Control antibodies

Control antibodies are a vital part of assay design and ensure signal detected during antibody-based applications, can be attributed to specific antigen binding, rather than non-specific background staining.

Negative isotype controls function by determining the level of non-specific binding of the primary antibody to target. This is achieved by matching the control antibody host species, isotype, and if applicable fluorochrome, to that of the experimental specific antibody. A matched fluorochrome is required if the experimental primary antibody is directly conjugated. Negative control antibody is deliberately generated against a chemical or target not expressed by the cell or tissue of interest, so any binding will be non-specific in nature. This non-specific binding is most frequently caused by Fc receptors expressed on the surface of the target sample. These receptors bind the antibody’s Fc region independently of their Fab epitope binding domains.

Control antibodies are most commonly used in flow cytometry or immunohistochemistry to determine levels of background staining, but can also be utilized as blocking agents before addition of primary antibody.