Comprehensive analysis of monoclonal antibodies against detergent-insoluble membrane/lipid rafts of HL60 cells.

Glycosphingolipids and cholesterol are principal components of plasmamembrane microdomains, i.e. lipid rafts. Recent studies revealed the possible presence of a variety of microdomains that distinctly differ in terms of their molecular composition and functions. To understand their precise structures and functions, we produced monoclonal antibodies (MAbs) by immunizing mice to the microdomains prepared from a fraction of detergent-insoluble membrane (DIM) of HL60 cells. Biochemical characterization of the antigen epitopes led to classification of the MAbs into two groups. One group consists of MAbs that react with lipids such as phosphatidylglucoside, lysophosphatidylinositol, and gangliosides (GM1a and GD1b), and the other consists of MAbs that react with proteins such as annexin I, aminopeptidase N and acrogranin. Immunofluorescence staining of HL60 cells with the MAbs, except for the MAbs that recognize lysophosphatidylinositol or annexin I, resulted in patchy-like images of the cell membranes. Interestingly, MAbs belonging to the former group had the potential to induce cell proliferation/differentiation in vitro. Our MAbs against the DIM fraction of HL60 cells can be valuable tools for the study of membrane microdomains.