Non-competitive ELISA with broad specificity for microcystins and nodularins

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Sultana Akter *
Markus Vehniäinen
Jussi Meriluoto
Lisa Spoof
Urpo Lamminmäki
(*) Corresponding Author:
Sultana Akter | suakte@utu.fi

Abstract

Simple and cost-effective methods with sufficient sensitivities for preliminary screening of cyanobacterial toxins are in high demand for assessing water quality and safety. We have recently developed a highly sensitive and rapid time-resolved fluorometry based non-competitive immunoassay for detection of microcystins and nodularins. The assay is based on a synthetic broad-specific anti-immunocomplex antibody SA51D1 capable of recognizing the immunocomplex formed by a generic anti-Adda monoclonal antibody (mAb) bound to either microcystins or nodularins. Using the same antibody pair, here we describe a very simple and cost-efficient non-competitive ELISA test for microcystins and nodularins based on conventional alkaline phosphatase (AP) activity measurement. The recombinant SA51D1 single-chain fragment of antibody variable domain (scFv) was produced as a fusion with bacterial alkaline phosphatase in Escherichia coli. After one step affinity purification through His-tag, the scFv-AP fusion protein could directly be used in the assay. For the assay, toxin standard/sample, biotinylated anti-Adda mAb and the scFv-AP were incubated together for one hour on streptavidin-coated microtiter wells, washed and AP activity was then measured by incubating (1 h at 37 ˚C) with chromogenic substrate para-nitrophenylphosphate (pNPP). The assay was capable of detecting all the eleven tested toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, LA -LY, -LF -LW, -WR, and nodularin-R) below WHO guide line value of 1 µg L-1. The detection limit (based on blank+3SD response) for microcystin-LR was ~0.2 µg L-1. The assay was verified using spiked (0.25 - 4 µg L-1 of microcystin-LR) tap, river and lake water samples with recoveries from 64 to 101%. The assay showed good correlation (r2>0.9) with four reference methods for its performance in detecting extracted intracellular microcystin/nodularin from 17 natural surface water samples. The described easy-to-perform assay has a high potential to be used in resource-poor settings as quantitative measurements can be obtained using a simple ELISA reader or easy-to-interpret qualitative results by visual readout. Based on the non-competitive format, the assay does not need any chemical toxin conjugates and offers robustness as compared to the currently available competitive format assays.

 


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Author Biographies

Sultana Akter, University of Turku

Molecular Biotechnology and Diagnostics, Department of Biochemistry

Markus Vehniäinen, University of Turku

Molecular Biotechnology and Diagnostics, Department of Biochemistry

Jussi Meriluoto, Åbo Akademi University

Biochemistry, Faculty of Science and Engineering,

Lisa Spoof, Åbo Akademi University

Biochemistry, Faculty of Science and Engineering

Urpo Lamminmäki, University of Turku

Molecular Biotechnology and Diagnostics, Department of Biochemistry