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).