Multihapten Approach Leading to a Sensitive ELISA with Broad Cross-Reactivity to Microcystins and Nodularin

Abstract

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp3]MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 μg/L, well below the WHO's maximum recommendation of 1 μg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs.

Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS)

“…(Gago, 2007). Broadly specific immunoassays have been developed as well, including 67 the use of antibodies (monoclonal and polyclonal) raised against the Adda structure of 68 microcystins and a multihapten approach, where antibodies recognize the general cyclic 69 peptide structure of microcystins (Fischer et al, 2001;Samdal et al, 2014;. 70 A commercially available polyclonal Adda ELISA kit was used in this study for the broadly 71 specific determination of microcystins.…”

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water

“…However, this broadly specific mAb also recognized free Adda, which is not toxic [27]. One pAb generated using the multihapten approach exhibited similar affinity to most of the common MCs [28]. However, the immunogen synthesis process was complicated and costly, making it unsuitable for popularization and applications.…”

Isolation and Characterization of [D-Leu1]microcystin-LY from Microcystis aeruginosa CPCC-464

“…Both types of variants would be readily detected if they were targeted in the LC-MS/MS method, or if untargeted LC-MS methods were used. Protein phosphatase inhibition assays, or immunoassays with appropriate cross-reactivities [28,29], can also be expected to detect both D-Ala 1 -and D-Leu 1 -containing MCs although they cannot indicate which type of variant is present.…”