Rare Earth Labels Based Time-resolved Fluoroimmunoassay Detection Systems

Stable fluorescent labels BHHCT-Eu3+, BPTA-Tb3", and europium nanoparticle were used to develop sandwich-type time-resolved fluoroimmunoassays for HBsAg. Fluorescence intensity was directly detected on the surface of the solid phase. The detection limits using four labels BHHCT-Eu3+-HBsAb, BHHCT-Eu3+-SA-BSA, BPTA-Tb3+-SA, and europium nanoparticle-HBsAb were 0.2 ng/mL, 0.05 ng/mL, 0.06 ng/mL and 0.03 ng/mL respectively. The calibration curves were in the range of 0-100 ng/mL (BPTA-Tb3+-SA-TrFIA was 0-50 ng/mL). These results obviously showed improved quantification performance of TrFIA compared with enzyme-linked immunosorbent assay, of which the detection limit was 0.5 ng/mL and the dynamic range was within 0-10 ng/mL. The within-run coefficient variations were all less than 10%. The analytical recoveries of the latter three methods were within 85%-110%. Totally 31 patient samples were detected by europium nanoparticle-HBsAb TrFIA and conventional ELISA, and the correlation coefficient was 0.98. Using magnetic microparticle as the solid phase and europium nanoparticle as the label, a new TrFIA of magnetic separation was developed to detected HBsAg. The detection limit was 0.5 ng/mL and the calibration curve was in the range of 0-100 ng/mL. The within-run coefficient variations for standard samples were less than 10%. One distinguished feature of this approach was its nearly homogeneous reaction and solution phase detection, which made the detection rapid and reproducible. Time-resolved fluoroimmunoassays using these rare earth labels are characteristic of high sensitivity, least interference,good precision and wide dynamic range. The labeling procedures are simple and the labels are stable. In conclusion, TrFIA using these labels will find wide applications in clinical diagnosis.