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Location: Home > Information Center > Technical FAQs > Antibody Technology Column > How to select a secondary antibody?

How to select a secondary antibody?

Date: 2015-04-21 Author: Leading Biology Click: 5641


The secondary antibody is an antibody prepared in another host that binds to the primary antibody or the fragment of the primary antibody. The above-mentioned enzyme or fluorescein is usually attached as the label. Due to the advantages of the secondary antibody, it is widely used in immunological experiments, such as western blot (identification of proteins by binding to specific antibodies), ELISA (detection of specific proteins, especially corresponding antigens or antibodies by labeling enzyme-conjugated antibodies or antigens), immunohistochemistry ( detection of specific antigens in tissues), immunocytochemistry (detection of antigenic composition of cells by immunological methods), flow cytometry (detection of fluorescence by excitation of lasers to identify different types of cells) and immunoprecipitation (by antigen and Specific binding of antibodies to isolate the corresponding antigen).

The secondary antibody is specific for antibodies of particular species (such as mice), so the use of labeled secondary antibodies eliminates the need to label each primary antibody, which saves time and money. In addition, a primary antibody molecule can be combined with several secondary antibodies at the same time, which greatly enhances the signal and improves the sensitivity of the experiment. The secondary antibody can be coupled to several different labels, which can be enzymes, fluorescein, or biotin.

How to choose a secondary antibody? 

At normal circumstances, there may be several secondary antibodies available for selection in a particular experiment. How to choose the secondary antibody that is suitable for the experiment most? The following aspects need to be considered. 

1. The source of the primary antibody

The secondary antibody should be of the same species source as the primary antibody. For example, if your primary antibody is a mouse-derived monoclonal antibody, the secondary antibody is selected as an anti-mouse secondary antibody (goat anti-mouse or rabbit anti-mouse). If the primary antibody is a rabbit-derived polyclonal antibody prepared from rabbit serum, the corresponding secondary antibody needs to select a secondary antibody against rabbit. That is, the corresponding secondary antibody against the species is selected according to the species source of the primary antibody.

2. The class or subclass of the primary antibody 

In addition to being consistent with the source of the primary antibody, the secondary antibody also needs to match the class or subclass of the primary antibody, which usually applies to monoclonal antibodies. Polyclonal antibodies are generally IgG-like immunoglobulins, so the corresponding secondary antibody is the anti-IgG antibody. Monoclonal antibodies are relatively complex. Because monoclonal antibodies have different classes and subtypes, the corresponding secondary antibodies also need to be selected based on these subtypes.

The classes and subclasses of monoclonal antibodies are usually described in the product manual. If your primary antibody is mouse IgM, then the corresponding secondary antibody should be an anti-mouse IgG antibody. If the monoclonal primary antibody is a subclass of mouse IgG (IgG1, IgG2a, IgG2b, IgG3), then almost all anti-mouse IgG can be combined with it, or you can choose to specifically target this subclass. Secondary antibodies, for example, if your primary antibody is mouse IgG1, then you can choose a secondary antibody against IgG1, which is especially suitable for double-labeling experiments. In cases where the primary antibody is not known for its type/subclass, IgG against the corresponding species can be used because such antibodies recognize most types of IgG immunoglobulins.

3. The source of the secondary antibody

In general, there is no inevitable connection between the source of different species and the quality of the secondary antibody. The secondary antibody derived from goats and the secondary antibody derived from sputum are not much different in general experiments. However, in some special experiments, such as double-label experiments, if one of the primary antibodies is of goat origin and one is of mouse origin, the corresponding secondary antibodies are resistant to goat and anti-mouse secondary antibodies, respectively. The secondary antibody cannot be selected from goat or mouse sources. Choosing the corresponding secondary antibody from the sputum source is very suitable for a double-labeled immunoassay. Two of the two markers were resistant, so the antibody from the third source was selected.

4. Which form of secondary antibody is selected, the entire IgG molecule, the F(ab')2 fragment, or the Fab fragment?

Whole IgG molecule: This antibody is suitable for most situations.

F(ab')2 fragment: This antibody is composed of two Fab fragments linked by disulfide bonds for binding antigens. These antibodies are used in specific cases, such as the experiment that  need to avoid antibodies and cells with Fc receptors combining.

Fab fragments: They have only one binding site and are generally used to block endogenous immunoglobulins.

