A3_4 Conjugation Methods

The Antibody-Analyte-Antibody sandwich of an immunometric assay is useless without a signal generating entity, which can be conjugated to one of the antibodies. This is one of several situations in immunoassay where an antigen or antibody needs to be chemically conjugated with another molecular entity. It is a specialized area of immunoassay that requires its own chapter.

This chapter explains the relevance of conjugation methods in preparing reagents for immunoassays and the concepts involved. Protein-protein coupling methods are described, grouped by chemistry and category. The chemistry of specific conjugation methods, such as the periodate method, are explained. Genetic engineering options are also highlighted. Protein-small molecule conjugation methods are also described, including biotinylation. There are separate sections on conjugate purification and characterization.

Alastair Dent FRSC is Managing Director of Fleet Bioprocessing Ltd., a contract R&D company specialising in the development of immunoassays and the reagents required to put them together. He has particularly strong expertise in the development of conjugates for immunoassay use, based on a 30-year career in this field with industry leaders such as Amersham International, Kodak and Johnson & Johnson. This expertise covers not only the chemistry of protein conjugation, but also the approaches required to achieve robust manufacturing of immunoassay reagents in an industrial environment. As well as contributing to the last three editions of The Immunoassay Handbook, he was co-editor and lead author of Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences, a major conjugation reference work, and has authored many other publications in this field. He is a reviewer for journals such as Clinical Chemistry and is on the committee of the Royal Society of Chemistry’s Analytical Biosciences Group.