Secondary antibodies, shown in Fig.1, can bind to the primary antibody in order for using in detection, sorting, and purification of target antigens. To enable detection, the secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used and is commonly conjugated.
Fig.1 Secondary antibodies.
They are similar in structure and similar classes/subclasses. About nomenclature, secondary antibodies are named according to their host, format, reactivity, target antigen, specificity, label and purification grade. Moreover, these should be similar to their primary antibodies. Compared to the primary antibody, secondary antibodies sometime need an extra purification process, known as pre-adsorption, to improve specificity and minimize non-specific. Through this process, non-specific antibodies among the solution will be removed and highly specific secondary antibodies are left. Primary antibodies can bind to the antigen directly, whereas secondary antibodies usually bind to primary antibodies by their Fc domain.
|Heavy Chains||IgG (gamma)||IgM (mu)||IgA (alpha)||IgD (delta)||IgE (epsilon)|
Table 1. Antibody classes and subclasses.
Applications of secondary antibodies mainly depend upon research objectives. Western Blot (WB), ELISA, immunofluorescence and flow cytometry (FC) are commonly required secondary antibodies. For immunofluorescence and FC, a conjugated secondary antibody is required.
A secondary antibody can be conjugated to enzymes, biotin, and fluorescent dyes/proteins. The conjugate choice depends on the experimental application and requirement.
|Immunoassay experiment||Secondary antibody label|
|Immunocytochemistry, IHC-Fr||Alexa Fluor, DyLight|
|Immunohistochemistry||Biotin, HRP or HRP-polymer|
|FC||Alexa Fluor, Phycoerythrin, FITC|
|WB||Enzymes - HRP or AP|
|ELISA||Enzymes - HRP or biotin|
Table 2. Conjugations of secondary antibodies.
Questions to Ask:
Should be same with the primary antibody.
|Ig Classes||IgG, IgM, IgA, IgE, IgD|
|Human Ig Subclasses||IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2|
|Mouse Ig Subclasses||IgG1, IgG2a, IgG2b, IgG3|
|Light Chains||Kappa, Lambda|
|Heavy Chains||IgG, IgM, IgA, IgD, IgE|
Table 3. Summary of immunoglobulin (Ig) classes and subclasses.
Most researchers select affinity isolated secondary antibodies because of the lowest amount of non-specific binding. When the antigen of interest is rare or present in low abundance, IgG fractions is recommended.
This part is described above and see Conjugations of Secondary Antibodies.
If using human tissues, secondary antibodies need to be pre-adsorbed.
If working with tissues or cells that contain Fc receptors, select a F(ab) or F(ab')2 fragment when possible to eliminate non-specific binding.
Creative Biolabs provides secondary antibodies with different conjugates to give our customers full flexibility in their experimental design. Besides produced antibodies, we also offer viroantibody discovery, engineering and customized services to help our customers achieve their research goals. If you are interested in these services and antibodies, please get in touch with us for more information.
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