What are secondary antibodies?
Secondary antibodies are essential components in various immunoassays and immunohistochemistry techniques.
In general antibodies are proteins known as immunoglobulins, produced by plasma cells, tasked with identifying and neutralizing potentially harmful foreign molecules called antigens (‘the non self’). The specific region of an antibody that recognizes the antigen is called the “paratope,” which specifically binds to the antigen’s “epitope.” This binding triggers various immune responses, including the deployment of macrophages to destroy ‘the non self’.
To fully grasp the role and importance of secondary antibodies, it’s essential to distinguish them from primary antibodies.
Primary antibodies, directly produced by the immune system, both as polyclonal and monoclonal varieties, are directly used in diagnosing diseases and detecting specific biomarkers associated with health conditions like diabetes, cancer, and neurological disorders such as Alzheimer’s and Parkinson’s diseases. They bind directly to antigens, but they must be detected to highlight the pathology.
In contrast, secondary antibodies have a different role. They do not bind directly to antigens. Instead, they bind to primary antibodies, creating an indirect linkage to the antigen. This unique interaction is critical as it amplifies the detection signal, enhancing the visualization and quantification of the primary antibody, bound to the target antigens. This amplification is vital in diagnostic assays where sensitivity and specificity are paramount.
Understanding the pivotal interactions between primary and secondary antibodies can significantly enhance the diagnostic accuracy and provide deeper insights into immune system functioning and disease pathology. These secondary antibodies are not just components of the immune response but are powerful tools that enable researchers and clinicians to achieve more precise and reliable results in various immunological assays.
What are Secondary Antibodies Used For?
Secondary antibodies are vital tools in both research and diagnostic settings. They are generated against the host species and isotype of a primary antibody. For example, to detect a human IgG polyclonal primary antibody, a goat anti-human IgG secondary antibody (targeting Fcγ or light and heavy chains) is typically used. These antibodies fulfill several key roles:
Signal Amplification: Secondary antibodies are often conjugated with labels such as enzymes (e.g., peroxidase or alkaline phosphatase for colorimetric detection) or fluorescent dyes (e.g., Alexa Fluor for fluorescence-based assays). This conjugation amplifies the primary antibody-antigen signal , enhancing the detectability of the primary antibody across various assay types.
Increased Selectivity and Sensitivity: Secondary antibodies provide high selectivity when binding to primary antibodies present in very low concentrations in complex biological fluids. This property is crucial for the accurate isolation or quantification of specific antibodies.
Enhancing Specificity: By using isotype-specific secondary antibodies, researchers can minimize non-specific binding and background noise, thereby improving the specificity and clarity of experimental results. This is particularly important in studies requiring high specificity and reliability.
Fields of Application:
- Assays: Secondary antibodies are integral to various laboratory techniques, including Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), immunofluorescence, and flow cytometry. Each technique benefits from the specific properties of the conjugated secondary antibodies.
- Clinical and Diagnostic Applications: They are crucial in diagnosing and studying diseases, particularly in detecting antibodies linked to tissue antigens and autoantibodies in autoimmune disorders. Additionally, they are used to isolate and quantify specific mouse monoclonal immunoglobulin classes such as IgG1, IgG2a, IgG2b, IgG3, and light or heavy chains.
These applications highlight the indispensable role that secondary antibodies play in enhancing the accuracy, sensitivity, and specificity of immunodetection methods across a wide array of biological and medical sciences.
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Examples of application of secondary antibodies
1° Example: Indirect ELISA assay
Let’s start by describing the indirect ELISA assay.
An indirect ELISA is one where the primary antigen-specific antibody is recognized by a secondary conjugated antibody.