Abstract
Nanobodies have several advantages, including great stability, sensibility, and ease of production; therefore, they have become important tools in immunoassays for chemical contaminants. In this manuscript, nanobodies for the detection of the toxin Nodularin-r (NOD-R), a secondary metabolite of cyanobacteria that could cause a safety risk for drinks and food for its strong hepatotoxicity, were for the first time selected from an immunized Bactrian camel VHH phage display library. Then, a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for NOD-R, based on the nanobody N56 with great thermostability and organic solvent tolerance, was established under optimized conditions. The results showed that the limit of detection for NOD-R was 0.67 µg/L, and the average spike recovery rate was between 84.0 and 118.3%. Moreover, the ic-ELISA method was validated with spiked water sample and confirmed by UPLC–MS/MS, which indicated that the ic-ELISA established in this work is a reproducible detection assay for nodularin residues in water samples.
Highlights
Nodularins (NODs) are harmful cyanobacteria toxins produced by Nodularia spumigena, especially when blooms brake out in lakes or rivers [1]
NOD-R is a characteristic cyclic peptide composed of five amino acids (-D-MeAsp–L-Y–Adda–D-Glu–Mdhb), in which the Adda structural region ((2S,3S,8S,9S)-3-amino- 9-methoxy-2,6,8-trimethyl-10-phenyl deca-4,6-dienoic acid) is found in microcystins (MCs), another group of cyanobacteria toxins composed of heptapeptides
The serum titer was up to 1:64,000, and inhibition of NOD-R was higher than 80%
Summary
Nodularins (NODs) are harmful cyanobacteria toxins produced by Nodularia spumigena, especially when blooms brake out in lakes or rivers [1]. Different forms of NOD variants have been found. Among these variants, NOD-R is the most abundant and the only one commercially available [2]. NOD-R is a characteristic cyclic peptide composed of five amino acids (-D-MeAsp–L-Y–Adda–D-Glu–Mdhb), in which the Adda structural region ((2S,3S,8S,9S)-3-amino- 9-methoxy-2,6,8-trimethyl-10-phenyl deca-4,6-dienoic acid) is found in microcystins (MCs), another group of cyanobacteria toxins composed of heptapeptides. NOD-R is similar to MCs in structure (Figure 1), it penetrates more into hepatocytes than MCs and exhibits stronger acute toxicity [3]. It has been found that NOD-R can inhibit the catalytic subunit of seine/threonine-specific protein phosphatases (PPs) 1 and 2A (PP1 and PP2A) and lead to severe hepatotoxicity [4,5,6]
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