Secondary Antibodies

Enzymes, like Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), can conjugate to an antibody via covalent linkages. Enzyme conjugated antibodies are used in conjunction with a substrate, either fluorogenic, colorimetric, or chemiluminescent, to create a detectable signal. The non-detectable, soluble substrate is converted by the enzyme into a detectable (and normally insoluble) form. Enzyme conjugated antibodies enable signal amplification; as the enzyme is able to convert many molecules of the substrate, the length of time the reaction is allowed to continue determines the strength of the signal.

Horseradish Peroxidase

Horseradish peroxidase (HRP) is an enzyme derived from the root of Armoracia rusticana (a.k.a. the horseradish plant) that is commonly used as a reporter protein. HRP conjugated secondary antibodies can either be used for colorimetric detection, where the HRP catalyzes the conversion of a chromogenic substrate to a colored precipitate, or chemiluminescent detection, where the HRP produces a signal by oxidizing a chemiluminescent substrate to a form which emits light.

Multiple HRP molecules can bind to a single secondary antibody, as such, HRP conjugated secondary antibodies can be utilized for signal enhancement and enable detection of proteins expressed at low levels. HRP also has a high turnover rate which enables HRP conjugated secondary antibodies to generate a strong signal in a short amount of time (often within five minutes).

Alkaline Phosphatase

Alkaline phosphatase (AP) conjugated secondary antibodies are commonly used for ELISA and western blotting. They are also sometimes used for immunohistochemistry, however, their large size may limit penetration into tissues. It is worth noting, that whilst HRP generates a maximum signal quickly, often within five minutes, the signal from AP gradually increases and peaks at around an hour. AP generates a very stable signal which can last several days; this is particularly useful if multiple exposures are needed, especially over a matter of days.

Trying to decide between HRP and AP for your blot?

Use our table below to help with the decision.

Biotin

Biotin is a small, water-soluble B vitamin that can be bound by avidin, NeutrAvidin, and streptavidin with high affinity. Due to its small size, binding with biotin does not typically affect a protein’s biological activity. Multiple biotin molecules can conjugate to a single secondary antibody, as such, biotin conjugated secondary antibodies can be utilized for signal enhancement and enable detection of proteins expressed at low levels. Visualization happens through biotin / streptavidin interaction whereby the streptavidin is bound to either HRP or a fluorescent probe. By utilizing a biotin conjugated secondary antibody, the same secondary antibody can be used in multiple applications simply by switching the streptavidin used.

Fluorescent-dye conjugated secondary antibodies allow for a brighter signal and provide the ability to distinguish between multiple proteins in a single sample, as such, they are a valuable tool for identifying proteins in immunocytochemistry, western blotting, immunofluorescence, immunohistochemistry, and many more applications. Common fluorescent dyes include the Alexa Fluor range, AMCA, Cy3, FITC, PE, and TRITC.

Examples of fluorescent detection:

An antibody is a "Y-shaped" glycoprotein that is capable of binding to specific antigens. Each antibody is composed of four polypeptide chains, two identical heavy chains and two identical light chains, which vary in sequence and length between species and between isotype classes. An antibody's structure can be broken down into: two F(ab) regions, the top sections of the "Y" which contain the variable region which binds specifically to a particular epitope on the antigen; a hinge region; and an Fc region, the bottom of the "Y" which provides a binding site for endogenous Fc receptors (and secondary antibodies).

In mammals, antibodies are classified into five main classes or isotypes according to the heavy chain they contain. These are: IgA (alpha), IgD (delta), IgE (epsilon), IgG (gamma), and IgM (mu). Each class differs in the sequence of constant domains, the number of constant domains, the hinge structure, and the valency of the antibody. The light chains of an antibody are classified as either kappa or lambda based on their polypeptide sequence. Typically, the two light chains in an individual antibody are the same type.

Whole antibodies can be digested by papain or pepsin to form F(ab), F(ab’) and F(ab’)₂ fragment antibodies, which have no Fc portion or a significantly reduced Fc portion. The structures of F(ab), F(ab’) and F(ab’)₂ fragments differ: F(ab) fragments are single antigen-binding F(ab) portions, F(ab’) fragments are single F(ab) portions with the hinge region present, and F(ab')₂ fragments are two F(ab) portions linked via the hinge region. F(ab), F(ab’) and F(ab’)₂ fragment secondary antibodies are used to prevent the non-specific binding between the Fc region of an antibody and the Fc receptor on a cell.

Antibody Classes and Subclasses

The species of the secondary antibody is dependent on the host species of the primary antibody that you are using. Secondary antibodies are developed against an immunoglobulin class or subclass from a specific species. You need to select a secondary antibody that was raised in a different species against the host species and isotype of the primary antibody. For example, if you used a polyclonal primary antibody raised in goat, you will require an Anti-Goat IgG (Heavy & Light chains) secondary antibody created in a different species.

Affinity purified secondary antibodies undergo a process to remove non-class specific secondary antibodies; as a result, these secondary antibodies produce less background signal and are a popular choice amongst researchers. For example, a rabbit anti-mouse IgG2a secondary antibody would be affinity purified using immobilized mouse IgG2a to purify all rabbit antibodies that bind to mouse IgG2a. These anti-IgG2a antibodies may then be further purified by passage through chromatography column(s) containing mouse IgE, IgG1, IgG2b, IgG3, and IgG4 (etc.); this removes any antibodies that also cross-react with isotypes other than IgG2a.

