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Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides

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Abstract

Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N′-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins. Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for simazine (detection limit 0.11 ± 0.02 μg L−1, IC50 0.88 ± 0.04 μg L−1), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC50 value of 0.35 ± 0.04 μg L−1 with a detection limit of 1.3 ± 0.9 ng L−1 and a dynamic range from 0.010 to 7.5 μg L−1 simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence.

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Abbreviations

ACN:

acetonitrile

ATR:

atrazine

BSA:

bovine serum albumin

CPGA:

controlled-pore glass covalently bound to protein A

2,4-D:

2,4-dichlorophenoxyacetic acid

DAD:

diode-array detection

ET:

enzyme tracer

FIIA:

flow-injection immunoassay

GC:

gas chromatography

HPLC:

high-performance liquid chromatography

HPPA:

3-(p-hydroxyphenyl)propanoic acid

HRP:

horseradish peroxidase

KLH:

hemocyanin from keyhole limpets

MALDI:

matrix-assisted laser desorption ionization

MS:

mass spectrometry

NPD:

nitrogen–phosphorous detection

PBS:

phosphate-buffered saline

PBST:

phosphate-buffered saline plus 0.2% Tween 20

RSD:

relative standard deviation

SIM:

simazine

SPE:

solid-phase microextraction

SPFIA:

solid-phase fluorescence immnunoassay

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Acknowledgements

The authors thank the Madrid Community Government (reference S-0505/AMB/0374), the ESF, the ERDF and the Ministry of Science and Education (reference CTQ2006-15610-C02). S.H. thanks the Ministry of Science and Education for a predoctoral grant.

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Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, 28040, Madrid, Spain

    Sonia Herranz, Elena Benito-Peña, María Dolores Marazuela & María Cruz Moreno-Bondi

  2. Applied Molecular Receptors Group (AMRg) IIQAB-CSIC CIBER of Bioengineering, Biomaterials and Nanomedicine Jordi Girona 18–26, 08034, Barcelona, Spain

    Javier Ramón-Azcón & María Pilar Marco

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  1. Sonia Herranz
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  2. Javier Ramón-Azcón
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  3. Elena Benito-Peña
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  4. María Dolores Marazuela
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  6. María Cruz Moreno-Bondi
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Correspondence to María Cruz Moreno-Bondi.

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Herranz, S., Ramón-Azcón, J., Benito-Peña, E. et al. Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides. Anal Bioanal Chem 391, 1801–1812 (2008). https://doi.org/10.1007/s00216-008-1895-1

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  • DOI: https://doi.org/10.1007/s00216-008-1895-1

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