This chapter highlights the importance of separation in heterogeneous immunoassays, including the difference in impact between competitive and immunometric formats. Solid-phase separations are reviewed in detail. Protein binding to plastic and its effects on antibody performance are explained, as is the control of the plastic and the molding process. Different types of solid phase media are reviewed. Washing is investigated step by step as a series of mechanical processes. Liquid-phase separations are reviewed in less detail as they are now seldom used. There is a special section on microarrays.

David Wild’s career spans 40 years in diagnostics, medical devices, pharmaceuticals and biotechnology. As a University of London undergraduate, he won the Driver Prize for Biochemistry after discovering an extra electron transfer step in photosynthesis. As leaders of the University Biochemistry Society, he and his girlfriend Cindy (now wife) hosted lectures by Sir Hans Krebs and Roger Ekins. After graduation, he worked first as a molecular biologist at a pharmaceutical company R&D laboratory, then, for 25 years, in immunodiagnostics, managing product development and industrial engineering projects. More recently he managed large medical device R&D and operational projects. He worked for Amersham, Kodak, Johnson and Johnson, Bristol-Myers Squibb and ConvaTec. His Director level positions include Research and Development, Design Engineering, Global Manufacturing, and Strategy. He indulges his passion for immunoassays as Managing Editor of The Immunoassay Handbook and lectures, trains and consults on development, manufacturing and marketing strategies, and how to integrate and execute them.

This chapter also contains material from Wlad Kusnezow, from the third edition of The Immunoassay Handbook.

Heterogeneous immunoassay, homogeneous immunoassay, immunometric immunoassay, competitive immunoassay, separation efficiency, solid phase, liquid phase, protein adsorption, antibody binding, capture protein, non-specific binding, background, polystyrene, coated tube, microtiter plate, microtiter well, latex particles, magnetizable particles, washing, streptavidin, second antibody, membrane filtration, immunochromatography, lateral flow, western blot, electrophoresis, interference, dilution, soaking, solubilization, stabilization, thermal enhancement, agitation, decantation, aspiration, sedimentation, resuspension, capillary action, gel filtration, charcoal, precipitation, double antibody, polyethylene glycol, microarray.