Determination of the dissociation constant of phosvitin-anti-phosphoserine interaction by affinity capillary electrophoresis.

We used affinity capillary electrophoresis (ACE) to study the interaction of a monoclonal anti-phosphoserine antibody (mAb) to a homopolyvalent antigen (hpAg), phosvitin. A model system, which allows the measurement of the true dissociation constant (Kd) in Ag excess based on measurement of migration shifts of mAb-hpAg complexes at different Ag concentrations in solution, is presented for the study of the interactions between a mAb and an Ag that has identical determinants. The experimental value of Kd (22.4 x 10(-6) M) obtained by ACE is shown to be in close agreement with the value (17.8 x 10(-6) M) obtained by the conventional immunoassay based on indirect competition enzyme-linked immunosorbent assay (ELISA). Moreover, the Kds of mAb-hpAg complexes were measured and shown to be independent of the applied electrical field strength. Thus, under conditions where the total Ag concentration is in large excess over the total Ab concentration and when certain requirements are fulfilled, this method offers the advantage of dealing with the determination of Kd for unlabeled mAb and homopolymeric Ag molecules in free solution rather than at the liquid-solid interface.

Light-induced fluorescence spectroscopy: a potential diagnostic tool for oral neoplasia.

For the early detection of oral neoplasia, light-induced fluorescence spectroscopy was used to measure the fluorescence emission of malignant (squamous cell carcinoma & verrucous carcinoma) and premalignant (epithelial dysplasia, hyperkeratosis & lichen planus) oral tissues as well as normal oral mucosa ex vivo to assess the ability of this technique to distinguish neoplastic from normal oral tissues. The emission spectra of histologically normal and neoplastic oral tissues were obtained under excitation wavelengths varied from 270 nm to 400 nm at 10-nm intervals. At 300-nm excitation, the most intensely fluorescent peak occurred at 330-nm and 470 nm emission. At 330-nm emission, the spectrum of the malignant oral tissue was significantly stronger than that of the normal oral mucosal tissue after area normalization. However, at 470-nm emission, the spectrum of the malignant oral tissue was significantly weaker than that of the normal oral mucosal tissue. A diagnostic algorithm based on the ratio of relative intensities of 330 nm to 470 nm emission within the +/-5 nm peak area of each sample was calculated and paired. The histogram of ratios showed that histologically neoplastic oral tissues could be distinguished from normal oral mucosal tissues using the 300 nm excitation wavelength. The average ratio of malignant or premalignant oral samples was significantly greater than that of the normal oral mucosal samples (p < 0.001). This ex vivo study indicated that fluorescence spectroscopy may be useful in differentiating malignant or premalignant oral tissue from normal oral mucosa.