Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay.
Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.
D000818:Animals; D000906:Antibodies / Q000502:physiology; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology*; D001667:Binding, Competitive; D004134:Dimyristoylphosphatidylcholine / Q000276:immunology; D004140:Dinitrophenols; D005452:Fluoresceins; D006021:Glycophorins / Q000032:analysis; D006241:Haptens / Q000276:immunology; D006801:Humans; D007158:Immunologic Techniques; D008027:Light; D008081:Liposomes / Q000032:analysis / Q000276:immunology*; D010316:Particle Size; D010714:Phosphatidylethanolamines / Q000276:immunology*; D011817:Rabbits; D012542:Scattering, Radiation; D012710:Serum Albumin, Bovine / Q000276:immunology
C021015:2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine; D000906:Antibodies; D000941:Antigens; D004140:Dinitrophenols; D005452:Fluoresceins; D006021:Glycophorins; D006241:Haptens; D008081:Liposomes; D010714:Phosphatidylethanolamines; C034638:dinitrophenyl-bovine serum albumin; D012710:Serum Albumin, Bovine; D004134:Dimyristoylphosphatidylcholine; C007740:fluorexon