Generation and characterization of polyclonal antibodies against microcystins—Application to immunoassays and immunoaffinity sample preparation prior to analysis by liquid chromatography and UV detection

• Published in: Talanta (Oxford) 2006-09, Vol.70 (2), p.225-235
• Main Authors: Mhadhbi, Houcine, Ben-Rejeb, Samuel, Cléroux, Chantal, Martel, Annie, Delahaut, Philippe
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Blue-green algae; Chemistry; Chromatographic methods and physical methods associated with chromatography; Enzyme immunoassay; Exact sciences and technology; Immunoaffinity chromatography; Microcystins; Miscellaneous; Other chromatographic methods; Sample preparation; Solid phase extraction; Spectrometric and optical methods
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2006.02.029

Polyclonal antibodies against microcystin-LR (MC-LR), a cyclic heptapeptide toxin, were generated in rabbits using MC-LR-BSA. An enzyme-linked immunosorbent assay (ELISA) was developed for the characterization of the antibodies and their potential use for analytical purposes. The concentration of MC-LR that inhibits 50% of antibody–antigen binding (IC 50) was 0.5 μg L −1 for the indirect ELISA format and 0.9 μg L −1 for the direct ELISA, using MC-LR-horseradish peroxidase conjugate. The limit of detection corresponding to IC 80 was found to be 0.06 μg L −1, well below the Word Health Organization level for drinking water of 1 μg L −1. The direct competitive ELISA was applied to water samples and was shown useful for screening purposes. The developed anti-microcystin antibodies were immobilized on solid supports for use in selective solid phase extraction (SPE) systems, prior to liquid chromatography (LC) quantification. An immunoaffinity cartridge (IAC), a Sepharose ®-based cartridge incorporating 2 mg of antibodies allowed the selective and quantitative recovery of a mixture of 0.2 μg of MCs showing potential use in sample preparation of real matrices. When applied to water and green algae samples, average recoveries from Sepharose ®-based cartridges were in the range of 86–113% for water samples and 85–92% for blue-green algae samples. Selectivity of the IAC clean-up was proven by comparison with non-specific solid phase extraction using octadecylsilica (ODS) sorbent. Results obtained using LC/UV after IAC clean-up agreed well with results obtained using liquid chromatography and mass spectrometry detection (LC/MS and LC/MS/MS) after SPE-C18 clean-up, allowing therefore to validate the resulting technique.