Generation and characterization of polyclonal antibodies against microcystins-Application to immunoassays and immunoaffinity sample preparation prior to analysis by liquid chromatography and UV detection.
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 0.5mugL(-1) for the indirect ELISA format and 0.9mugL(-1) for the direct ELISA, using MC-LR-horseradish peroxidase conjugate. The limit of detection corresponding to IC(80) was found to be 0.06mugL(-1), well below the Word Health Organization level for drinking water of 1mugL(-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((R))-based cartridge incorporating 2mg of antibodies allowed the selective and quantitative recovery of a mixture of 0.2mug of MCs showing potential use in sample preparation of real matrices. When applied to water and green algae samples, average recoveries from Sepharose((R))-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.
Mhadhbi
Houcine
H
Laboratoire de Chimie et Biochimie des Complexes Moléculaires, UMR 7576, Ecole Nationale Supérieure de Chimie de Paris, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France.
Ben-Rejeb
Samuel
S
Cléroux
Chantal
C
Martel
Annie
A
Delahaut
Philippe
P