Development of homologous enzyme-linked immunosorbent assays to quantify two forms of vitellogenin in guppy ( Poecilia reticulata )

Abstract

Guppy (Poecilia reticulata) is a promising model organism in toxicological studies, and vitellogenin (Vtg) is a commonly used biomarker for environmental estrogens. Although an ELISA for guppy Vtg has been developed previously, we found that guppy had two forms of Vtgs. In this study, two Vtgs were characterized and enzyme-linked immunosorbent assays (ELISAs) for each Vtg were developed. Two Vtgs purified from 17β-estradiol (E₂)-exposed guppy were characterized as phospholipoglycoproteins with molecular weights of ~ 520 and ~ 480 kDa, respectively. In SDS-PAGE, one purified Vtg appeared as three major bands of ~ 210, ~ 126, and ~ 102 kDa, and the other revealed a clear band of ~ 68 kDa. Matrix-assisted laser desorption/ionization–time of flight/time of flight mass spectrometry analysis showed that they were VtgAb and VtgC. Using purified Vtgs and their corresponding antibodies, two sandwich ELISAs with working ranges of 7.8~1000 and 15.6~500 ng/mL were developed. Precision tests showed that intra- and inter-assay coefficients of variations of both ELISAs were below 10%. Parallelism between Vtg standard curves and serial dilutions of whole body homogenate from E₂-exposed guppy confirmed that two ELISAs could quantify guppy Vtgs. Furthermore, two ELISAs were used to measure Vtg inductions in liver, caudal fin and whole body of male guppy exposed to 17a-ethinylestradiol to validate their use for detecting estrogenic effects of exogenous chemicals. These homologous Vtg ELISAs will promote the use of guppy as a model organism to study estrogenic chemicals.