F(ab), F(ab’) and F(ab’)₂ fragment antibodies are used to eliminate non-specific binding between the Fc region of an antibody and the Fc receptor on a cell. These fragment antibodies still bind to antigens but either have no Fc portion or a significantly reduced Fc portion (as a result of being digested by papain or pepsin). The structure of F(ab), F(ab’) and F(ab’)₂ fragments differ: F(ab) fragments are single antigen-binding F(ab) portions, F(ab’) fragments are single F(ab) portions with the hinge region present, and F(ab')₂ fragments are two F(ab) portions linked via the hinge region. It is a good idea to use a F(ab), F(ab’) or F(ab’)₂ fragment antibody when working with samples that express high levels of Fc receptors.

Pre-adsorption is used to increase the specificity of a secondary antibody in order to minimise any non-specific binding. If you are planning on staining with several primary antibodies and several secondary antibodies simultaneously, you should consider using pre-absorbed secondary antibodies. Pre-adsorption reduces the cross-reactivity between the secondary antibody and any endogenous immunoglobulins present in the sample.

In order to create pre-adsorbed secondary antibodies, secondary antibodies are passed through a matrix containing immobilised serum proteins from a range of species; any antibody that cross-reacts is caught by the matrix and removed from the sample. As a result, the pre-absorbed secondary antibodies only contain antibodies which specifically recognize the desired primary antibody.

A primary antibody directly binds to specific antigens, with high specificity and affinity, for the purposes of purifying or detecting and measuring the antigens. Primary antibodies can either be developed as monoclonal antibodies, which bind to one specific epitope on the antigen, or they can be polyclonal antibodies, which bind to multiple different epitopes on the antigen. Primary antibodies can either be supplied unconjugated or directly conjugated with an enzyme (like HRP or AP) or a fluorophore (like Cy3 or FITC) to enable visualization. Secondary antibodies are developed to bind specific isotypes of primary antibodies. If a primary antibody is unconjugated it cannot be visualized without the addition of a conjugated secondary antibody which binds to the primary antibody. The secondary antibody will be conjugated either to an enzyme or fluorophore to enable visualization.

A secondary-only control is where samples are incubated with PBS or antibody diluent, instead of the primary antibody. It is important that the rest of the protocol is identical to the other samples. This control helps to determine the specificity of the staining / signal observed. No staining in the secondary-only control implies that the observed staining in the other samples is specific and generated from the secondary antibody binding only the primary antibody. Considerable staining in the secondary-only control implies that the staining (for all samples) is non-specific and originates from the secondary antibody binding to endogenous immunoglobulins present in the tissue or to some other non-target antigens.

Secondary antibodies enable signal amplification which can improve the sensitivity of an assay and enable detection of proteins expressed at low levels. Signal amplification can either be the direct result of multiple secondary antibodies binding to a single primary antibody or the result of using an enzyme conjugated secondary antibody (i.e. the longer a reaction is allowed to continue, the greater the signal).

At a high concentration, some antibodies may form aggregates which can result in a speckled signal in immunostaining assays. Centrifuging the antibody vial at high speeds for between 10-15 minutes and carefully pipetting the antibody solution (it is important to be careful not to touch the bottom of the vial) can help remove any aggregates which may have formed. It is also worth double checking that the slides and coverslips are clean, as this can also give the appearance of speckling. If they look dirty under a microscope, give them a clean using 100% alcohol.

When performing a multi-label experiment, you should aim to only use secondary antibodies raised in the same species. This ensures that the secondary antibodies will only recognise the intended primary antibody and not other secondary antibodies.

The type of label you choose will depend upon the technique you are performing. For example, enzyme conjugated secondary antibodies (such as HRP or AP) are commonly used for ELISA and immunoblotting. HRP is both an economical and stable enzyme, however, AP can be more sensitive in certain situations (i.e. for colorimetric analysis).

For flow cytometry, immunohistochemistry, and immunofluorescence the ideal labels may either be enzymes or fluorochromes. DAB staining is effective as a single label or as a second color for the labelling of multiple antigens in IHC. Alternatively, a two-step biotin / avidin system may for used for signal enhancement and to enable detection of proteins expressed at low levels.

The structure of your primary antibody and secondary antibody may or may not be the same depending on the host, clonality, and isotype of the antibodies you select.

An antibody is a 'Y-shaped' glycoprotein that is capable of binding to specific antigens. Each antibody is composed of four polypeptide chains, two identical heavy chains and two identical light chains, which vary in sequence and length between species and between isotype classes. An antibody's structure can be broken down into: two F(ab) regions, the top sections of the 'Y' which contain the variable region which binds specifically to a particular epitope on the antigen; a hinge region; and an Fc region, the bottom of the 'Y' which provides a binding site for endogenous Fc receptors (and secondary antibodies).

In mammals, antibodies are classified into five main classes or isotypes according to the heavy chain they contain. These are: IgA (alpha), IgD (delta), IgE (epsilon), IgG (gamma), and IgM (mu). Each class differs in the sequence of constant domains, the number of constant domains, the hinge structure, and the valency of the antibody.

The light chains of an antibody are classified as either kappa or lambda based on their polypeptide sequence. Typically, the two light chains in an individual antibody are the same type. Therefore, the structural differences between primary and secondary antibodies depend on the species, isotype, clonality, and target specificity of the two antibodies.