Simple and rapid methods are required for screening and analysis of
water samples to detect cyanobacterial cyclic peptide hepatotoxins:
microcystin/nodularin. Previously, we reported a highly sensitive
non-competitive heterogeneous assay for microcystin/nodularin utilizing a
generic anti-immunocomplex (anti-IC) single-chain fragment of antibody
variable domains (scFv) isolated from a synthetic antibody library
together with a generic adda
((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic
acid)-specific monoclonal antibody (Mab) recognizing the common adda
part of the microcystin/nodularin. Using the same antibody pair, here we
report a homogeneous non-competitive assay for microcystin/nodularin
based on TR-FRET (time-resolved Förster resonance energy transfer)
measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab
labeled with europium enabled the FRET process to occur in the presence
of microcystin/nodularin. The TR-FRET signal is proportional to the
toxin concentration in the sample. The rapid (15 min) homogeneous assay
without requiring any washing step detected all the tested nine toxin
variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and
nodularin-R). Very good signal to blank ratio (~13) was achieved using
microcystin-LR and the sample detection limit (blank+3SD of blank) for
microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The
practical application of the TR-FRET assay was demonstrated with water
samples spiked with microcystin-LR as well as with environmental water.
The average recoveries of microcystin-LR from spiked water ranged from
65 to 123%. Good correlation (r² = 0.73 to 0.99) with
other methods (liquid chromatography-mass spectrometry and previously
reported heterogeneous assay) was found when environmental samples were
analyzed. The developed wash-free assay has the potential to play as a
quick screening tool to detect microcystin/nodularin from water below
the World Health Organization’s guideline limit (1 μg/L of
microcystin-LR).