Time-resolvable fluorescent conjugates for the detection of pathogens in environmental samples containing autofluorescent material

Abstract

Water is routinely monitored for environmental pathogens such a Cryptosporidium and Giardia using immunofluorescence microscopy (IFM). Autofluorescence can greatly diminish an operators capacity to resolve labeled pathogens from non-specific background. Naturally fluorescing components (autofluorophores) encountered in biological samples typically have fluorescent lifetimes (τ) of less than 100 nanoseconds and their emissions may be excluded through use of time-resolved fluorescence microscopy (TRFM). TRFM relies on the large differences in τ between autofluorescent molecules and long-lived lanthanide chelates. In TRFM, targets labeled with a time-resolvable fluorescent immunoconjugate are excited by an intense (UV) light pulse. A short delay is imposed to permit the decay of autofluorescence before capture of luminescence from the excited chelate using an image intensified CCD camera. In our experience, autofluorescence can be reduced to insignificant levels with a consequent 30-fold increase in target visibility using TRFM techniques. We report conjugation of a novel europium chelate to a monoclonal antibody specific for Giardia lamblia and use of the immunoconjugate for TRFM studies. Initial attempts to conjugate the same chelate to a monoclonal antibody directed against Cryptosporidium parvum led to poorly fluorescent constructs that were prone to denature and precipitate. We successfully conjugated BHHCT to anti-mouse polyvalent immunoglobulin and used this construct to overcome the difficulties in direct labeling of the anti-Cryptosporidium antibody. Both Giardia and Cryptosporidium were labeled using the anti-mouse protocol with a subsequent 20-fold and 6.6-fold suppression of autofluorescence respectively. A rapid protocol for conjugating and purifying the immunoconjugate was found and methods of quantifying the fluorescence to protein ratio determined. Performance of our TRFM was dependent on the quality and brightness of the immunoconjugate and optimization of the conjugation process is necessary to reap the full benefit of time-resolved techniques.

Long-lived visible luminescence of UV LEDs and impact on LED excited time-resolved fluorescence applications

“…Xenon flashlamps and nitrogen lasers are two of the conventional pulsed excitation sources employed for TRF, however, both have serious drawbacks. In flashlamps, a luminous plasma remains for many tens of microseconds after the primary discharge and this remnant glow competes with probe luminescence to significantly degrade SNR [9,17]. N 2 lasers on the other hand suffer from low repetition rate, are expensive, have a limited lifetime and emit electromagnetic intereference unless carefully shielded.…”

Flash lamp-excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver an 11-fold increase in signal-to-noise ratio

“…However, using similar techniques we have successfully labeled Giardia cysts and Cryptosporidium oocysts in situ with time-resolvable immunofluorophores. 25…”