Homogeneous chemiluminescent immunoassay based on complement-mediated hemolysis of red blood cells

A novel method was developed for the homogeneous chemiluminescent immunoassay using complement-mediated hemolysis of sheep red blood cells. The chemiluminescent reaction of luminol and H2O2 was catalyzed by hemoglobins leaked from hemolyzed sheep red blood cells. The chemiluminescence was measured by counting photons. When using nonhemolyzed cells, chemiluminescence was virtually not observed. Only hemoglobin released from the cells was chemiluminescent, so that the extent of hemolysis could be measured without separating hemolyzed and nonhemolyzed cells. The immunoassay was done for the immunoagents: complement, hemolysin (anti-sheep red blood cell antibody), and anti-human albumin antibody. In the assay of complement or anti-human albumin antibody, sheep red blood cells used were bound with hemolysin or human albumin, respectively. The cell was hemolyzed by the action of antigen-antibody binding and subsequent activation of complement. The extent of hemolysis depended on the concentration of the antibody or complement. The calibration curves were obtained by chemiluminometric measurements on the added diluted antibody or complement. The sensitivity was 0.047 CH50 unit/mL for complement and below 1.0 micrograms/mL of anti-human albumin antibody.