title: Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR.
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 microg 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.
authors.affiliation: Facultad de Química, Instituto de Higiene, Universidad de la República, Av. A. Navarro 3051, piso 2, Montevideo, 11600, Uruguay.
authors.forename: Natalia
authors.initials: N
authors.lastname: Ferraz
authors.forename: Daniel
authors.initials: D
authors.lastname: Carnevia
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Nande
authors.forename: Martin
authors.initials: M
authors.lastname: Rossotti
authors.forename: María N
authors.initials: MN
authors.lastname: Miguez
authors.forename: Jerold A
authors.initials: JA
authors.lastname: Last
authors.forename: Gualberto
authors.initials: G
authors.lastname: Gonzalez-Sapienza
mesh_terms: D000818:Animals; D000941:Antigens / Q000235:genetics; D015415:Biomarkers / Q000032:analysis; D023681:Cichlids; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004527:Egg Proteins / Q000032:analysis*; D052244:Endocrine Disruptors / Q000032:analysis*; D004703:Endocrine System / Q000187:drug effects / Q000378:metabolism*; D004784:Environmental Monitoring / Q000379:methods; D005260:Female; D006727:Hormone Antagonists / Q000032:analysis*; D007118:Immunoassay / Q000379:methods*; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000032:analysis*; D011994:Recombinant Proteins / Q000235:genetics; D013997:Time Factors; D014819:Vitellogenins / Q000032:analysis*
chemical_list: D000941:Antigens; D015415:Biomarkers; D017931:DNA Primers; D004527:Egg Proteins; D052244:Endocrine Disruptors; D006727:Hormone Antagonists; D011498:Protein Precursors; D011994:Recombinant Proteins; D014819:Vitellogenins; C104950:choriogenin H