Time-resolved fluorescence microscopy using an improved europium chelate BHHST for the in situ detection of Cryptosporidium and Giardia

Fluorescent immunoconjugates prepared with the europium chelate BHHCT (4,4′-bis(1″,1″,1″,2″,2′,3″, 3″-heptafluoro-4″,6″-hexanedion-6″-yl) -chlorosulfo-o-terphenyl) have previously been reported as suitable labels for time-resolved fluorescence applications. BHHCT is limited by a tendency to destabilize immunoglobulins when covalently bound to the protein at moderate to high fluorophore to protein ratios (F/P). We report a new derivative of BHHCT prepared by appending a short hydrophylic tether to the chlorosulfonate activating group on BHHCT. The new derivative, BHHST (4,4′-bis-(1″, 1″,1″,2″,2″,3″,3″-heptafluoro-4″, 6″-hexanedion-6″-yl)sulfonylaminopropyl-ester-N-succinimide-ester-o- terphenyl), was activated to bind at the tether terminus with a succinimide leaving group that displayed less aggressive coupling activity and improved storage stability. BHHST has been used to prepare a stable and useful immunoconjugate with the anti-Cryptosporidium monoclonal antibody CRY104. The BHHST immunoconjugate provides more than a 10-fold enhancement in the signal to noise ratio (SNR) of labeled oocyst fluorescence over background when observed using TRFM techniques. An immunoconjugate was also prepared with BHHST and (goat) anti-mouse that effectively labeled Giardia cysts in situ. Detection of cysts with the TRFM was achieved with an 11-fold increase in SNR when a gate-delay of 60 μs was employed. The storage half-life of both immunoconjugates is extended more than 20-fold when compared to immunoconjugates prepared with BHHCT.