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 micros was employed. The storage half-life of both immunoconjugates is extended more than 20-fold when compared to immunoconjugates prepared with BHHCT.
Department of Biological Sciences, Division of Environmental and Life Sciences, Macquarie University, Sydney, NSW 2109, Australia. rconnall@ics.mq.edu.au
Russell
R
Connally
Duncan
D
Veal
James
J
Piper
D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D002614:Chelating Agents / Q000378:metabolism*; D003458:Cryptosporidium / Q000276:immunology / Q000302:isolation & purification*; D005063:Europium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005872:Giardia / Q000276:immunology / Q000302:isolation & purification*; D006588:Hexanones / Q000378:metabolism*; D018796:Immunoconjugates; D008856:Microscopy, Fluorescence; D013730:Terphenyl Compounds / Q000378:metabolism*; D005466:Fluorocarbons
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D002614:Chelating Agents; D005063:Europium; D005456:Fluorescent Dyes; D006588:Hexanones; D018796:Immunoconjugates; D013730:Terphenyl Compounds; C110071:4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedione-6''-yl)chlorosulfo-o-terphenyl; D005466:Fluorocarbons