Wolf D. Kuhlmann
Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
Laboratory Diagnostics & Cell Science, 56112 Lahnstein, Germany
Enzymes, fluorochromes and other marker molecules can be conjugated to antibodies, avidin, protein A or G and other proteins in many ways. For such conjugation procedures, reagents are preferred which form stable covalent linkage. Conjugation can be carried out in so-called one-stage, two-stage or multiple stage reactions. The choice of a coupling reagent is often a matter of personal preference. In certain instances, however, there are important advantages to choosing a particular procedure. The goal of protein-protein coupling is limited by the type of functional groups available in the given molecules. Because unconjugated antibodies will interfere with labeled antibodies by competing for antigenic sites, analytical applications of conjugates rely on both (a) the purity of the conjugates and, in the case of enzymes as markers, (b) its enzymatic activity. Thus, purification of conjugates is strongly recommended. Apart from the use of conjugates, immunological bridge techniques which do not require covalent conjugation of antibody with marker may be employed; an example is the use of soluble peroxidase-antiperoxidase complexes (PAP) according to L A STERNBERGER.