For those who are new to working in this area of work, it may prove useful to outline a few tips on choosing antibodies.
Immunoglobulins are purchased as primary or secondary, and mono or polyclonal proteins. Primary antibodies bind directly to the antigen, and can be conjugated (labelled) with an enzyme or fluorescent dye, to produce a visible signal when the appropriate substrate is added. This allows direct detection of the antigen.
Alternatively, the primary can be used unconjugated, and probing done with a secondary antibody (conjugated or unconjugated) that binds the primary.
Secondary antibodies have several advantages over primary (10) immunoglobulins. Firstly, it makes immunoassays less expensive, as unconjugated 10 immunoglobulins can be bought in bulk and used as stock reagents for different lab processes. Secondly, you can label immunoproteins to specific epitopes on the antigen molecule, thus amplifying the signal.
Immunochemistry assays make wide use of antibody fragments, for example Fab fragments, to which secondary Igs bind. For example, using the Fab fraction rather than the entire molecule as the10 Ig will boost the signal-to-noise ratio, as the smaller Fab fragment can bind more easily to its target protein. A Fab-specific secondary antibody will reduce the background signal that might occur from Fc binding. Secondary fragments can also be used to bind to primary fragments; for example, secondary F(ab´)2 fragments specific to Fab primaries can be employed for even greater sensitivity.
We at Novus Biologicals produced a wide range of antibodies with very specific antigen-binding requirements. Many are available in a range of conjugated and modified forms, targeted to specific epitopes and protein fragments.
