Comparison of ELISAs for detecting vitellogenin in the fathead minnow ( Pimephales promelas)

Abstract

Measurement of vitellogenin (VTG) concentrations in the fathead minnow ( Pimephales promelas) is currently being considered and evaluated for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp ( Cyprinus carpio), was recently evaluated in an inter-laboratory ring test using whole body homogenates from juvenile fathead minnows. The objective of the current study was to compare the performance of three different ELISAs for measuring fathead minnow VTG: (1) a heterologous carp VTG (cVTG) ELISA used in the ring test, (2) a homologous fathead minnow VTG (fVTG) ELISA, and (3) a hybrid ELISA with the antibody developed for cVTG, but using fVTG for coating the plates and preparing standard curves. VTG was measured in whole body homogenates from juvenile fathead minnows exposed to 17α-ethynylestradiol (EE 2; 10 ng/l) and whole body homogenates and plasma from adult fathead minnows exposed to 17β-estradiol (E 2; 5 mg/kg; i.p.). The cVTG assay showed lower specificity for fathead minnow VTG in whole body homogenates and plasma from treated fish, compared to the fVTG assay. VTG concentrations in juvenile fathead minnow homogenates from the EE 2-exposed group were approximately 50-fold higher when measured using the fVTG method compared to the cVTG method. Use of the homologous fVTG in the hybrid cVTG assay yielded VTG concentrations in the range of the fVTG assay but the low specificity persisted. The homologous fVTG assay is recommended to achieve accurate quantification of VTG levels in fathead minnows.