To induce the production of antibodies, exogenous antigens are taken up and degraded in antigen presenting cells in vivo. Since this process inevitably lead to distort antigen¡¦s structure, it is likely that some arising antibodies following immunization may not react appropriately with native protein. In the present study, comparative studies on the reactivity of cobrotoxin and recombinant cobrotoxin toward anti-cobrotoxin antibodies were carried out. CD spectra and acrylamide quenching of Trp fluorescence showed that global structure of recombinant cobrotoxin was different from that of native toxin. Results of ELISA and dot blotting assay revealed that recombinant cobrotoxin had a superior reactivity toward anti-cobrotoxin antibodies than native toxin did. Reactivity with antibody fractions specifically against N-terminal region or C-terminal region of cobrotoxin also showed the same results. The binding of recombinant cobrotoxin with antibodies was stronger than that of cobrotoxin as revealed by ammonium thiocyanate elution assay. Recombinant protein was susceptible to reduce its antigenicity after tryptic digestion compared to cobrotoxin. Distorting disulfide linkages at C-terminus caused a marked decrease in immunoreactivity of recombinant cobrotoxin, indicating that anti-cobrotoxin antibodies mostly recognized conformation-dependent epitopes. Moreover, cobrotoxin and recombinant cobrotoxin showed a similar immunoreactivity under denaturing condition. Taken together, these results suggest that native conformation with cobrotoxin may unfavorably impede the interaction of some epitope(s) with anti-cobrotoxin antibodies.

The flow of polymer solutions in the high Elasticity number, El, regime in complex geometries may lead to strong viscoelastic behaviour and eventually become unstable as the Weissenberg number, Wi, is increased beyond a critical level. So far, the success of numerical simulations in predicting the highly non-linear behaviour of polymer solutions in complex flows has been limited. In this thesis, selected constitutive models are evaluated under the high El flow regime in the cross-slot and contraction benchmark flows using a numerical technique based on the finite volume method. The numerical technique is implemented within the OpenFOAM framework and thoroughly validated in the benchmark flow. A modification to the FENE dumbbell model based on the non-affine deformation of polymer solutions is proposed, which enabled the prediction of some non-linear material functions and also enhanced numerical stability, allowing a higher Wi to be attained. Asymmetric flow instability in the cross-slot flow has been studied. Time-dependent stability diagrams were constructed based on Wi and the strain, ε, both of which govern the stretching of a polymer chain. In the contraction flow, elastic instability is simulated for the first time in this geometry. Substantial time-dependent asymmetric flow patterns were predicted as seen in experiments. The effect of the contraction ratio is investigated through a stability diagram. Three-dimensional finite element simulations were also carried out to study the effect of the aspect ratio in the contraction flow of a Phan-Thien-Tanner fluid. The simulations suggest that a lip vortex mechanism is a signature for the onset of strong viscoelastic behaviour.