A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications. Selection of the best secondary antibody can improve application results and reduce false positive or negative results. Thus, choosing suitable secondary antibodies is a key factor in research work. Since secondary antibodies target primary antibodies, we are going to introduce more about primary antibodies.
Primary antibodies are antibodies raised against an antigenic target of interest (a protein, peptide, carbohydrate, or other small molecule) and are typically unlabeled. Since primary antibodies are the immunogen for secondary antibody production, we should know more about the structure of the antibodies. Primary antibody structure could be defined in many ways; Abnova provides secondary antibodies against different structure and regions. Before choosing the correct secondary antibodies, there are several things you need to understand:
Host species of the primary antibody If you are using the primary antibody raised in mouse, you will need an anti-mouse secondary antibody.
Isotype of the primary antibody
Secondary antibody must be directed against the correct Ig isotype.
Typical secreted antibodies (immunoglobulins) have a basic four-peptide structure of two identical heavy chains (α, δ, ε, γ and μ) and two identical light chains (κ and λ) joined together by interchain disulfide bonds, forming a "Y"shaped molecule. The disulfide bonds are positioned within a flexible region called the hinge region, which separates the lobes of the antibody from one another and provides ample flexibility to bind antigens effectively. Following are isotypes of human immunoglobulin:
Besides looking at antibodies by their structure, the immunoglobulin can be broken down into regions by using enzyme treatment.
F(ab) x 2 +F(c)
Target region of the secondary antibody We have secondary antibodies available with specificity for whole Ig molecules (H & L) or antibody fragments such as the Fc or Fab regions.
Reacts with both the heavy and light chains of the IgG molecule, i.e. it reacts with both the Fc and F(ab')2 portions of IgG. It also reacts with other immunoglobulin classes (e.g. IgM, IgA, etc.) since all immunoglobulins share the same light chains (eitherκ or λ). This is a commonly used secondary antibody.
Reacts with the Fc portion of the IgG heavy chain. In each case, it has been adsorbed against F(ab')2 fragments. In some cases, the antibodies are additionally adsorbed to minimize possible cross-reactivity to IgM and IgA, or to IgM alone.
Reacts with the Fab portion of IgG. Since it reacts with light chains, it also reacts with other immunoglobulins sharing the same light chains. This antibody is used for specific applications such as double labeling methods or for tissues or cells that have Fc receptors (thymus, spleen).
Conjugation for downstream analysis The secondary antibodies can be conjugated with enzymes, fluorophores or biotin for further assay analysis. We summarized the common usage of different conjugations in the table below:
Peroxidase is economical, rapid and a more stable enzyme
Alkaline phosphatase is more sensitive than
Commonly used for double or multiple staining methods
It's used to amplify the signal and resulting in greater sensitivity than with an enzyme or fluorescence conjugated secondary antibody