A conjugated antibody is a polyclonal or monoclonal antibody that has a molecule attached which can be used to create a detectable signal. Detection can be visualized by color-generation, fluorescence, or other signals. Conjugated antibodies are used in a wide range of applications including flow cytometry, immunohistochemistry, immunocytochemistry, western blot, and ELISA.
Types of Detection Methods
There are two types of detection methods used with antibodies, direct and indirect.
Direct detection uses a primary antibody that is directly conjugated to a label. By conjugating primary antibodies, you can directly detect the target without use of secondary antibodies. The advantages of direct detection include ease of use for multicolor staining and eliminating the concerns regarding non-specific binding of the secondary antibody. It is ideal to use directly conjugated antibodies when the target of interest is in high abundance, or the conjugate signal can compensate for low target expression levels.
Indirect detection uses a primary antibody that is unconjugated and a conjugated secondary antibody that is specific for the host species of the primary antibody. Indirect detection is the preferred method if the target of interest has a lower level of expression. By using secondary antibodies, this allows signal amplification due to the secondary antibodies carrying multiple labels that can bind to the primary antibody. In general, indirect detection methods generally have a higher level of sensitivity and generate a more intense signal.