A fluorescence detection system for the analytical ultracentrifuge and its application to proteins, nucleic acids, and viruses

Abstract

A fluorescence detection system was developed for the analytical ultracentrifuge Spinco model E. Fluorescence is excited by a laser beam which is focussed into the cell and illuminates an area with a dimension of 60 μm in radial direction. For scanning the laser beam is moved in radial direction. After passing the cell, the laser beam is quenched by a carbon light trap and a set of optical filters. Fluorescence emission intensity is monitored by a photomultiplier located behind the light trap and the set of filters. The sensitivity of the detection system was tested by applying it to the sedimentation analysis of proteins and nucleic acids. Bovine serum albumin (BSA) was covalently labelled with the fluorescence-dye fluorescein-isothiocyanate (FITC), and its sedimentation coefficient could be determined even if BSA was analyzed in a concentration as low as 10⁻¹⁰ M. Nucleic acids were labelled non-covalently by the intercalating dye ethidium bromide. Only 8 ng RNA were needed for the determination of the sedimentation coefficient. The particular advantages of the fluorescence detection system were exploited for the establishment of a new method for quantitative virus detection. To tobacco mosaic virus (TMV) a monoclonal anti-TMV antibody from mouse was bound, and to this a second, anti-mouse antibody that carried the fluorescence-label FITC was attached. Either by UV-irradiation or by incubation with glutaraldehyde, the first antibody was covalently crosslinked to TMV, and the second antibody to the first. In CsCl density centrifugation with fluorescence detection as little as 3.2 ng virus/80 μl or 6×10⁸ virus particles/ml were recorded in a well expressed band at the corresponding buoyant density. Tenfold lower concentration would result still in a significant band. The sensitivity compares well with those of the most advanced techniques from immunology. Due to the specific labelling of viruses by antibodies it will be possible to carry out quantitative physical characterization of virus containing samples without purifying the virus. Future applications of the fluorescence detection system and of the virus detection technique are discussed.