A NOVEL DUAL-CHANNEL PHOSPHORIMETER FOR STUDIES OF ANTIGEN-ANTIBODY COMPLEXES

A phosphorescence anisotropy selective technique is proposed as a new method for real-time monitoring of immunological reactions and formati on of antigen-antibody complexes. To assess the utility of the techniq ue, a novel phosphorimeter has been developed. The phosphorimeter cons ists of a pulsed, frequency-doubled Nd:YAG laser, as a sample excitati on source, a T-format dual-channel signal detection system, a digitize r and a microcomputer for data processing. Nonlinear regression softwa re has also been developed, using a Marquardt curve-fitting procedure to extract time-resolved information on the resulting phosphorescence induced from the sample by the laser pulse. Two oppositely positioned photomultiplier tubes are used to collect the orthogonally polarized p hosphorescence components, which are digitized by a digital storage ad apter, and then transferred to an IBM microcomputer and the intensity or depolarization (anisotropy) of the emission is calculated. The rang e of emission lifetimes or rotational correlation times that can be ro utinely determined with this instrument extends from a few microsecond s to milliseconds. The instrument has an 8 bit magnitude resolution an d a signal sampling rate of 20 mega-samples per second. The averaging function of the instrument yields a single measurement with an average of over 2 to 255 excitation transients. To demonstrate the utility of the new instrument, bovine serum albumin has been employed as an anti gen, which is added to a monoclonal IgG anti-bovine serum albumin anti body and the formation of the antigen-antibody complex is studied usin g the technique. Enhancement of the emission anisotropy signal by immo bilization of the antibody on a large surface before addition of the a ntigen was also observed using this phosphorimeter.