Acid Bit

By Brigette Federico

 Immunoassays such as Western blotting and ELISA often rely on a two-stage technique. A primary antibody (1o Ab) is used to bind directly to the protein of interest. A secondary antibody (2o Ab), labelled with a detection tag such as a fluorescent dye or colourmetric enzyme, is then applied. This binds to a site on the primary Ab, allowing indirect detection of the antigen.

Single stage assays have been developed, in which primary immunoglobulins are tagged, allowing both probing and detection to take place from the same molecule. However, the indirect approach is still preferred, for many reasons. One-step 1o Abs are custom-built and thus expensive. In addition, more than one 2o Ab can target the primary protein producing clearer results.

Primary Abs are typically purchased from antibody suppliers in monoclonal form. These are derived from a single cell-line and are totally homogenous. By contrast, most secondary proteins are polyclonal. Here, animals are injected with a specific peptide antigen of a particular species, a secondary immune response is triggered and in this way a whole plethora of proteins are produced unique for that antigen.

The difference between monoclonal and polyclonal proteins is that, while a monoclonal only recognises one specific epitope, polyclonals can recognise several. The primaries are raised against a specific antigen, and are unlabelled. 2o Abs are raised against primaries, and are labelled, or conjugated, with a reporter enzyme or one of the new fluorescent products such as Dylight.

Sometimes best results are obtained by using antibody fragments, such as an Fab fragment or gamma light chain, as the primary Ab rather than the entire protein. This can improve signal-to-noise ratio in assays, and improve binding to the antigen. The 2o Ab will be specific to Fab.

The secondary antibodies supplied by us at Novus Biologicals are available for a wide range of specie and primary proteins.

The Article is written by novusbio.com providing antibody database and antibodies Services. Visit http://www.novusbio.com for more information on novusbio.com Products & Services___________________________Copyright information

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