Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR

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Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR

Tema

INMUNO ENSAYO
VITELLOGENIN
CHORIOGENIN
BIBLIOGRAFIA NACIONAL QUIMICA
2007

Abstract

Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L−1 of 17-beta-estradiol, 100 ng L−1 of ethynylestradiol, and 6.6 μg L−1 of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.

Autor

Ferraz, Natalia
Carnevia, Daniel
Nande, Gonzalo
Rossotti, Martín
Miguez, Maria N.
Last, Jerold A.

Fuente

Analytical and Bioanalytical Chemistry v. 389, no. 7-8, 2007. -- p. 2195-2202

Editor

Springer

Fecha

2007

Derechos

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Artículo

Identificador

DOI 10.1007/s00216-007-1630-3
Fecha de agregación
October 14, 2014
Colección
Bibliografía Nacional Química
Tipo de Elemento
Document
Etiquetas
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Citación
Ferraz, Natalia, “Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR,” RIQUIM - Repositorio Institucional de la Facultad de Química - UdelaR, accessed March 28, 2024, https://riquim.fq.edu.uy/items/show/1956.
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