Title page for ETD etd-04172008-111655


Document Type Master's Dissertation
Author Van der Merwe, Hermanus Daniel
Email manie@tuks.co.za
URN etd-04172008-111655
Document Title A resonant mirror biosensor approach to understand antibody-antigen interactions in Guillain Barré Syndrome
Degree MSc (Biochemistry)
Department Biochemistry
Supervisor
Advisor Name Title
Prof J A Verschoor Committee Chair
Keywords
  • antibody-antigen interactions
  • antibodies
Date 2007-09-06
Availability unrestricted
Abstract
Guillain Barré Syndrome in humans is characterised by ascending paralysis. It is often associated with preceding infections two to four weeks prior to nadir and is fatal in five percent of cases. Antibodies specific to several nerve components are frequently associated with clinical symptoms in GBS. These antibodies were found to be specific to various gangliosides and ganglioside complexes. It was also found that antibody reactivity to gangliosides is affected by membrane components. The most prevalent (20-30%) immunoglobulin in GBS is anti-GM1 (20-30%), which also binds to the LPS of the PEN O:19 Campylobacter jejuni serotype. This is the most common infectious agent associated with GBS and emphasizes the importance of infection and anti-ganglioside antibodies in disease development.

Intravenous infusion of pooled immunoglobulin from healthy donors, also called intravenous immunoglobulin (IVIg), halves the severity of disease manifestation. The action mechanism of IVIg in curing GBS is not clear, but intravenous immunoglobulin was shown to neutralize anti-ganglioside binding activity and its pathogenic effects. It was further found that anti-idiotypic antibodies in IVIg inhibit anti-ganglioside antibody activity. Treatment with IVIg is not equally effective in all GBS cases, which might be due to the inability of IVIg to neutralize anti-ganglioside antibodies in all patients adequately. Therefore, the treatment of GBS with IVIg needs to be better understood in order to improve its use as a cure for GBS.

This study confirmed previous findings that the interaction of patient serum anti-GM1 antibodies and ganglioside auto-antigens is greatly impaired by components in healthy serum. Bound anti-GM1 antibodies could be displaced by (presumably) anti-idiotypic antibodies from healthy donor serum. This study found that the displacement potential between donor sera differs. Anti-GM1 antibody displacement was found to be dependent on the character of both anti-GM1 and anti-idiotypic antibody. This demonstrated the feasibility of improving the efficiency of treatment by IVIg by sourcing it from only those sera that test best for displacing auto-antibodies from their ganglioside antigens in ELISA. IVIg selection may therefore greatly benefit from the use of recombinant phage display antibodies to distinguish between the various types of GBS for treatment.

To develop a method to characterize anti-ganglioside antibodies sensitively, an evanescent field biosensor was employed in which gangliosides were presented in a liposome environment. This provided a more physiological way of antibody antigen recognition. The optimized method determined the ganglioside binding specificity of purified IgG from a GBS patient, and mouse monoclonal anti-GM1 and anti-GD1a antibodies accurately. The results compared well with those from ELISA. The results obtained with purified IgG were far better than that obtained with whole serum analysis. This could be due to non-specific binding or the presence of inhibiting anti-idiotypic antibodies in patient sera. The biosensor method for antibody detection in GBS may allow the detection of anti-idiotypic antibodies in patients in future, because it requires no prior labelling of antibodies. Anti-idiotypic interaction may be detected by displacement of Ab1 from antigen, or by capturing Ab2 on Ab1 immobilized on the biosensor surface.

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