A broad-spectrum noncompetitive immunoassay allowing sensitive and
simple detection of a group of similar compounds would be an ideal tool
for screening low-molecular weight analytes (<2000 Da) having many
variants. However, the development of an essential antibody pair capable
of sandwich-type recognition of the analytes’ small generic core
structure is a demanding task due to limited space available for
simultaneous binding of two different antibodies. We report here a
generic noncompetitive assay for cyanobacterial microcystins (MCs) and
nodularins (Nod), a group of structurally related small cyclic peptides
(∼1000 Da) with more than 100 naturally occurring analogs. The assay is
based on the unique combination of a generic anti-immunocomplex
(anti-IC) single-chain fragment of antibody variable domain (scFv) and a
monoclonal antibody capable of binding to an Adda-group
(3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic
acid) present in all MCs/Nod. The anti-IC scFv was isolated from a
large synthetic antibody library with phage display and used to develop a
single-step sandwich-type noncompetitive immunocomplex assay. The
sensitive time-resolved immunofluorometry-based assay is capable of
detecting all the 11 tested commonly occurring hepatotoxins (MC-LR,
-dmlR, -RR, -dmRR, -LA, -LY, -LF, -LW, -YR, -WR, and Nod-R) at
concentration below 0.1 μg/L in a 1 h assay. Using MC-LR, the most
studied toxic and widely distributed of the toxins, the calculated
detection limits (based on blank + 3SD response) are ∼0.026 μg/L in 1 h
and ∼0.1 μg/L in 10 min assay time. This is by far the fastest reported
immunoassay for MCs and Nod with a detection limit far below the World
Health Organization’s guideline limit (1 μg/L of MC-LR equivalent in
drinking water). The assay was validated with spiked tap and lake water
as well as with environmental surface water samples. The developed assay
provides a simple, rapid, and highly sensitive tool for the
quantitative detection of MCs/Nod with the additional benefit of
automation and high-throughput possibilities for large scale screening
of drinking and environmental surface water samples. Furthermore, the
study describes the first demonstration of the assay intended for the
detection of an analyte group comprising similar low-molecular weight
compounds exhibiting the benefits of a reagent excess type assay.