Antibodies are widely employed in the quantification of antigens in complex biological samples. Using techniques such as Western blotting, ELISA, and immunohistochemistry researchers are able to measure a single antigen, or perhaps a limited number of antigens, in each sample. In the post-genomics era, advances in multiplex immunoassay technologies now allow scores or even hundreds of antigens to be measured simultaneously.
While almost all antibody-based detection techniques require a label of some description, which confers measurability, the vast majority of commercially available antibodies are not labeled. Only antibodies with the greatest commercial value might be offered in conjugate form by suppliers, and then perhaps only with a few key labels.
In order to use unlabeled antibodies it is necessary to adopt an indirect detection method. In this approach, the primary antibody binds to its target antigen and is then detected with a secondary reagent, commonly another antibody that bears the required label. In multiplex assays it becomes increasingly difficult to create a panel of secondary reagents with the desired selectivity and lack of unwanted cross-reactions if there are more than two or three primary antibodies.
By covalently attaching the label directly to the primary antibody it is possible to overcome these difficulties and to reduce the complexity of immunoassays. Historically, antibody conjugation has been conducted by those with specialist knowledge of chemical-modification techniques. Most strategies for conjugating two molecules, A and B, involve chemical modification of each entity to introduce reactive groups.
The resulting derivatives of A and B are separated from excess chemical reagents by column chromatography and then mixed, with the aim of creating soluble AB conjugates. One difficulty is that the introduction of too many reactive groups can lead to the formation of polymers and insoluble aggregates. Today, because of significant advances in conjugation technology, the production of labeled antibodies is one of the simplest procedures performed in a research lab.