A new europium beta-diketone chelate for ultrasensitive time-resolved fluorescence immunoassays.
4,4'-Bis(1",1",1"-trifluoro-2",4"-butanedione-6"-yl)-chlorosulfo-o-terphenyl (BTBCT) was synthesized by modifying the structure of the reported BHHCT. In comparison with the original BHHCT, the detection sensitivity of BTBCT-Eu chelate in aqueous solution was improved approximately 8 times by time-resolved fluorescence measurement. To construct sensitive TRFIAs with the use of BTBCT-Eu chelate as the fluorescent label, streptavidin-BSA conjugate was prepared by the maleimide-thiol method and labeled by BTBCT. The streptavidin-BSA conjugate and its BTBCT-labeled complex were affinity-purified using 2-iminobiotin-agarose as binding reagent. With streptavidin-BSA-BTBCT-Eu complex as signal generation reagent, a highly sensitive indirect serum hTSH TR-IFMA was developed. The low limit of detection (LLD) of the TSH TR-IFMA was 0.011 mIU/L with 10 microl of sample volume, corresponding to approximately 337,900 molecules per test. To evaluate the utility of BTBCT-Eu label in direct TRFIAs, a competitive serum T4 TRFIA was developed with T4-BSA-BTBCT-Eu complex as competing tracer. The measurements obtained by the present TSH TR-IFMA or T4 TRFIA correlated well with those obtained by commercial Wallac TSH DELFIA Ultra or T4 DELFIA, respectively. Primary results show that BTBCT can be employed as a powerful labeling material for constructing ultrasensitive TRFIAs.
Wu
Feng-Bo
FB
Department of Isotope, China Institute of Atomic Energy, PO Box 275-39, Beijing 102413, People's Republic of China. wufb@etang.com
Zhang
Chao
C
D000818:Animals; D002138:Calibration; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry*; D005063:Europium / Q000737:chemistry*; D015200:Fluoroimmunoassay / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007659:Ketones / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013223:Statistics as Topic; D019809:Streptavidin / Q000737:chemistry; D013972:Thyrotropin / Q000097:blood*; D013974:Thyroxine / Q000097:blood*
D002614:Chelating Agents; D007659:Ketones; D009942:Organometallic Compounds; D012710:Serum Albumin, Bovine; D005063:Europium; D013972:Thyrotropin; D019809:Streptavidin; D013974:Thyroxine