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Title: The study of cell surface phenotypes by high performance liquid chromatography
Author: Wu, Zhiwei
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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Size-exclusion high performance liquid chromatography was developed to study surface membrane proteins of malignant lymphocytes. The membrane proteins were separated in accordance to their molecular sizes or complexed sizes and displayed, in conjunction with SDS-PAGE, as two-dimensional matrices on which the location of proteins is determined by both retardation in the HPLC column and the mobility on the SDS-PAGE gels. Any association or induced-interaction between proteins are reflected by predictable changes of the retention times, thus by the location on the two-dimensional matrix. Individual membrane proteins in the mixture were identified or separated by the changes in their retardation in the column when mAb-Ag complexes formed by applying specific monoclonal antibodies to the extracts of vectorially labelled cells. A number of surface markers have been identified and several new components are characterized. This study has demonstrated that (1) SE-HPLC is a potent method for analysis of complicated mixtures, particularly when a complete separation is not necessary (2) the technique has advantages for the study of non-covalentinteractions between proteins, particularly valuable for antibodies of low affinity (3) the method is powerful for compositional study and initiative investigation of the complexity of mixtures. By using this technique in conjunction with SDS-PAGE, B-CLLs were found to have heterogeneous surface phenotypic presentations with respect to both quantity and quality. The HPLC-SDS PAGE study revealed that the expression levels of individual surface components in B-CLL patients were at a correlated fashion and, across the patient panel, present a continuous spectrum of such relationships, confirmed the earlier reports of immunological studies by Maddy that individual B-CLL patients can be ranked in a sequence according to their expression level of CD45 isoforms and that this sequential variation of CD45 is in correlation with levels of sIg and CD21. In addition, this study discovered a group of uncharacterized proteins, band 4.1 (160KD), band 4.3 (135KD) and band 2 (300KD) which are also correlated with CD45RA, CD21 and sIg. A comprehensive repertoire of B-CLL phenotypes has been established with respect to the membrane protein expression.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available