Broad-Spectrum Noncompetitive Immunocomplex Immunoassay for Cyanobacterial Peptide Hepatotoxins (Microcystins and Nodularins)

A broad-spectrum noncompetitive immunoassay allowing sensitive and simple detection of a group of similar compounds would be an ideal tool for screening low molecular weight analytes (<2000 Da) having many variants. However, the development of an essential antibody pair capable of sandwich-type recognition of the analytes' small generic core structure is a demanding task due to limited space available for simultaneous binding of two different antibodies. We report here a generic noncompetitive assay for cyanobacterial microcystins (MCs) and nodularins (Nod), a group of structurally related small cyclic peptides (similar to 1000 Da) with more than 100 naturally occurring analogs. The assay is based on the unique combination of a generic antiimmunocomplex (anti-IC) single-chain fragment of antibody variable domain (scFv) and a monoclonal antibody capable of binding to an Adda-group (3-amino-9-methoxy-2,6,8-trimethy1-10-phenyldeca-4(E),6(E)-dienoic acid) present in all MCs/Nod. The anti-IC scFv was isolated from a large synthetic antibody library with phage display and used to develop a single-step sandwich-type noncompetitive immunocomplex assay. The sensitive time-resolved immunofluorometry-based assay is capable of detecting all the 11 tested commonly occurring hepatotoxins (MC-LR,-dm1R,-RR,-dmRR,-LA,-LY,-LF,-LW,-YR,-WR, and Nod-R) at concentration below 0.1 mu g/L in a 1 h assay. Using MC-LR, the most studied toxic and widely distributed of the toxins, the calculated detection limits (based on blank + 3SD response) are, similar to 0.026 mu g/L in 1 h and similar to 0.1 mu g/L in 10 min assay time. This is by far the fastest reported immunoassay for MCs and Nod with a detection limit far below the World Health Organization's guideline limit (1 mu g/L of MC-LR equivalent in drinking water). The assay was validated with spiked tap and lake water as well as with environmental surface water samples. The developed assay provides a simple, rapid, and highly sensitive tool for the quantitative detection of MCs/Nod with the additional benefit of automation and high-throughput possibilities for large scale screening of drinking and environmental surface water samples. Furthermore, the study describes the first demonstration of the assay intended for the detection of an analyte group comprising similar low-molecular weight compounds exhibiting the benefits of a reagent excess type assay.