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. 2017 Sep 12;5(3):58.
doi: 10.3390/microorganisms5030058.

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

Sultana Akter  1 , Markus Vehniäinen  2 , Harri T Kankaanpää  3 , Urpo Lamminmäki  4
Affiliations

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

Sultana Akter et al. Microorganisms. .

Abstract

Nodularin (NOD) is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) monoclonal antibody (Mab). One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R) over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR). It was expressed in Escherichia coli as a single chain antibody fragment (scFv) fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD) of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R) water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain.

Keywords: anti-immunocomplex binder; cyanotoxin; hepatotoxin; nodularin; non-competitive immunoassay; quantification of nodularin; synthetic antibody phage library.

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Conflict of interest statement

S.A., M.V. and U.L. are inventors in a pending patent application PCT/FI2016/050911 concerning the anti-immunocomplex antibody described in the manuscript. The assignee of the application is University of Turku.

Figures

Figure 1
Figure 1
Cross-reaction profile of the anti-immunocomplex binder scFv-AP SA32C11 towards different cyanotoxins. The error bars represent the standard errors of means in the time-resolved fluorescence signals from duplicate measurements. The sample “10 MC-Mix” consists of an equimolar mixture of all the individually tested MCs (i.e., NOD not included). TRF: Time-resolved fluorescence. Cps: counts per second.
Figure 2
Figure 2
The dose-response curves for the single-step non-competitive assay with different reaction times. Each data point represents the average of duplicate measurements and the standard errors of the means are shown with error bars. TRF: Time-resolved fluorescence. Cps: counts per second.
Figure 3
Figure 3
The dose-response curves for the single-step non-competitive assay with different reaction volume. Each data point represents the average of duplicate measurements and the standard errors of the means are shown with error bars. TRF: Time-resolved fluorescence. Cps: counts per second.
Figure 4
Figure 4
Concentration of NOD and total toxin (MCs and NOD) in environmental water and surface bloom samples. The total toxin concentration was measured with the generic assay [41] using NOD-R as standard.
See this image and copyright information in PMC

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