Providing Better Immunology Tools For Better Healthcare ::
Applications
Epitope Characterization
Super Type Analysis.
Determination of allele overlapping peptide-binding specificities has become an important issue in vaccine design with direct implications concerning population coverage. Depending upon allelic composition, it is import to identify the binding capacity of single peptides in order to determine their useful ness for treating a larger subset of patients who express the MHC allele product that is capable of binding that specific peptide. Unfortunately, direct experimental validation of cross-reactivities for single peptides has been tremendously difficult because of lack of high quality HLA molecules.
The Supertype Analysis approach is capable of screening panels of different sHLA alleles for binding with a peptide of interest. For screening purposes, a FITC-labeled peptide candidate is incubated with activated sHLA molecules and peptide/HLA interactions are monitored over time. Our direct biochemical approach allows the identification of broad cross-reactive peptide epitopes, thus accurately revealing population coverage of a given peptide candidate thereby greatly enhancing the effectiveness of future vaccine designs.
There is currently a significant interest in quantifying peptide/HLA interactions for the better understanding of immunity. Exact definition of molecular parameters involved in such interactions are of practical use in the identification, validation and engineering of peptides with desired binding properties and T cell reactivity.
Kinetic Analysis
Several studies have reported good correlation between binding kinetics and the degree of T cell activation. Because FP is a simple solution-based assay, we are able to obtain specific kinetic binding profiles to further assess the peptides capacity to stabilize HLA complexes by determining association and dissociation parameters. Kinetic experiments deliver apparent rate constants (kob). Extrapolation procedures allow to calculate absolute rate constants for association (kon) and dissociation (koff) as well as equilibrium dissociation constants (Kdkin).
Equilibrium Analysis
FP is also used for equilibrium analysis as alternative to kinetic studies providing information on the strength of the MHC/peptide interaction. FP readings are plotted as a function of pFITC concentrations and fitted by nonlinear regression analysis using a dose-response model. Unlike the classical binding curve profile, the FP signal is greater for low ligand concentration because both bound and free fluorescent ligand contribute to the signal. To determine the equilibrium dissociation constants (KdTitrK) and maximal binding (Bmax) of the fluorescent peptides designed, original data sets are transformed using the recently described FP Kd model.
