An enzyme-linked immunosorbent assay for rare minnow ( Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish ( Danio rerio)

Abstract

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow ( Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp ( Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17α-ethinylestradiol (EE 2) of rare minnow and zebrafish ( Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE 2 l − 1 for rare minnow and 4 ng EE 2 l − 1 for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC–MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment.