Surface Properties of Antibodies and their Complexes with Antigens Studied by LLPC
Abstract: The main objective of this thesis was to gain further insight into the relationship between the exposed surfaces and the functional properties of antibodies and their complexes with antigens in solution. To study this relationship, a new technique, liquid-liquid partition chromatography (LLPC) in aqueous two-phase systems, was developed further with respect to sensitivity, selectivity and reproducibility. The capacity of LLPC to compare the surface properties of antibodies in relation to their antigen specificity was explored. The usefulness of LLPC for examination of the events upon binding of antigen and for analysis of surface properties of antigen-antibody complexes in relation their structural/functional properties were studied. In this study, it was shown that LLPC in aqueous PEG-dextran two-phase systems offers the unique possibilities of comparing the overall surface properties of intact antibodies and antigen-antibody complexes in solution. A remarkable relationship between the surface of an antibody´s combining site and its chromatographic behaviour was found. LLPC provided the means to screen the antigen-binding sites of unliganded antibodies for differences/similarities in exposed surfaces even in those cases when the specificities of the antibodies were unknown. Our data suggest that antibodies may be regarded as being constructed of highly solvent-exposed, dominant CDRs of variable shape and size which, depending on the actual isotype, are grafted onto different scaffoldings of basically conserved structure. Moreover, different conformational (isomeric) forms of antibodies and enzymes could be detected and even separated using LLPC. Furthermore, the results demonstrated that LLPC could be used to examine the events upon binding of ligand by both antibodies and enzymes. The surface properties of antigen-antibody complexes were found to be dependent on the variable regions of the antibodies, the nature of the antigen, and/or possible conformational changes induced by ligand-binding.
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