Universal Assay of Vitellogenin as a Biomarker for Environmental Estrogens

Environmental Health Perspectives 103, Supplement 7, October 1995

Universal Assay of Vitellogenin as a Biomarker for Environmental Estrogens

Scott A. Heppell,¹ Nancy D. Denslow,² Leroy C. Folmar,³ and Craig V. Sullivan¹

¹Department of Zoology, North Carolina State University, Raleigh, North Carolina;
²Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida;
³U.S. Environmental Protection Agency, Gulf Breeze, Florida

Abstract
Vitellogenin (VTG), the serum phospholipoglycoprotein precursor to egg yolk, is potentially an ideal biomarker for environmental estrogens. This study was undertaken to develop antibodies against conserved regions on the VTG molecule that could form the basis for establishing bioassays to detect estrogen exposure in any oviparous vertebrate. We developed monoclonal antibodies (mAbs) generated against purified rainbow trout (Oncorhynchus mykiss) VTG and selected for the property of specifically recognizing VTG purified from two phylogenetically distant vertebrates, trout and striped bass (Morone saxatilis). Results of enzyme-linked immunosorbent assay and Western blotting indicated that these mAbs specifically recognize purified VTG and VTG or other estrogen-inducible proteins in plasma or serum from representative species of four vertebrate classes (fish, amphibians, reptiles, and birds). All of the mAbs generated were IgM class. A polyclonal antiserum was raised against a synthetic consensus peptide representing the conserved N-terminal amino acid sequence of VTG. The results of Western blotting indicate that this antiserum specifically recognizes VTG in plasma or serum from teleost fish of diverse families. It was used to detect VTG in Western blots of serum from brown bullhead (Ameiurus nebulosus) with cancer (hepatocellular and cholangio-carcinoma) collected from a contaminated industrial site outside of their normal vitellogenic season. Our results indicate that it is feasible to generate antibodies capable of recognizing VTG without regard to species and that development of a universal VTG assay is an achievable goal. -- Environ Health Perspect 103(Suppl 7):9-15 (1995)

Key words: vitellogenin, universal assay, environmental estrogens, xenobiotics, monoclonal, immunoassay, synthetic peptide, biomarker, polyclonal, hormone mimics

This paper was presented at the Symposium on Estrogens in the Environment, III: Global Health Implications held 9-11 January 1994 in Washington, DC. Manuscript received: March 15, 1995; manuscript accepted: April 4, 1995.
We thank several people for assistance in obtaining either plasma samples, fish, or both. The following people donated plasma from vitellogenic and nonvitellogenic animals: M. Brown, tuatara; P. Licht, snake; E. Casillas, pollock and white croaker; T. Siopes, F. Edens, and A. Youngblood, chicken; J. Hinshaw donated the rainbow trout. We are grateful to A. Hara for the gift of homologous antiserum to trout VTG and to L. Greene for the antihepatitis B-core protein mAbs. We thank B. Parten for microsequencing. Protein sequencing was carried out in the Protein Chemistry Core Facility in the Interdisciplinary Center for Biotechnology Research at the University of Florida. C. Bidwell provided the sequence for white sturgeon vitellogenin cDNA. This work was supported by the University of Florida, an Electric Power Research Institute Graduate Research Fellowship (to SAH), a North Carolina Biotechnology Center grant (#9113ARIG 0803 to CVS) and a cooperative agreement between the U.S. Environmental Protection Agency and the University of Florida (#CR821437 to NDD).
Address correspondence to Dr. Craig V. Sullivan, Department of Zoology, Campus Box 7617, North Carolina State University, Raleigh, NC 27695. Telephone: (919) 515-7186. Fax: (919) 515-2698. E-mail: craig_sullivan@ncsu.edu
Abbreviations used: ABC, avidin-biotin complex; DAB, diaminobenzidene; ELISA, enzyme-linked immunosorbent assay; E₂, 17ß-estradiol; Fab, antibody binding fragment; FMOC, flourenylmethoxycarbonyl; IgG, immunoglobulin G; IgM, immunoglobulin M; KLH, keyhole limpet hemocyanin; mAb, monoclonal antibody; M_r, relative mobility; OVA, ovalbumin; PAH, polynuclear aromatic hydrocarbons; PBS, phosphate-buffered saline; PITC, phenylisothiocyanate; PVDF, polyvinilydene fluoride immunoblot membranes; rpHPLC, reverse-phase high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCSN, tissue culture supernatant; TFA, trifluoroacetic acid; VTG, vitellogenin.

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