A new microsphere-based immunofluorescence assay for antibodies to membrane-associated antigens.
The screening of panels of hybridoma supernatants for specific secreted monoclonal antibodies is often achieved by cellular immunofluorescent staining and flow cytometric analysis. In some circumstances such assays are difficult because the required antigen-bearing cell population is not suitable for use in flow cytometry, has limited cell cycle expression or poor in vitro growth. A method is presented here that provides a solution to these difficulties. A system was developed, using polyacrylamide microspheres coupled with cell membranes, which permitted the production of an easily stored, standardised antigen source which could be used in subsequent flow cytometric assays. Studies comparing the binding of antibodies to whole cells and cell membrane-coupled microspheres indicate a strong qualitative and quantitative correlation in the expression of surface antigens. It is shown here that membrane antigens can be stored coupled to microspheres for months and still retain good reactivity with the appropriate antibodies. This same technique could be used in studies of antigens other than those of the mammalian cell membrane - for example, membrane antigens of sub-cellular organelles such as the mitochondrion and endoplasmic reticulum, plant or bacterial membrane antigens, or antigens not associated with membranes such as viral proteins.
Wilson
M R
MR
Clinical Immunology Research Centre, University of Sydney, NSW, Australia.
Mulligan
S P
SP
Raison
R L
RL
D000911:Antibodies, Monoclonal / Q000032:analysis*; D000954:Antigens, Surface / Q000276:immunology*; D001402:B-Lymphocytes / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008863:Microspheres
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface