target protein.
  • Choose a host species of the primary antibody that is different from the host species in which your samples were collected.
  • Make sure the host species in which the secondary antibody was produced is different from the one produced the primary antibody.
  • Find a secondary antibody that will recognize the primary antibody. For example, a mouse primary antibody needs an anti-mouse secondary antibody produced in another host species.
  • Choose a secondary antibody that matches the class or subclass of the primary antibody. If the primary monoclonal antibody is in the mouse IgM class, choose an anti-mouse IgM secondary antibody. When the immunoglobulin classes or subclasses of a primary antibody is unknown, you can choose an anti-mouse IgG secondary antibody.
  • Choose a secondary antibody with high specificity and less cross-reactivity with non-target antibodies.


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