Primary and secondary antibodies are two groups of antibodies that are classified based on whether they bind to ''antigens or proteins'' directly or target another (primary) antibody that, in turn, is bound to an ''antigen or protein''.

Primary

A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease and they are used for the study of absorption, distribution, metabolism, and excretion (ADME) and multi-drug resistance (MDR) of therapeutic agents.

Secondary

Secondary antibodies provide signal detection and amplification along with extending the utility of an antibody through conjugation to proteins. Secondary antibodies are especially efficient in

immunolabeling Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These an ...

. Secondary antibodies bind to primary antibodies, which are directly bound to the target antigen(s). In immunolabeling, the primary antibody's Fab domain binds to an antigen and exposes its

Fc domain The fragment crystallizable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune ...

to secondary antibody. Then, the secondary antibody's Fab domain binds to the primary antibody's Fc domain. Since the Fc domain is constant within the same animal class, only one type of secondary antibody is required to bind to many types of primary antibodies. This reduces the cost by labeling only one type of secondary antibody, rather than labeling various types of primary antibodies. Secondary antibodies help increase sensitivity and signal amplification due to multiple secondary antibodies binding to a primary antibody. Whole Immunoglobulin molecule secondary antibodies are the most commonly used format, but these can be enzymatically processed to enable assay refinement. F(ab')₂ fragments are generated by pepsin digestion to remove most of the Fc fragment, this avoids recognition by Fc receptors on live cells, or to Protein A or Protein G. Papain digestion generates Fab fragments, which removes the entire Fc fragment including the hinge region, yielding two monovalent Fab moieties. They can be used to block endogenous immunoglobulins on cells, tissues or other surfaces, and to block the exposed immunoglobulins in multiple labeling experiments using primary antibodies from the same species.

Applications

Secondary antibodies can be conjugated to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); or

fluorescent dyes A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with sev ...

such as

fluorescein isothiocyanate Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. First described in 1942, FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group ( ...

(FITC),

rhodamine Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and inks ...

derivatives, Alexa Fluor dyes; or other molecules to be used in various applications. Secondary antibodies are used in many biochemical assays including: *

ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presen ...

, including many

HIV test HIV tests are used to detect the presence of the human immunodeficiency virus (HIV), the virus that causes acquired immunodeficiency syndrome (AIDS), in serum, saliva, or urine. Such tests may detect antibodies, antigens, or RNA. AIDS diagnos ...

s *

Western blot The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...

Immunostaining In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by A ...

* Immunohistochemistry *

Immunocytochemistry Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visu ...

References

{{reflist Glycoproteins Immune system Biochemistry methods