Abstract
A lipovitellin (Lv) based sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in marine medaka (Oryzias melastigma). Lv and Vtg were purified from the unfertilized eggs and the whole body homogenates (WBH) of estradiol (E2)-exposed fish. The purified Lv sample appeared as three clear bands (118, 112 and 100 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and was identified as an Lvs mixture from VtgAa and VtgAb by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Polyclonal antibody against marine medaka VtgAa was also raised. Compared with Vtg, Lv was more stable to heat stress (37 °C for 8 h or 4 °C for a week) and repeated freeze/thaw stress. In addition, western blot analysis revealed that marine medaka Vtg and Lv had similar immunogenicity. Therefore, in this study, Lv was applied instead of Vtg as the standard to establish an ELISA. The Lv standard curve was parallel to serial WBH dilutions of E2-exposed fish, and the absorbance values were very low in control male samples, suggesting the specificity and feasibility of the method for Vtg quantification. The developed assay was sensitive with the detection limit of 3.1 ng/mL and had a working range between 15.6 and 500 ng/mL. The intra- and inter-assay coefficients of variation were both below 5%. Moreover, the standard curves of Lv antigen treated under different stresses were almost identical, indicating high robustness of the assay. Overall, our study provides an important methodology reference for quantification of marine medaka Vtg.
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