What types of labels can be attached to antibodies?

Antibodies can be labeled with small molecules, enzymatic proteins, radioisotopes, and fluorescent dyes. There are two different chemistries involved to label antibodies covalently with small molecules. The first type of chemistry is making the label reactive to primary amines and used to label lysine residues in the antibody. The other type of chemistry is reducing the disulfide bonds within the antibody chains and then reacted with a thiol-reactive label. It is important to note that novel methods have been developed such as the non-covalent addition of labels to the Fc fragment of the antibody. The type of label being attached is dependent on the downstream applications. Biotin tagging forms a strong non-covalent interaction with antibodies, and its small size seldom disrupts the activity of antibodies. It is used in western blot, ELISA, IHC, flow cytometry and biotin labeling is frequently used to increase the sensitivity of an assay when an antigen is hard to detect. Enzyme labels are larger than biotin, but they seldom disrupt antibody function. The main enzyme labels used are horseradish peroxidase, alkaline phosphatase, and glucose oxidase. To use these, samples incubated with an enzyme-specific substrate that is catalyzed by the enzyme to create a colored product or light. Fluorescent tags can also be labeled to antibodies. Fluorescent dyes are directly conjugated to the antibody and thus no enzyme/substrate is required for detection. The quantity of fluorescent signal observed is proportional to the amount of target protein in the sample. Fluorescent labels are used in fluorescent western blot assays, flow cytometry and IF experiments.