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.
Highlights
Cyanobacterial toxins pose a threat for humans, and toxin-contaminated water bodies have accidentally been utilized for recreational use, as a supply for drinking water and production of seafood
Using NOD-R to form the immunocomplex with the anti-ADDA specific monoclonal antibody (Mab) and phage display selection from the synthetic antibody library, we show here the isolation of NOD-specific immunocomplex binders and the development of the first non-competitive NOD-specific immunoassay
An in-house synthetic antibody phage library scFvP [45] was exploited to isolate the antibodies recognizing the immunocomplex (IC) of NOD-R bound to anti-ADDA Mab
Summary
Cyanobacterial toxins pose a threat for humans, and toxin-contaminated water bodies have accidentally been utilized for recreational use, as a supply for drinking water and production of seafood. Domestic and wild animals have been affected and even killed because of cyanobacterial toxins (for a review, see [1]). The bioaccumulation of cyanotoxins like nodularin (NOD) in the aquatic food web to seafood such as flounder, cod, clam and mussel has been confirmed [2,3,4,5]. The first identified and scientifically reported cyanobacteria to cause animal poisonings was Nodularia spumingena from Australia in 1878 [6], the plausible toxin, NOD, was identified and its structure resolved about 110 years later [7,8]. NOD is a non-ribosomally composed cyclic pentapeptide and shares a structural similarity, like a non-proteinogenic amino acid, with ADDA side chain, to microcystins (MCs)—the most frequently occurring and widespread cyanotoxin group. Though about 10 analogues of NOD are reported in the literatures [22], NOD-R is the most abundant and often referred as NOD
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