Non-competitive ELISA with broad specificity for microcystins and nodularins

  • Sultana Akter | suakte@utu.fi University of Turku, Finland.
  • Markus Vehniäinen University of Turku, Finland.
  • Jussi Meriluoto Åbo Akademi University, Finland.
  • Lisa Spoof Åbo Akademi University, Finland.
  • Urpo Lamminmäki University of Turku, Finland.

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.

 

Dimensions

Altmetric

PlumX Metrics

Downloads

Download data is not yet available.

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
non-competitive ELISA for cyanotoxins
Published
2017-05-03
Info
Issue
Section
Toxic cyanobacteria / cyanotoxin detection techniques
Supporting Agencies
This work has been financially supported (grant 823/31/2014) by the National Technology Agency of Finland (TEKES). Sultana Akter received grants from MVTT, Maa- ja vesitekniikan tuki (grant 24808) and from Turku University Foundation (grant 10146)
Keywords:
Immunocomplex assay, sandwich-type ELISA, cyanotoxin, microcystin, nodularin, harmful algal bloom.
Statistics
  • Abstract views: 2129

  • PDF: 629
  • Supplementary: 211
  • HTML: 349
How to Cite
Akter, S., Vehniäinen, M., Meriluoto, J., Spoof, L., & Lamminmäki, U. (2017). Non-competitive ELISA with broad specificity for microcystins and nodularins. Advances in Oceanography and Limnology, 8(1). https://doi.org/10.4081/aiol.2017.6349