BHHST : An Improved Lanthanide Chelate for Time-Resolved Fluorescence Applications

摘要

The unique discriminatory power that arises from the use of immunofluorescence techniques within the field offluorescence microscopy has ensured the widespread adoption of this method. However the utility ofimmunofluorescence methods can at times be greatly diminished due to the ubiquity of naturally occurring, nativelyfluorescent substances (autofluorophores).Time resolved fluorescence techniques have proven useful in reducing or eliminating the difficulties caused byautofluorescence. These technique rely on pulsed excitation to excite fluorescence from probe and autofluorophoresalike, however the capture of fluorescence is delayed until the short-lived (nanosecond regime) autofluorescence hassubstantially decayed. Fluorescence from the long-lived probe however, decays only marginally within this short hold-offperiod and may be captured using an appropriately gated image-intensified CCD camera.Discrimination of probe fluorescence is enhanced when probe τ is several orders of magnitude greater than typicalautofluorescence τ and several lanthanide chelates have been identified that offer long-lived and intense fluorescence.BHHCT (4,4'-bis(1',1',1',2',2',3',3'-heptafluoro-4',6'-hexanedion-6'-yl)-chlorosulfo-o-terphenyl) is a recentlysynthesized europium chelate that is strongly fluorescent and has a characteristic τ of greater than 600 μs. The compoundhas been employed as the reagent for ultrasensitive time-resolved fluorescence assays and also for the in-situ detection ofGiardia cysts within strongly autofluorescent water concentrates.Significant difficulties can be encountered in the preparation of immunoconjugates with BHHCT because the chelate isstrongly hydrophobic and the resultant conjugates have poor stability and were often inactive at higher fluorophore toprotein (F/P) ratios. To overcome this difficulty, we appended a short hydrophilic tether to the molecule via achlorosulfonate group and then activated the new compound, BHHST (4,4’-bis-(1”,1”,1”,2”,2”,3”,3”-heptafluoro-4”,6”-hexanedion-6”-yl)sulfonylamino-propyl-ester-N-succinimide-ester-o-terphenyl), to bind to protein via a mildersuccinimide activating group. Previous attempts to prepare a BHHCT immunoconjugate against Cryptosporidium hadmet with no success, however a strongly fluorescent conjugate was successfully prepared when BHHST was employed.The F/P ratio of the immunoconjugate was determined to be about 6:1 and stability of the preparation was significantlyimproved over BHHCT conjugates with a half-life of 6 months. Cryptosporidium oocysts were labeled in-situ against astrongly autofluorescent matrix of algae and the signal to noise ratio of the detection system (epifluorescencemicroscope) was improved more than 8-fold with a 12-fold reduction in autofluorescence intensity. BHHST was alsoconjugated to (goat) anti-mouse immunoglobulin for use in indirect immunofluorescence studies with Giardia cysts togive an 8-fold increase in SNR over conventional epifluorescence mode. BHHST is more easily purified than BHHCTand the succinimide activating group is far less labile than the chlorosulfonate group of the parent compound.