Non-competitive ELISA with broad specificity for microcystins and nodularins

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 noncompetitive 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 (r 2 >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.

A Mini-Review on Detection Methods of Microcystins

“…In subsequent studies, IC-ELISA based on anti-Adda monoclonal antibody (mAb2G5), bifunctional single chain variable fragment-alkaline phosphatase fusion protein (scFv−AP), anti-MC-LR scFv7-scFv (MscFv7-scFv), as well as anti-MC-LR polyclonal antibodies and scFv (PAbs and scFv) were constructed for high MC-LR specificity and sensitivity [ 79 , 80 , 95 , 98 ]. It is of interest that the novel fluorometry noncompetitive ELISA based on synthetic broad-specific anti-immunocomplex antibody SA51D1, Fluorescent ELISA (FELISA) based on silane-doped carbon dots and Norwegian ELISA successfully detected varying variants of MC below the WHO’s guideline value of 1 µg/L [ 87 , 97 , 108 ]. Moreover, to detect MCs in animal cells and tissues, a direct monoclonal ELISA (DM-ELISA) has been developed for rapid and easy detection [ 119 ].…”

“…Generally, ELISA is capable of yielding repeatability, reproducibility and variability results of MCs concentrations compared to the other methods [ 86 , 87 , 107 , 118 ]. Besides, no sample cleanup is needed, detection limits are often below the WHO’s 1 µg/L guideline value, and it is sensitive to low pH (formic acid), MeOH or MeCN [ 79 , 87 , 88 , 114 , 115 ]. ELISA can be used to determine the biological evidence of human exposure to MCs.…”

Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin

“…For these samples extracted total toxin concentrations (MC-LR equivalent) had been previously analyzed by LC-MS and published earlier [ 47 ]. Also the toxin analogues in these samples were known [ 41 , 48 ]. The lakewaters contained no NOD but most of them contained MCs.…”

“…Furthermore, there was no indication of significant interferences with different water matrices. The assay was then challenged by a set of sea and lakewater samples, most of which contained various MCs as revealed by the previous LC-MS analysis [ 41 , 47 , 48 ]. Among a total of 15 samples, the assay was able to correctly detect the two NOD-containing samples in a good accordance with the concentrations measured by LC-MS ( Table 3 ).…”

“…As a control, thirteen lake water samples and two seawater samples from Finland and Estonia ( Table 1 ) were tested by the NOD-specific immunoassay using 100 µL total volume (50% sample and standard). For these samples, total toxin concentrations (cell extracted) as well as the toxin variants identified by LC-MS were already known [ 41 , 47 , 48 ]. All lake water samples were NOD-negative and most of them were positive for microcystins.…”