The selective detection of cell surface determinants by means of antibodies and acetylated avidin attached to highly fluorescent polymer microspheres.
Procedures are described for the synthesis of 500 A-diameter polymer microspheres containing a novel fluorescent cross-linking agent. These microspheres have very high fluorophore concentration without quenching of the fluorescence and show very low nonspecific interaction with cells. When monoclonal anti-Thy-1.2 is attached to the fluorescent microspheres, specific binding results in 10(4) spheres being attached per thymocyte while non-specific binding is less than 1%. Similar values are obtained for an indirect staining procedure. The high non-specific binding of cationic avidin to negative cell surfaces is shown to be decreased to negligible levels by acetylation of the amine groups of the protein without decreasing its high-affinity binding to biotin. The use of acetyl-avidin (pI = 6.7) directly, or when attached to fluorescent microspheres, resulted in a highly selective detection of biotinyl groups on the erythrocyte or lymphocyte cell surface. Attachment of biotinyl groups to the hinge carbohydrates of antibodies did not affect their specificity. It allowed their detection by means of microspheres-acetyl-avidin conjugates.
D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface / Q000032:analysis*; D001360:Avidin; D002462:Cell Membrane / Q000276:immunology*; D000939:Epitopes / Q000032:analysis*; D004910:Erythrocyte Membrane / Q000276:immunology*; D004912:Erythrocytes / Q000276:immunology*; D005452:Fluoresceins / Q000138:chemical synthesis; D005456:Fluorescent Dyes; D006801:Humans; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008863:Microspheres
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D000939:Epitopes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D001360:Avidin; C037251:di(methylene diallylamine)fluorescein diacetate