Detection of soluble antigens by the reverse passive haemagglutination assay

A series of parameters that influence coupling of antibodies to the surface of erythrocytes with Cr3+ as well as the sensitivity and specificity of the reverse passive haemagglutination (RPH) assay in various antigen-antibody systems have been studied. The optimum coating of erythrocytes with antibodies was achieved at a final concentration of 30 micrograms CrCl3/ml with a protein antibody/CrCl3 ratio of 3.66 for most antibody IgG tested (anti-human IgG, anti-human albumin, anti-human hemoglobin and anti-rabbit IgG) and of 3.16 for anti-human myoglobin. The temperature and incubation time were of 30 degrees C and 60 min, respectively. Five soluble antigens were assayed by RPH: myoglobin, hemoglobin, IgG, human albumin and rabbit IgG. The minimum detectable analyte amount ranged between 1.75 ng/ml for human myoglobin and 17.16 ng/ml for human albumin. The reaction specificity was demonstrated by the absence of cross-reactions against heterologous antigens and the RPH inhibition reaction with immune sera. By stabilization with glutaraldehyde of antibody-coated erythrocytes the shelf-life was prolonged from seven days (non-stabilized erythrocytes) up to 300 days. The reverse passive haemagglutination assay is quite simple, rapid, easy in execution and by its high sensitivity is comparable to RIA and ELISA.