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

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.

Development of Time-Resolved Fluoroimmunoassay for Detection of Cylindrospermopsin Using Its Novel Monoclonal Antibodies

“…In this way, a TRFIA was established to detect CYN in environmental samples. This immunoassay has been applied previously to detect microcystins and nodularin, and some advantages such as a wide detection range and high sensitivity have been demonstrated [ 33 , 45 , 46 ]. In our study, the TRFIA also exhibited a wide quantitative range between 0.1 and 50 ng mL −1 and maintained good reliability.…”

Cyanobacterial Akinete Distribution, Viability, and Cyanotoxin Records in Sediment Archives From the Northern Baltic Sea

“…The same sample was highly positive in the generic assay, indicating that most of the hepatotoxins were of microcystin origin. As the nodularin-specific assay has slight cross-reactivity with other microcystins such as microcystin-YR ( Akter et al, 2017 ), it is also likely that the weak positive result arose from the cross-reactivity of the assay with other microcystins.…”

“…The detection limit (expressed in microcystin-LR equivalents) of the immunoassay was 0.04 μg L –1 , based on the average + 3 SD of the blank measurements. Similarly, each sample was also analyzed to detect any possible nodularin-R according to the method described by Akter et al (2017) in reaction volume of 75 μL well –1 using 25 μL well –1 of sample. The detection limit of the nodularin-R immunoassay was 0.011 μg L –1 .…”

Single-step noncompetitive immunocomplex immunoassay for rapid aflatoxin detection

“…Though the discovery of such binders is problematic by traditional immunization-based techniques, phage display methodology provides a more controlled strategy for generating antibody binders beyond the polyclonal and monoclonal ones. Owing to the remarkable characteristics of anti-IC antibodies, also referred to as antimetatype antibodies (Voss & Mummert, 1997), they have been applied to the development of sensitive immunoassays for several small molecules in various assay formats, for example, morphine (Pulli, Höyhtyä, Söderlund, & Takkinen, 2005), microcystins (Akter et al, 2016), nodularin (Akter, Vehniäinen, Kankaanpää, & Lamminmäki, 2017), HT-2 toxin (Arola, Tullila, Nathanail, & Nevanen, 2017;Arola et al, 2016), and estradiol (Leivo, Kivimäki, Juntunen, Pettersson, & Lamminmäki, 2019).…”