Figure 1: Schematic representation of the mechanism of action of a secondary antibody.
Figure 2: Immunofluorescence analysis of U-2OS cells, using MATN3 antibody (CAB15072) as the primary antibody at dilution of 1:100. The cells were incubated with the primary antibody overnight at 4°C. Secondary antibody: FITC Goat Anti-Rabbit IgG (H+L) (CABS011).Blue: DAPI for nuclear staining.
|What are Secondary Antibodies?||Why are Secondary Antibodies Used?|
|Types of Secondary Antibodies||Top Secondary Antibodies|
What are secondary antibodies?
Primary antibodies are antibodies which bind to a specific target antigen or protein. Secondary antibodies, on the other hand, bind to the primary antibody targeting the antigen/protein of interest. Secondary antibodies bind to the heavy chains of primary antibodies; this ensures that there is no interference between the binding of the primary antibody and the target antigen/protein.
Secondary antibodies are used primarily for the detection and purification of target analytes. Secondary antibodies often have labels or tags added to their structure. Via the detection of such conjugates (or byproducts of such conjugates e.g. TMB product of HRP enzymatic reaction), information can be gathered about the protein of interest. For example, the concentration of a protein/proteins within a sample may be determined, a protein may be visualized microscopically, or a protein may be purified from a complex solution.
Why are secondary antibodies used?
There are two main reasons for the use of secondary antibodies. Firstly, secondary antibodies must only be specific to the species and isotype (most often IgG) of the primary antibody. Primary antibodies on the other hand must be specific against each individual antigen or protein it targets. For this reason, secondary antibodies are much more versatile than individually labelled primary antibodies.
Secondly, secondary antibodies aid in signal amplification. This is because several secondary antibodies can bind to one primary antibody. This greatly improves the sensitivity of laboratory experiments for which secondary antibodies are used.
Types of Secondary Antibodies
Secondary antibodies vary in structure depending on their species of origin, isotype, and conjugate.
As explained previously, for a secondary antibody to bind to a primary antibody, it must be specific to the species and isotype of the antibody. Secondary antibodies must be made in a species different than both those of the primary antibody or the specimen. This is to avoid cross-reactivity between the secondary antibody and other naturally-occurring antibodies within the sample.
For example, if a sample being tested has been obtained from a mouse, the primary antibody used must be from a species other than mouse e.g goat igG molecule. Additionally, if a secondary antibody is added to the sample, this antibody must be from a different species to the primary antibody and sample e.g a rabbit IgG molecule may be used.
Therefore, the type of secondary antibody chosen depends on the source of the sample and primary antibody.
The most common isotype of secondary antibodies is immunoglobulin G (IgG). This is because IgG molecules from different species are good at binding to one another. Subclasses of immunoglobulin molecules IgG include: IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4. Other immunoglobulin isotypes include IgA, IgD, IgE, and IgM. These are less commonly used as secondary antibodies, however when they are the same applies; only similar immunoglobulin molecules bind to one another.
Another variable of the secondary antibody structure is its conjugate. The type of conjugate chosen depends on the use of the secondary antibody. For example, for experiments in which the concentration of an antigen or protein must be determined (ELISA, Western blot), enzymatic tags may be used e.g. HRP and colour development is measured. The colour produced as a result of the enzymatic reaction is proportional to the amount of protein within the sample. Another type of commonly used class of conjugate are fluorescent conjugates. Fluorescent conjugates may be used in experiments such as immunofluorescence and immunohistochemistry to visualize proteins within samples or establish if a protein is present within a sample.
Common secondary antibody conjugates include:
- Horseradish peroxidase (HRP) - Enzyme
- Alkaline phosphatase (AP) - Enzyme
- Biotin - Enzyme
- FITC- Fluorophore
- Aminomethylcoumarin Acetate (AMCA) - Fluorophore
- Rhodamine - Fluorophore
- Cy3 - Fluorophore
- Enzyme-linked immunosorbent assay (ELISA)
- Western Blot
- Flow Cytometry
Top Secondary Antibodies
Cy3 Goat Anti-Rabbit IgG (H+L)
FITC Goat Anti-Rabbit IgG (H+L)
HRP Donkey Anti-Goat IgG (H+L)
HRP Goat Anti-Mouse IgG (H+L)
Rhodamine (TRITC) Goat Anti-Rabbit IgG (H+L)
HRP-conjugated Goat Anti-Rabbit IgG Heavy Chain
TRITC Goat Anti-Mouse IgG (H+L)
AMCA Goat Anti-Rabbit IgG (H+L)
Biotin Goat Anti-Rat IgG (H+L)
HRP Goat Anti-Mouse IgG1
HRP Goat Anti-Mouse IgG2a