Broad-Spectrum Noncompetitive Immunocomplex Immunoassay for Cyanobacterial Peptide Hepatotoxins (Microcystins and Nodularins)

Abstract

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.

Non-competitive ELISA with broad specificity for microcystins and nodularins

“…Allowing reliable analysis outside well-equipped (Savela et al, 2014);°main toxin variants are highlighted in bold; # corresponding raw water samples collected from these sources were used in spiking experiment; § commercial immunoassay, PPIA, HPLC and the LC-MS results for this sample was published earlier (Akter et al, 2016). MC, microcystin; Nod, nodularin; nd, not detected.…”

“…The toxin content in the lake water was undetectable by the non-competitive ELISA and the commercial ELISA. However, it contained a low amount of toxin (0.03 µg L -1 ), as revealed by the TRF assay (Akter et al, 2016). The recovery values ranged from 64% to 101% and the coefficient of variation % (CV%) values of the measurements were below 10.5 for the spiking concentrations of 0.5 to 4 µg L -1 .…”

“…1). The isolation and characterization of the binder antibody was described in detail earlier (Akter et al, 2016).…”

“…The spiked samples and the corresponding unspiked samples were measured by the noncompetitive ELISA in duplicates. The unspiked samples were also measured by a commercial immunoassay (Microcystins-ADDA ELISA, Abraxis, PA, USA) and by the time resolved fluorescent measurement based non-competitive immunoassay (Akter et al, 2016). Presence of toxin in the unspiked samples by any of the three tested methods was taken into consideration for recovery calculation.…”

“…We have recently reported isolation of a unique generic anti-immunocomplex binder from our in-house synthetic antibody library and development of a broad-spectrum noncompetitive immunocomplex immunoassay for microcystins and nodularins (Akter et al, 2016). The described time-resolved fluorometry (TRF) based assay is highly sensitive and rapid.…”