Factors to Consider When Selecting a Secondary Antibody for ELISA

An enzyme-linked immunosorbent assay (ELISA) is a plate-based immunoassay technique used to detect and quantify unknown analytes in a biological sample. To perform an ELISA, researchers select a primary (detection) antibody with a high binding affinity and selectivity for the target antigen and measure the resulting antigen-antibody reaction. In an indirect ELISA, researchers use a secondary (capture) antibody that is conjugated with a reporter enzyme and designed to target the primary antibody that binds to the antigen of interest. The binding of these matched antibody pairs converts undetectable substances into measurable products, allowing researchers to detect, amplify, and quantify the generated signal.

Indirect ELISA is the most popular format and offers numerous advantages. Secondary antibodies are easy and cost-effective to obtain, simpler to use than conjugated primary antibodies, and enable accurate highly sensitive signal detection. They are also extremely versatile because one species can generate multiple different types of primary antibodies, all of which can implement the same labeled secondary antibody for detection. Labeling a secondary antibody allows the primary antibody to generate a maximum immunoreactive signal and reduce potential detection interference that could result from a conjugated primary antibody's undesired response to the target antigen.

Before running an experiment with an ELISA kit, it is important to avoid potential cross-reactivity by carefully selecting a secondary antibody for your specific application and research needs. The following factors are the most important for obtaining accurate, meaningful results:

• The Host Species Used to Generate the Primary Antibody

The first step is to determine the host species that was used to create the primary antibody, then select a secondary antibody with the ability to detect the primary antibody species. When a host animal is immunized against a target antigen, its immune system generates specific antibodies to identify and neutralize the antigen. To initiate the proper reaction, the species used to create the secondary antibody must be different from the host species that raised the primary antibody. 

• The Class and Subclass of the Primary Antibody

The secondary antibody must also match the primary antibody's class or subclass. Antibodies are categorized into five distinct classes or isotypes based on the specific heavy chains of their molecular composition and binding valency. Immunoglobulin G (IgG) molecules have gamma-chains, immunoglobulin A (IgA) molecules have alpha-chains, immunoglobulin D (IgD) molecules have delta-chains, immunoglobulin E (IgE) molecules have epsilon-chains, and immunoglobulin M (IgM) molecules have mu-chains. IgG and IgA are further divided into subclasses depending on variations in their amino acid sequences and carbohydrate structure. IgA has two subclasses (IgA1 and IgA2), while IgG has four subclasses (IgG1, IgG2, IgG3, and IgG4). Polyclonal antibodies typically belong to the IgG class, so the secondary antibodies are mainly anti-IgG and may also belong to multiple subclasses. 

• The Immunoassay Format

After choosing a host species to source the secondary antibodies, select an appropriate detection method based on the immunoassay you plan you perform. The effectiveness of antibody labels in detecting target antigens is highly dependent on application. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most popular enzymes to conjugate secondary antibodies in ELISA, but AP is often preferred for standard colorimetric detection, and HRP is more popular for chemiluminescent detection. 

• Whether the Secondary Antibody Is a Whole Immunoglobulin or a Fragment

Secondary antibodies are available in whole antibody and antibody fragment formats. Because they contain both heavy and light chains, whole antibodies offer strong covalent binding to the primary antibody's variable regions as well as a constant region for attaching signal-generating enzymes or dyes. However, this can lead to lower specificity due to cross-reactivity between various immunoglobulins and high background noise. 

• The Degree of Purification Required for the Experiment

Secondary antibodies can be affinity-purified by separating the monospecific antibodies of interest from other proteins or non-specific immunoglobulins in the sample. Affinity purification increases sensitivity, decreases background noise, reduces non-specific binding, and generates antibodies with high lot-to-lot consistency, allowing for more reproducible assays. Affinity purification is performed by passing an animal-derived serum that contains antibodies through chromatographic columns featuring an immobilized ligand (such as Protein G). After the antibody binds to the affinity ligand, you can wash away the other serum components and recover the purified antibody with a buffer solution that decouples them. 

Consider Cross-Adsorption of the Secondary Antibody

Cross-adsorption (or pre-adsorption) of the secondary antibody is valuable for increasing specificity and reducing unwanted cross-reactions, especially if you are using multiple primary antibodies from different species at the same time. In this step, all antibodies that were not generated from the target species are immobilized on a column, and a mixture of serum antibody-protein from several species is passed through the column matrix. 

The secondary antibodies that recognize antibodies or serum proteins from other species will bind to the column, and those that do not cross-react will flow through. This eliminates cross-reactivity from undesired immunoglobulins, antibody fragments, and the cell or tissue sample.