A semiquantitative rosetting assay for detection of cell-surface class I major histocompatibility complex molecules

Abstract

A method for detection of cell-surface antigens, referred to as cell-bead immunoassay (CBIA), has been developed by cross-linking monoclonal antibodies specific for cell-surface antigens to protein G-agarose beads. In this case, the antibodies were specific for different murine class I major histocompatibility complex (MHC) antigens and murine beta 2 microglobulin. The antibody-conjugated beads were incubated with cells expressing the relevant MHC molecule and observed microscopically for rosette formation. The number of cells bound per bead correlated with the amount of class I MHC expressed per cell, as measured by fluorescence activated cell sorting (FACS) analysis. In addition, changes in the amount of surface antigen expressed after induction could be followed by CBIA. The advantages of CBIA over other commonly used techniques, such as FACS and immunofluorescence, are that it requires only a few minutes incubation after beads are prepared, and no further manipulations are needed after the cells and beads are mixed together. Although CBIA is primarily a qualitative technique, it can also be used semiquantitatively by determining the number of cells bound per bead.