ANALYTE DETECTION WITH DNA-LABELED ANTIBODIES AND POLYMERASE CHAIN-REACTION

We demonstrate immuno-polymerase chain reaction (PCR) assays for two c linical analytes-human thyroid-stimulating hormone and chorionic gonad otropin (hTSH, hCG)-using DNA-labeled antibodies and PCR for amplifica tion of assay response. DNA-antibody conjugates were synthesized by us ing heterobifunctional cross-linker chemistries to covalently attach s ingle- or double-stranded DNA labels through amine or sulfhydryl group s on the analyte antibodies. These approaches yielded molecular chimer as possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demons trated for immuno-PCR assays of both analytes in a microtiter plate-ba sed, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exce eded those of conventional enzyme immunoassays by 2-3 orders of magnit ude. We also evaluated various DNA design factors influencing label am plification and assay performance, such as primer sequence, strand num ber, and DNA length. Our findings, in concert with previous reports, s uggest that this hybrid technology could provide the basis for a new g eneration of ultra-sensitive immunoassays offering multianalyte capabi lities.