Secondary Antibodies

Antibodies (by target):
I K L

What is Secondary Antibodies?

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.

Secondary antibodies. Figure.1 Secondary antibodies.

Similarity and Difference of Primary and Secondary Antibodies

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.

Secondary Antibody Classes and Subclasses

Immunoglobulin Classes IgA IgD IgE IgG IgM  
Ig Subclasses-Human IgG1 IgG2 IgG3 IgG4 IgA1 IgA2
Ig Subclasses-Mouse IgG1 IgG2a IgG2b IgG3    
Light Chains Kappa Lamba        
Heavy Chains IgG (gamma) IgM (mu) IgA (alpha) IgD (delta) IgE (epsilon)  

Table.1 Antibody classes and subclasses.

Applications of Secondary Antibodies

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.

Conjugations of Secondary Antibodies

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.

How to Choose a Secondary Antibodies?

Questions to Ask:

  • What species was the primary antibody developed?
  • Should be same with the primary antibody.

  • What is the class (isotype) and/or subclass of 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.

  • Choosing an affinity purified antibody or an IgG fraction?
  • 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.

  • What kind of label/conjugations do I choose?
  • This part is described above and see Conjugations of Secondary Antibodies.

  • Do I need a pre-adsorbed secondary antibody?
  • If using human tissues, secondary antibodies need to be pre-adsorbed.

  • Using a F(ab) or F(ab')2 fragment antibody?
  • 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.

All products and services are intended for Research Use Only, and NOT to be used in diagnostic or therapeutic procedures.

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