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AAT Bioquest

What are the major differences between primary and secondary antibodies?

Posted September 22, 2021


Answer

Primary antibodies and secondary antibodies are both involved in immune response. Both are structurally similar but that’s where the similarities end. These are some major differences between the two.

Basis for comparisonPrimary antibodySecondary antibody

Definition

Is an immunoglobulin that specifically binds to a specific protein or the biomolecule of research interest for the purpose of purifying or detecting and measuring.Is a type of antibody that binds to the primary antibody after the primary antibody attaches to the antigen in order to assist in detection, sorting, and purification of target antigens.

Binding mechanism

Has the ability to bind directly to the antigen.Does not have the ability to bind directly to the antigen. Interacts with the target antigen by binding to the heavy chains of the primary antibody.
Use in immunoassaysIs essential in all immunoassays to bind to the target antigen. For example, in Direct ELISA the primary antibody is labeled directly with an enzyme for enzymatic reaction and signal detection. A secondary antibody is not needed.Is needed only in some immunoassays, not all. For example, in Indirect ELISA, the primary antibody is not labeled directly. Instead, the secondary antibody is labeled with the enzyme.
SourceThe host species of the primary antibody must be different from the species of the test sample, to prevent cross-reactivity from the secondary antibody with sample immunoglobulins.The host species of the secondary antibody must be different from the host species of the primary antibody, as the secondary antibody should be directed against the species of the primary antibody.
Pre-adsorption requirementDo not need to be pre-adsorbedMay need to be pre-adsorbed to increase specificity and minimize non-specific antibodies.
ConalityCan be either monoclonal or polyclonal.Can be either a monoclonal or polyclonal antibody that binds to the primary antibody or its fragments.
FunctionUsed as biomarkers to detect diseases such as diabetes, cancer, Parkinson’s Disease, and Alzheimer’s.Used in immunolabeling.
Additional resources

Domain-Specific Antibodies Reveal Differences in the Membrane Topologies of Apolipoprotein L1 in Serum and Podocytes

HRP-labeled goat anti-rabbit IgG (H+L)