Surface plasmon resonance as a tool in the functional analisys of an immunodominant site in foot-and-mouth disease virus

A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates, medium antibody surface densities and high peptide concentrations. The method was applied to an extensive analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total agreement with previous competition ELISA experiments. Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21 residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features previously correlated with antigenicity were more pronounced in the cyclic peptide. Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicity-enhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide ? mAb 4C4 complex showed paratope ? epitope interactions identical to all FMDV peptide ? mAb complexes studied so far. Key residues for these interactions are those directly involved in epitope ? paratope contacts (141Arg, 143Asp, 146His) as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds, delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif).

Cristalização de Biomateriais Vitrocerâmicos e Mineralização em Meio Fisiológico Simulado

We propose new theoretical models, which generalize the classical Avrami-Nakamura models. These models are suitable to describe the kinetics of nucleation and growth in transient regime, and/or with overlapping of nucleation and growth. Simulations and predictions were performed for lithium disilicate based on data reported in the literature. One re-examined the limitations of the models currently used to interpret DTA or DSC results, and to extract the relevant kinetic parameters. Glasses and glass-ceramics with molar formulation 0.45SiO2? (0.45-x)MgO?xK2O?0.1(3CaO.P2O5) (0?x?0.090) were prepared, crystallized and studied as potential materials for biomedical applications. Substitution of K+ for Mg2+ were used to prevent devritification on cooling, to adjust the kinetics of crystallization and to modify the in vitro behaviour of resulting biomaterials. The crystallization of the glass frits was studied by DTA, XRD and SEM. Exothermic peaks were detected corresponding to bulk crystallization of whitlockite-type phosphate, Ca9MgK(PO4)7, at approximately 900ºC, and surface crystallization of a predominant forsterite phase (Mg2SiO4) at higher temperatures. XRD also revealed the presence of diopside (CaMgSi2O6 in some samples. The predominant microstructure of the phosphate phase is of the plate-type, seemingly crystallizing by a 2-dimensional growth mechanism. Impedance spectroscopy revealed significant changes in electrical behaviour, associated to crystallization of the phosphate phase. This showed that electrical measurements can be used to study the kinetics of crystallization for cases when DTA or DSC experiments reveal limitations, and to extract estimates of relevant parameters from the dependence of crystallization peak temperature, and its width at half height. In vitro studies of glasses and glass-ceramics in acelular SBF media showed bioactivity and the development of apatite layers The morphology, composition and adhesion of the apatite layer could be changed by substitution of Mg2+ by K+. Apatite layers were deposited on the surface of glass-ceramics of the nominal compositions with x=0 and 0.09, in contact with SBF at 37ºC. The adhesion of the apatite layer was quantified by the scratch test technique, having been related with SBF?s immersion time, with composition and structure of the glass phase, and with the morphology of the crystalline phase of the glass-ceramics. The structure of three glasses (x=0, 0.045 and 0.090) were investigated by MAS-NMR ( 29Si and 31P), showing that the fraction of Q3 structural units increases with the contents of Mg, and that the structure of these glasses includes orthophosphate groups (PO43-) preferentially connected to Ca2+ ions. Mg2+ ions show preference towards the silicate network. Substitution of Mg2+ by K+ allowed one to change the bioactivity. FTIR data revealed octacalcium phosphate precipitation (Ca8H2(PO4)6.5H2O) in the glass without K, while the morphology of the layer acquires the shape of partially superimposed hemispheres, spread over the surface. The glasses with K present a layer of acicular hidroxyapatite, whose crystallinity and needles thickness tend to increase along with K content.