5. Coupling mark of secondary antibody

In general, the probes coupled to the secondary antibody are mainly enzymes (horseradish peroxidase HRP and alkaline phosphatase AP or its derivatives APAAP, PAP), fluorescent groups (FITC, RRX, TR, PE) and biotin. Which secondary antibody with a different probe is selected depends on the specific experiment. For western blot and ELISA, the most commonly used secondary antibody is the enzyme-labeled secondary antibody, while fluorophore-labeled secondary antibodies are commonly used in cell or tissue labeling assays (cyto-immunochemistry, tissue immunochemistry, flow cytometry), immunological groups. Horseradish peroxidase or alkaline phosphatase-labeled secondary antibodies can also be used in the treatment. If you want to amplify the detection signal to a greater extent, the biotin/avidin detection system can be selected. In some fluorescence detection schemes, different fluorescent labels need to be selected; while gold particle labeled secondary antibodies are more commonly used in immunoelectron microscopy.

6. Purity of the antibody

Affinity-chromatized antibodies are generally more popular because they have the highest purity and the least non-specific background. However, in some cases, the lost IgG during purification have high affinity, so it should be considered, especially when the amount of antigen of interest is small, the high-affinity IgG moiety is particularly important.

Summary of several secondary antibodies with different specificities

Specific for the entire antibody molecule (H+L)Such as anti-IgG (H + L), such antibodies can bind to the heavy chain and the light chain of the antibody, that is, react with the Fc, F (ab') 2 / Fab part of the antibody molecule, anti-IgG (H+L) can also react with other immunoglobulin families (such as IgM and IgA) because all immunoglobulins have the same light chain (kappa chain or lambda chain).

Specific for Fab fragmentsSuch antibodies can bind to heavy chain light chains, and since they can react with light chains, they can also react with other types of immunoglobulins having the same light chain.

Specific for Fc fragments or heavy chainsSuch antibodies react with the Fc portion of the heavy chain, and thus they are class-specific. That is, the gamma chain-specific antibody reacts only with IgG, the mu chain-specific antibody recognizes only IgM, and so on.

Light chain (lambda, kappa) specificityReacts with all classes of antibodies because all classes of antibodies have the same lambda chain or kappa chain

Adsorbed secondary antibodyImmunoglobulins from different sources contain similar structures, and antibodies against one species may cross-react with other species, so some secondary antibodies are treated with animal or human IgG to reduce non-specific background. For example, in the case of mouse tissues or cells, the selection of secondary antibodies that have been treated with mouse serum or IgG adsorption can reduce non-specific binding. However, the number of such antibody recognition epitopes is greatly reduced.

Advantages and Disadvantages of Secondary Antibody Probes and Their Applicable

EnzymeThere are two main enzyme couplings, HRP and AP. In comparison, the former is more economical, faster, and more stable, while the latter is more sensitive than the former. Usually, HRP is widely used in immunohistochemistry, western blot, and ELISA, and alkaline phosphatase (AP) is more suitable for solid. Phase immunoassay experiments such as ELISA and western blot.

Fluorescent groupFITC is a widely used fluorophore, but its biggest disadvantage is its fast quenching the utilize of the anti-quenching agents will reduce the effect.

AMCA: commonly used in multi-label experiments, such as immunofluorescence, flow cytometry. The disadvantage is fast quenching, so the AMCA-coupled secondary antibody is more suitable for detecting a large amount of antigen present because of the hard detection of the blue fluorescence by the naked eye. 

Cyanine dyes (Cy2, Cy3, Cy5): Cy2 is more stable than FITC, and Cy2 fluorescence is stronger under surface fluorescence microscopy. Cy3 and Cy5 are brighter, more stable, and have a weaker background than most fluorophores. Both are often multi-labeled in confocal microscopy. However, the disadvantage of Cy5 is that it cannot be observed with a surface fluorescence microscope and is therefore usually observed with a confocal microscope with a far-infrared detector.

TRITC, RRX, TR: TRITC is often used in conjunction with FITC for dual-labeling experiments, and RRX or TR can also be used, but TR produces a brighter background. RRX is especially useful when using a laser confocal scanning microscope equipped with a hydrogen lamp for tri-labeling experiments. It can be used with Cy2 (or FITC) and Cy5 because the emission wavelength of RRX is between Cy2 and Cy5, and there is little overlap.

PE: often used in conjunction with FITC for flow cytometry double-labeling experiments.

Biotin: Biotin can be tightly bound together with an antibiotic (avidin) irreversible reaction. First, a biotin-labeled secondary antibody is added, and then avidin, ExtrAvidin, or streptavidin coupled to an enzyme or a fluorescent group is added, and the latter can bind to a plurality of sites on the surface of the secondary antibody, therefore the detection signal can be greatly amplified.

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