Novel solution-phase immunoassays for molecular analysis of tumor markers

Molecular forms of serum prostate-specific antigens (PSA-free, PSA-total, P SA-complex) were conjugated with an N-hydroxysuccinimide ester of a rutheni um(ii) tris-bipyridine (Ru(bpt)(3)(2+)-NHS) which provided both electrochem iluminescence (ECL) and phosphorescence. ECL intensity of labeled PSA, prod uced upon oxidation of Ru(bpy)(3)(2+) in a solution containing tri-n-propyl amine, was proportional to the concentration of PSA and inversely proportio nal to its mass and size of PSA. ECL intensity of the PSA-free antigen decr eased upon binding with its monoclonal antibody (MAB) due to the increased mass and size of the conjugated pair and the decrease in diffusion coeffici ent. The binding affinity of PSA-free antigen with its MAB at 1 x 10(10) M- 1 was determined. However, no binding of PSA-free antigen with the MAB of P SA-complex was observed. A similar approach was applied to study labeled PS A-complex indicating affinity of PSA-complex to its MAB at 3 x 10(9) M-1 an d a step-wise binding process with PSA-free MAB. Thus, this solution-phase quantitative ECL immunoassay allowed measurement of the affinity of serum P SAs with their MABs and screening of PSAs based upon their affinity to MABs . Unlike other immunoassays, this immunoassay demonstrated one-step rapid a nalysis while simultaneously eliminating immobilization, separation and was hing steps and detected PSA at a level of 1.7 pg mL(-1), which is 1000-fold more sensitive than current PSA immunoassays. Furthermore, single-molecule (SM) phosphorescence microscopy was developed to detect single serum PSA-f ree and PSA-complex molecules in solution with no use of antibody showing t hat PSA-free molecules diffused faster than PSA-complex molecules in soluti on. This finding is consistent with ECL measurements and implies the possib ility of screening individual analytes in a complex mixture using their dis tinct SM diffusion distance. This is the first report describing the detect ion of single protein molecules labeled with a metal-complex using phosphor escence microscopy and also the screening of serum tumor markers using ECL and SM phosphorescence solution-phase assays.