|
Phosphorescence anisotropy measurements have been proposed as a method of following immunological reactions. To assess the utility of the technique, bovine serum albumin (BSA) has been employed as an antigen and its motion detected by covalently labelling the protein with the triplet probe, eosin-5'-isothiocyanate. A monoclonal IgG anti-BSA antibody was added to a solution of the labelled antigen thereby creating a heterogeneous population of rotating species of different sizes. The rotational mobilities of the labelled antigen and the labelled antigen-antibody complex have been examined by measurement of time-resolved phosphorescence depolarization of the probe, excited by a brief laser pulse from a frequency-doubled Nd:YAG laser. The orthogonally-polarized emission components were collected simultaneously and the time-resolved phosphorescence anisotropy (r(t)) calculated using a Marquardt curve fitting procedure. In the initial experiment, the time-resolved anisotropy from each antigen-antibody mixture was found to consist of more than one exponential decay process. The initial anisotropy values r(O) were found to be population dependent. The initial anisotropy values r(O) obtained from the phosphorescence decay measurements were relatively small (less than 0.07), however it can be enhanced by conjugating one of the species to a relatively large polymer bead. The experimental results indicate that the time-resolved phosphorescence depolarization could be exploited to study the kinetics of antigen and antibody interaction. There is also scope for development of the method for optical immunoassay.
|
