Purification and biochemical characterization of immunotoxins

Written on a plastic bottle of liquid soap in one of our bathrooms (JML) is the phrase 'Absolute cleanliness is next to Godliness!'. Perhaps absolute purity for ITs does not rank so highly, but the availability of purified ITs that contain no nonconjugated antibody or toxin, and no material of very high Mr (aggregates) is essential for a proper comparison of the biological activities of the component proteins with their nonconjugated counterparts. Purified and biochemically well-defined ITs make it possible to perform quantitative binding assays, to evaluate receptor-mediated endocytosis and to do cytotoxicity tests in vitro and efficacy studies in vivo, without fear that competition by nonconjugated antibody is affecting the experimental result. This chapter illustrates some of the methods that in combination can be used to purify ITs, including affinity chromatography, gel filtration, and ion-exchange chromatography with buffers of carefully defined composition, using examples from our laboratory to illustrate the procedures. For further details concerning these methods, we would refer the reader to the excellent book by Scopes [40], Purification of Proteins: Principles and Practice, which contains much useful information of both a technical and a practical nature regarding methods for purification of proteins.