Development of highly fluorescent detection reagents for the construction of ultrasensitive immunoassays

We developed two kinds of highly fluorescent streptavidin-based conjugates for use as universal detection reagents in ultrasensitive immunoassays, The direct conjugate was produced by covalently linking streptavidin to poly(G lu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to t he conjugate of BSA-poly(Glu:Lys)-Eu chelate, Both direct and indirect conj ugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA), Of two monoclonal antibodies us ed in the assay, one was coated on the well surface of the microtitration s trips, and the other was biotinylated, When 10 muL of sample volume was use d, we found that the assay using the indirect conjugate had a detection lim it of 0.006 mug/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin, However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate, When 45 muL of sample volume was used, a detection limit of 0.001 mug/L was achieved by using the direct con jugate. This improvement in sensitivity should be equally obtainable for th e analytes other than PSA, We further demonstrated that the final immunoass ay performance was affected not only by the quality of the streptavidin-bas ed conjugate used but also by the quality of the biotinylated antibody reag ent. The universal detection reagents described here are believed to be par ticularly useful for the construction of ultrasensitive time-resolved fluor ometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection.