Enzyme flow immunoassay using a Protein G column for the screening of triazine herbicides in surface and waste water

A method for screening of triazine herbicides in surface and waste water is presented. The method is based on an enzyme flow immunoassay (EFIA) for th e detection of the free fraction of a horse radish peroxidase (HRP)-labelle d antigen (tracer). This was accomplished by trapping the bound tracer frac tion in a Protein G column, allowing the residual free tracer fraction to p ass and be detected spectrophotometrically after incubation with an enzyme substrate. As compared with detecting the bound tracer fraction this reduce s the regeneration requirements of the Protein G column used for capturing the bound fraction and, therefore, reduces assay time. A polyclonal antibod y directed against simazine showed no reactivity towards tracers that were thiopropionic acid derivatives of atrazine, simazine and terbutylazine. It had good sensitivity towards tracers using derivatives of 2-chloro-4,6-(alk ylamino)-s-triazines such as atrazine and simazine. The highest sensitivity was obtained with an Et/Cl/N-C-5-HRP tracer because this tracer could be u sed in combination with the lowest concentration of antibody. The detection limit was 0.1 mug l(-1) with a linear range between 0.1 and 10 mug l(-1) a nd an assay throughput of 12 h(-1). Natural water samples from various loca tions in Russia were analysed for triazines and the results were compared w ith a previously developed fluorescein how immunoassay for triazines. The r esults were further verified by supported liquid membrane (SLM) extraction combined with HPLC. The results show that the two immunoassays behave diffe rently and that the sample matrix influences their performance, however, no false negative results were obtained. The possible reasons for the differe nt results between the two immunoassays are discussed. (C) 2001 Elsevier Sc ience B.V. All rights reserved.