Detection of attograms of antigen by a high-sensitivity enzyme-linked immunoabsorbent assay (HS-ELISA) using a fluorogenic substrate.

In an attempt to increase the sensitivity of the ELISA technique three factors were examined: the use of a fluorogenic substrate (4MU-P), an increase in the antigen binding surface area, and the prolongation of the incubation time with the substrate. The use of the fluorogenic substrate and fluorometric detection method increased the sensitivity of the assay 16--39 times as compared to the colorigenic substrate (PNP-P). A 4-fold increase in the antigen binding surface area resulted in a 1000-fold increase in sensitivity, while prolonged incubation periods with the substrate at 10 degrees C yielded a further increase in sensitivity of 10--50-fold. Therefore, the present high sensitivity ELISA method (HS-ELISA) has the potential of increasing the sensitivity of the previously described method (Voller et al., 1974) by 1.6--19.5 X 10(5) times. HS-ELISA can detect as low as 3--10 attog/ml (3--10 X 10(-18) g/ml) antigen (Rb alpha M F(ab')2) or 24,000 molecules or purified mouse myeloma IgG. Our findings suggest that with simple modifications enzyme immunoassays can reach extreme levels of sensitivity.