Secondary Antibodies

Secondary antibodies, also called detector antibodies, are critical in immunological assays for detecting the presence of primary antibodies bound to target antigens. Appropriate selection of a secondary antibody, based on the species and class of the primary antibody and the intended detection method, enhances the sensitivity and specificity of the experiment. These antibodies are conjugated with detection molecules such as fluorescent dyes or enzymes to allow for visualization and quantification of the target antigen. Secondary antibodies are necessary in laboratory research for detecting primary antibodies and their associated target antigens.

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What is the difference between primary and secondary antibody?

Primary antibodies are specific proteins that recognize and bind to a target antigen. Secondary antibodies are non-specific antibodies that attach to the constant region of the primary antibody and are often conjugated with a detection molecule to facilitate detection in immunological assays. Secondary antibodies bind to multiple sites on the primary antibody in close proximity, forming a multi-valent complex that augments the assay signal.

How are secondary antibodies made?

Secondary antibodies are made by immunizing a host animal, such as a rabbit, goat, or mouse, with an antigen similar to the primary antibody used in the assay. The immune system produces antibodies that recognize and binds to the primary antibody. Protein A or G affinity columns are used to purify the antibodies from the serum. The purified antibodies are then conjugated with labels such as biotin, enzyme, or fluorescent molecule for detection. The primary antibody determines the specificity of the secondary antibody, while the assay determines the labeling technique. Host selection, purification strategy, and label conjugation are critical parameters that determine the sensitivity and specificity of the antibody.

Why use secondary antibodies?

Primary antibodies conjugated to a detection label, such as a fluorophore or enzyme, are a standard means of detecting target antigens. However, secondary antibodies offer several advantages over conjugated primaries. Secondary antibodies provide flexibility in choosing the detection label, regardless of the primary antibody used. For instance, a single primary antibody could be utilized with a secondary antibody conjugated to horseradish peroxidase (HRP) for Western blotting and with an alternative secondary antibody conjugated to a fluorophore for immunofluorescence. Such versatility in labeling options makes secondary antibodies valuable in many experimental applications. There are several reasons to use secondary antibodies:

1. Amplification- Secondary antibodies can provide a signal amplification step, allowing for the detection of low levels of primary antibody binding.
2. Prevents interference- Primary antibody binding site may be compromised when a reporter molecule is bonded to it; using a secondary antibody preserves primary antibody activity.
3. Cross-Reactivity- Secondary antibodies are often species-specific, allowing for the detection of primary antibodies derived from a specific species, such as a mouse or rabbit, without cross-reactivity with antibodies from other species.
4. Conjugate options- Primary antibodies have a limited range of conjugation available compared to secondary antibodies. Choosing a secondary antibody increases the flexibility of the experiment by providing multiple labeling options.

How to select your secondary antibody?

There are several factors to consider when selecting a secondary antibody.

1. Host species: The secondary antibody must be specific to the primary antibody. For example, a secondary antibody that recognizes rabbit immunoglobulins should be used if the primary antibody was produced in a rabbit.
2. Isotype: The secondary antibody must be specific to the isotype of the primary antibody. For example, if the primary antibody is an IgG antibody, a secondary antibody that recognizes IgG should be used.
3. Label: The secondary antibody can be labeled with detection labels such as enzymes, fluorophores, or biotin. The label should be compatible with the detection method being used.
4. Cross-reactivity: The secondary antibody should not cross-react with other proteins or molecules in the sample, which can lead to non-specific binding and false-positive results.
5. Sensitivity and specificity: The secondary antibody should have high sensitivity and specificity to the primary antibody to ensure accurate and reliable detection.