Monoclonal antibody and polyclonal antibody: monoclonal antibody is produced by a single clone of cells and recognizes a single epitope; while polyclonal antibodies are a mixture of antibodies that are derived from different cells, and recognize diverse epitopes.

Antibody structure:As shown in figure 1, each antibody consists of four polypeptides-two heavy chains and two light chains joined to form a “Y” shaped molecule. The amino acid sequence in the tips of the “Y” varies greatly among different antibodies (variable region), giving the antibody its specificity for binding antigen. The rest of the antibody is the constant region.

There are two types of light chains: lambda and kappa. Only one type is present in each antibody.

Secondary antibodies: Secondary antibodies are antibodies that bind to primary antibodies or antibody fragments. They are usually labeled with conjugates such as enzyme or fluorescein. They are widely used in immunodetection, immunopurification, or immunoisolation experiments, for example, western blot, ELISA (a sensitive immunoassay using an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody), immunohistochemistry (an assay that shows specific antigens in tissues), immunocytochemistry (the study of cell constituents by immunologic methods), flow cytometry (a technique used to identify and separate different types of cells based on detecting and measuring the fluorescence emitted with a laser light beam), and immunoprecipitation (the precipitation of antigen as the result of the interaction of antigen with a specific antibody in solution).

Several kinds of moeites can be conjugated to secondary antibodies, such as enzyme, fluorescein, and biotin.

The interactions between different types of secondary antibodies and primary antibodies are shown in figure2 and figure 3.

In most cases, several secondary antibodies work well for a particular application. Then, how can we find the best one? Here is some helpful information:

What species of animal was the primary antibody produced from?

The secondary antibody should be directed against the species in which the primary antibody was raised. For example, if the primary antibody was raised in mouse, an anti-mouse secondary antibody should be used. And if the primary antibody is raised in rabbit, an anti-rabbit secondary antibody should be used. As for the species of secondary antibody, there has been no evidence of species-specific difference in the quality of secondary antibodies.

Which class and/or subclass of the primary antibody?

The secondary antibody should match the class or subclass of the primary antibody. This is particularly important for monoclonal antibodies. Polyclonal antibodies are typically IgG class immunoglobulins, and the secondary antibodies are mainly anti-IgG.

If the primary monoclonal antibody is one of the mouse IgG subclasses (IgG1，IgG2a，IgG2b，IgG3), almost anti anti-mouse IgG can bind to it. Alternatively, a secondary antibody specific to this subclass can be used, for example, if the primary antibody is mouse IgG1, anti-mouse IgG1 secondary antibody should be selected. The point is particularly important for double labeling application.

If the class and/or subclass of the primary antibody are not known, the anti-mouse IgG may be used since they recognize most mouse IgG subtypes.

The following summarizes different specificities of antibodies:

• Whole molecule (H+L) specific: for example, anti-IgG (H+L). This antibody reacts with both the heavy and light chains. That is, it reacts with both the Fc and F(ab’)₂ portions (see Figure 5). Anti-IgG(H+L)can also react with other classes of immunoglobulin classes such as IgM and IgA, since all immunoglobulins share the same light chains (kappa or lambda).
• Fab fragment specific: this antibody reacts with only the Fab portion. Due to its reactivity with light chains, it can also react with other immunoglobulins with the same light chains.
• Fc or heavy chain specific: this antibody reacts with the Fc portion of heavy chains only, therefore, they are class specific (i.e. gamma chain specific reacts only with IgG, mu chain specific reacts with IgM only, etc.).
• Light chain (kappa, lambda) specific: this antibody can react with all classes, since they share the same kappa or lambda light chains.
• Adsorbed antibodies: antibodies against one secies may cross-react with other species, therefore, some secondary antibodies have been adsorbed with animal or human IgG to reduce non-specific background. For example, if working with mouse tissues or cells, you can choose a secondary antibody that has been adsorbed with mouse serum or IgG. However, their epitope recognition has been greatly reduced and they are recommended only when the possible presence of immunoglobulins from other species may interfere with correct labeling.

Which form of antibody to choose, the whole IgG, F (ab’) ₂ fragments, or Fab fragment?

• Whole IgG molecules: this is a commonly used antibody.
• F (ab’) ₂ fragments: it is composed of two Fab fragments joined by a disulfide bond, and can be used in applications where non-specific background caused by binding of the secondary antibody to Fc receptors on cell surfaces should be avoided.
• Fab fragments: they have only one binding site, and are usually used to block endogenous immunoglobulins.

Which kind of probe will be appropriate?

Generally, secondary antibodies can be labeled with enzyme (peroxidase, alkaline phosphotase or their derivatives APAAP or PAP), fluorochromes (FITC, AMCA, Cy2, Cy3, Cy5, TRITC, RRX, TR, PE), or biotin. The choice of the conjugate is very application-dependent. For immunoblotting and ELISA, enzyme-labeled secondary antibodies are the most popular. For cell or tissue staining (immunocytochemistry, immunohistochemistry and flow cytometry), fluorochrome-labeled antibodies are commonly used, and in immunohistochemistry, enzyme-labeled antibody can also be used. A two-step biotin/avidin system can also be used to increase the detection sensivity. The following describe briefly the advantages and disadvantages of several probes, as well as the applications they are suitable for.

• Enzyme: There are two different enzyme conjugates, HRP and AP. The former is economical, rapid and a more stable enzyme, and is applicable for immunohistochemistry, western blot and ELISA, while the latter is considered more sensitive and is suitable for solid-phase immunodetection such as ELISA and western blot.
• Fluorochromes:
• FITC: it is a widely used fluorophore, but its major disadvantage is rapid fading, which can be mitigated in the presence of an anti-fading reagent.
• AMCA: it is used mostly in multiple labeling, such as immunofluorecence and flow cytometry. Its disadvantage is also rapid fading and anti-fading reagent should be used, in addition, since the blue fuorescence is not well detected by the human eye, AMCA-labeled secondary antibodies should be used to detect abundant antigens in multiple labeling experiments.
• Cyanine dyes (Cy2, Cy3, and Cy5): Cy2 is more stable than FITC, and appears brighter than FITC in an epifluorescence microscope. Cy3 and Cy5 are brighter, more stable and cause less background than most other fluorophores, and they are often paired with each other for multiple labeling in confocal microscopy. However, Cy5 can not be detected with a conventional epifluorescence microscope. It is most commonly visualized with a confcal microscope equipped with an appropriate laser for excitation and a far-red detector.
• TRITC, RRX, and TR: TRITC is often used together with FITC in double labeling, RRX or TR can also be used, but TR may lead to higher background staining.

  RRX is particularly useful for triple labeling with Cy2 (or FITC) and Cy5 using a confocal laser scanning microscope equipped with a krypton/argon laser, because fluorescence from RRX lies about midway between that of Cy2 and Cy5, and shows little overlap with either.

• PE: PE-conjugated secondary antibodies are often used for double labeling with FITC in flow cytometry.
• Biotin: biotin binds to avidin with high affinity via an irreversible interaction. A biotinylated secondary antibody is applied first, then avidin, ExtrAvidin or streptavidin conjugated to an enzyme or fluorochrome binds to multiple sites on the biotinylated secondary antibody, thus greatly amplifying the signal (see figure 4).

Purity of the secondary antibody Affinity purified antibodies are popular to most people since they are the most purified and give the lowest amount of non-specific binding. However, in certain cases, IgG fractions should be considered because there are so many antibodies with high affinity, and they may be useful especially in situations that the antigen of interest is rare or present in low abundance.