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Secondary Antibodies. |
Secondary antibodies are other host antibodies that bind to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or sorting applications. Secondary antibodies may be polyclonal antibodies or monoclonal antibodies. Polyclonal antibodies lack the specificity of monoclonal antibodies but frequently have higher sensitivities because polyclonal antibodies are a mixture of antibodies that may include very high affinity antibodies. Cloning may not effectively select the highest affinity antibody from a polyclonal host. Secondary antibodies are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions.
Sigma’s polyclonal secondary antibodies are produced from the serum of host animals including mouse, rabbit, goat and sheep. Monoclonal secondary antibodies are produced from mouse hybridoma clones. Secondary antibodies are used in immunodetection, and immunoaffinity purification applications. Immunodetection applications include enzyme linked immunosorbent assays (ELISA); Western blotting; immunohistology, immunoblotting, immunostaining and cell based assays, such as cell-based immunochemical assays and high throughput cell-based screening assays (HTS). Secondary antibodies are useful for cell sorting, fluorescence activated cell sorting, FACS.
The specific utility of a secondary antibody depends upon its conjugated probe(s). Probes are molecules that support various detection technologies. The most common detection systems for conjugated secondary antibodies are colorimetric or fluorescent. Colorometric assays are typically based on the use of alkaline phosphatase (AP) or horseradish peroxidase (HRP) or derivatives of these, APAAP or PAP. The biotin avidin (streptavidin) conjugate binding system is often used to amplify the colorometric signal for AP or HRP. The most common fluorescent assays utilize Fluorescein (FITC) or Rhodamine or it derivative, TRITC. Newer fluorescent dyes such as Cyanine (Cy3) and Phycoerythrin (R-PE), which have unique advantages, are also used. A secondary antibody can also be conjugated with agarose or colloidal gold for special applications. |
