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A novel dual-channel phosphorimeter for studies of antigen-antibody complexes

Liqun Yang, D McStay, A J Rogers and P J Quinn

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A phosphorescence anisotropy selective technique is proposed as a new method for real-time monitoring of immunological reactions and formation of antigen-antibody complexes. To assess the utility of the technique, a novel phosphorimeter has been developed. The phosphorimeter consists of a pulsed, frequency-doubled Nd:YAG laser, as a sample excitation source, a T-format dual-channel signal detection system, a digitizer and a microcomputer for data processing. Nonlinear regression software 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 phosphorescence components, which are digitized by a digital storage adapter, and then transferred to an IBM microcomputer and the intensity or depolarization (anisotropy) of the emission is calculated. The range of emission lifetimes or rotational correlation times that can be routinely determined with this instrument extends from a few microseconds to milliseconds. The instrument has an 8 bit magnitude resolution and 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 antigen, which is added to a monoclonal IgG anti-bovine serum albumin antibody and the formation of the antigen-antibody complex is studied using the technique. Enhancement of the emission anisotropy signal by immobilization of the antibody on a large surface before addition of the antigen was also observed using this phosphorimeter.


PACS

87.80.-y Biophysical techniques (research methods)

87.15.R- Reactions and kinetics

33.50.Dq Fluorescence and phosphorescence spectra

87.15.K- Molecular interactions; membrane-protein interactions

42.62.Be Biological and medical applications

87.15.M- Spectra of biomolecules

Subjects

Atomic and molecular physics

Instrumentation and measurement

Optics, quantum optics and lasers

Medical physics

Biological physics

Dates

Issue 9 (September 1994)



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