Teresa Teixeira Lopes

Immunochemical characterization of type a botulinum neurotoxin in its purified and complexed forms

Immunochemical reactivities of type A botulinum neurotoxin to polyclonal antibodies raised against the neurotoxin complex toxoid have been investigated using enzyme-linked immunosorbent assay (ELISA) and with a fiberoptic immunosensor. The complex contains a group of complexing proteins in addition to the neurotoxin itself. Neurotoxin specific immunoglobulin G (IgG), purified from the IgG raised against the complex using an affinity column, showed a two-fold increase in reactivity with purified neurotoxin compared to the neurotoxin in the complex. Antibodies against the whole complex reacted approximately five times better with the complex than with the neurotoxin, suggesting that the detection of the neurotoxin complex may be more sensitive. Considering the fact that the amount of the complexing proteins and neurotoxin appears to be in a 4:1 ratio, a five-fold higher reactivity could suggest a 25-fold higher detectability of the neurotoxin in the complex. ELISA binding curves of complexing proteins and purified neurotoxin with antibodies raised against the whole complex indicated the complexing proteins to have significantly higher antigenicity. Furthermore, IgG fraction with or without the neurotoxin specific antibodies reacted almost equally to the neurotoxin complex, again suggesting higher immunogenicity of the complexing proteins. Increased binding of the complexing proteins versus the purified neurotoxin to antibodies against the complex and thus immunogenicity was also observed in the binding curves generated using a fiberoptic immunosensor.

USE OF AN OPTICAL-FIBER-BASED BIOSENSOR TO STUDY THE INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

An optical fiber-based laser biosensor has been used to investigate adsorption of blood lipoproteins and immunoglobulin G to the glass optical-fiber surface. While lipoproteins readily interact (adsorb) with unmodified optical-fiber surface, immunoglobulin G does not bind to the glass surface at a concentration of 5 μg/mL. However, upon glass surface modification with silane, immunoglobulin G binds significantly to the optical fiber. High-density lipoproteins and low-density lipoproteins show a differential interaction with the glass surface, and their interaction was altered upon modification of the glass surface with cholesterol. With these results in mind, the utility of the biosensor for the study of protein interaction with biorelevant surfaces has been outlined.