A homogeneous immunofluorescence assay based on dye-sensitized photobleaching.
A novel homogeneous immunoassay requiring only one incubation step, and applicable in principle to the determination of low- as well as high-molecular-weight substances, has been developed. The method is based on (a) photooxidation by singlet oxygen (1O2) of a fluorescent substrate (1,3-diphenylisobenzofuran, DPBF) embedded in unilamellar vesicles on the surface of which antibody to the analyte antigen is covalently attached (DPBF-immunoliposomes); (b) generation of singlet oxygen, upon illumination, by a chromophore (erythrosine) covalently attached to an antibody (Ab*) or antigen (Ag*); (c) formation of a "sandwich"- or "competition"-type complex whereupon the singlet oxygen-generating chromophore conjugate (Ab* or Ag*) and immunoliposome-embedded DPBF are brought into close proximity. Competition- and sandwich-type model assay systems for the detection of protein antigens and viruses were investigated. The detection range with protein antigens in competition- and sandwich-type assays was three (10(-10) - 10(-7) M) and two (10(-10) - 10(-8) M) orders of magnitude, respectively. With poliovirus using a sandwich-type assay, the detection range was 10(2) - 10(6) plaque-forming units per milliliter (pfu/ml).
Bystryak
S
S
Department of Human Genetics, Sackler Faculty of Medicine, Tel Aviv University, Israel.
Goldiner
I
I
Niv
A
A
Nasser
A M
AM
Goldstein
L
L
D000818:Animals; D000906:Antibodies; D000937:Antigen-Antibody Reactions; D001572:Benzofurans; D004923:Erythrosine; D005455:Fluorescent Antibody Technique; D006041:Goats / Q000276:immunology; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D007700:Kinetics; D008081:Liposomes; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010100:Oxygen; D010782:Photolysis; D011817:Rabbits / Q000276:immunology; D026082:Singlet Oxygen
D000906:Antibodies; D001572:Benzofurans; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008081:Liposomes; D026082:Singlet Oxygen; C011238:1,3-diphenylisobenzofuran; D004923:Erythrosine; D010100:Oxygen