A Homogeneous Immunofluorescence Assay Based on Dye-Sensitized Photobleaching

• Published in: Analytical biochemistry 1995-02, Vol.225 (1), p.127-134
• Main Authors: Bystryak, S., Goldiner, I., Niv, A., Nasser, A.M., Goldstein, L.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antigen-Antibody Reactions; Benzofurans; Erythrosine; Fluorescent Antibody Technique; Goats - immunology; Immunoglobulin G; Indicators and Reagents; Kinetics; Liposomes; Molecular Weight; Oxidation-Reduction; Oxygen; Photolysis; Rabbits - immunology; Singlet Oxygen
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1995.1118

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 ( 1O 2) 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).