DEVELOPMENT OF THE METHOD FOR STRUCTURAL ANALYSIS OF DOMAIN OF ANTIHAPTENIC ANTIBODY BY HYPHENATED MASS SPECTROMETRY

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Recently, many interests have been focused on the usefulness of mass spectrometry (MS) heyphenated with chromatographic methods such as liquid chromatography (LC) and capillary electrophoresis for the structural elucidation of proteins. On the other hand, it is keen needed elucidation of mechanisms of the intermolecular interaction between proteins and biologically active molecules. The purpose of this project was the development of the method for the structural elucidation of the domain of the anti-haptenic antibody bounded to the hapten.Initially, the monoclonal antibody for 11-deoxycortisol was prepared using the steroid having a bridge at C-4 on the nucleus as a hapten. Next, lysosome as a model enzyme for enzyme immunoassay was reacted with 11-deoxycortisol and the structure of the conjugated lysozyme was analyzed with electrospray ionization (ESI) mass spectrometry on the flow injection mede. Before and after reductive alkylation, the conjugated lysosome has been further subjected to hydrolysis with various enzyme followed by the LC/ESI-MS analysis to clarify the structure of the domain.