Secondary Antibody: Overview & Definition

Secondary Antibody: Overview & Definition

Overview of Secondary Antibodies

Antibodies (also known as immunoglobulin) play a crucial role in life science, research, and diagnostic centers to diagnose diseases and develop targeted drugs & treatments for many life-threatening diseases. The molecule has applications in a range of workflows, such as western blotting, ELISA, flow cytometry, and immunological assays.

These applications mainly involve two types of antibodies: Primary and Secondary antibodies.

Primary antibodies are applied before secondary antibodies in any workflow. They bind at a single epitope (to the Fab fragment or domain) of the target protein or antigen. They are of two types: monoclonal antibodies and polyclonal antibodies.

The secondary antibody is mainly used for labeling purposes. They are added to the surface or membrane, where they bind to the epitope (at the Fc fragment through the Fab domain) of the primary antibody.

Figure: An illustrative diagram of the binding of primary antibody to the antigen and labeled secondary antibody.

The secondary antibodies are conjugated to a range of fluorescent dyes (fluorophores or fluorochrome), enzymes, radioisotopes, and small molecules that help in the visualization of the target molecule.

A few examples of such conjugates are biotin (binds with avidin and streptavidin), horseradish peroxidase (HRP), glucose oxidase,  fluorescein isothiocyanate (FITC), alkaline phosphatase (AP), Alexa Fluor, and β-galactosidase.

However, unconjugated forms of secondary antibodies are also commercially available for specific lab workflows.

In this article, we will review different isotypes of antibodies, their working mechanism, preparation, and applications in different lab workflows.

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How Do Secondary Antibodies Work?

The fluorophore attached to secondary antibodies helps in studying the localization and distribution of the protein of interest or target antigen using the fluorescent microscopy technique.

And, based on the attachment of a label to the primary or secondary antibody, the detection method is of two types: direct and indirect detection technique.

Direct Detection

Direct detection only requires one labeling step. Here, the labeled primary antibody is added to the membrane, and the protein is detected only after a single incubation step. It reduces the cross-reactivity between different species (other than the target).  However, this method is less sensitive, and has poor reproducibility and increased background.

Indirect Detection

Indirect detection involves two incubation steps. Here, the membrane/surface is first incubated with the primary antibody that binds to the target protein. Then, secondary antibodies bind to the primary antibodies.

The secondary antibody used in the workflow must have specificity for the antibody host species and the isotype of the primary antibody.

Because of the involvement of both primary and secondary antibodies and the fact that multiple secondary antibodies bind to a single primary antibody, the indirect method helps increase the signal amplification and sensitivity of an assay.

Figure: An illustration of the difference between direct and indirect immunoassays.

Classes of Secondary Antibodies and Choosing the Right One

Commercially, more than 90 different types of secondary antibodies are available for R&D purposes. They are specifically designed to suit a variety of workflows.

Let’s take an example.

The polyclonal primary antibodies are generally IgG isotype and raised in host animals, such as goats, sheep, rabbits, and donkeys. Thus, an anti-IgG H&L (Heavy & Light chains) antibody will be the preferable antibody class for those workflows.

Similarly, monoclonal antibodies are raised in animal species like rabbits, mice, and rats. Thus, if you choose a mouse IgG1 monoclonal primary antibody for your workflow, an anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG will be required for the assay.

The five classes of secondary antibodies include IgG, IgM, IgD, IgE, and IgA antibodies. A series of subclasses of these antibodies are available to carry out specific lab procedures.

Selecting The Right Secondary Antibody

Secondary antibodies are raised in different animal species and conjugated with different enzymes and fluorophores. Thus, it’s essential to understand which antibody will be suitable for your experiment.

Figure: An illustrative diagram of the production of secondary antibodies in different hosts.

Here’s an example for better understanding.

If a goat is immunized with the mouse IgG, it will produce goat anti-mouse IgG antibodies, which will bind to all fragments and classes of IgG. Whereas, if the goat is immunized with IgG1 antibody, it will only generate mouse IgG1 specific antibodies.

Thus, a spectrum of antibodies raised in different host species is available for the lab workflows, such as anti-human IgG, anti-rabbit IgG, anti-rat IgG, and anti-bovine IgG.

The above-mentioned examples are just to name a few. The varieties of commercially available antibodies number is in the hundreds. So, how can one decide which antibody should they buy?

Here’re some factors to look for:

  • Host and Target Species: Consider in which host animal species the antibody is generated. It should be different from the host of primary antibodies and target species.
  • Targeted Reactivity: Ensure that the antibody binds with the target primary antibody, which is based on the target species and class and subclass of the antibodies.
  • Multiplexing: The use of distinct dyes to visualize a target protein is called multiplexing. Choose antibodies that are labeled with conjugates not containing overlapping emission spectra.
  • Antibody class and subclass: A range of classes and subclasses of antibodies are available for different purposes. Thus, ensure you choose antibodies suiting your workflow.
  • Cross-adsorption: Cross-adsorbed antibodies help in eliminating cross-reactivity or non-specific binding and background noise. Thus, ensure the antibodies are cross-adsorbed against a range of species.
  • Conjugates: The conjugate choice is based on the procedure of antibody detection and application. Thus, ensure the antibody you purchase supports running your experiment.
  • Whole antibodies vs. fragment formats: The secondary antibodies are found in the fragment or whole antibody formats. An example is F(ab’)2 fragments that are used with samples containing Fc receptors.

What Are Secondary Antibodies Used For?

Secondary antibodies have many applications in molecular biology and life science labs. Below are lab workflows that extensively involve the use of the product.

Western Blot

Western blot is an analytical technique used to identify and measure the protein of interest. Enzyme conjugated (HRP or AP) and fluorescently labeled secondary antibodies are commonly used antibodies in this workflow.


ELISA is a biomolecular technique used to measure and detect the antigens, such as hormones, peptides, and peptides, in the provided samples. The streptavidin-HRP (biotinylated secondary) antibody is the most preferred antibody for the assay compared to conventional enzyme-labeled secondary antibodies.

Immunological Assays

The immunological assays are used to better understand the immune response of the organisms. The types of the assay include:

  • Immunocytochemistry (ICC)
  • Immunoprecipitation
  • Immunohistochemistry (IHC)
  • Immunofluorescence

For such assays, enzyme-conjugated, biotinylated, or fluorescently labeled secondary antibodies are the most commonly preferred.  

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Secondary antibodies are one of the essential reagents used in the life sciences labs that facilitate workflows like western blotting, immunoassays, and ELISA.

They are generally conjugated with fluorochromes, enzymes, or small molecules that facilitate the visualization of the target protein. A few examples of such conjugates are fluorescein isothiocyanate (FITC), alkaline phosphatase, Alexa Flor, and horseradish peroxidase (HRP).

A spectrum of secondary antibodies is available in the market. Thus, it’s essential to consider factors like conjugates, host species, and antibody class and subclass, before purchasing the product.

Furthermore, a premium quality antibody with next-generation equipment boosts the reproducibility and reliability of your data. However, acquiring equipment makes the procedure too expensive.

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