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)sulfonylamino‐propyl‐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. Microsc. Res. Tech. 64:312–322, 2004. © 2004 Wiley‐Liss, Inc.