Lipovitellin as an antigen to improve the precision of sandwich ELISA for quantifying zebrafish (Danio rerio) vitellogenin

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a core biomarker for screening environmental estrogens in test guidelines of the Organization for Economic Cooperation and Development. To accurately quantify zebrafish Vtg, lipovitellin (Lv), the main Vtg-derived yolk protein, was used as the antigen to establish a sandwich enzyme-linked immunosorbent assay (ELISA). The purified Lv was a phospholipoglycoprotein with apparent molecular weight of ~ 445 kDa, and separated into three polypeptides corresponding to ~ 117, ~ 102, and ~ 23.8 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunological analysis confirmed the specificity of the anti-Lv antibody for Vtg and the immunological similarity between Vtg and Lv. Using the purified Lv and anti-Lv antibody, a sandwich ELISA with a detection limit of 4.3 ng/mL and a detection range from 7.8 to 250 ng/mL was developed. The intra- and inter-assay coefficients of variation were both below 10%. Moreover, the Lv standard curve was nearly identical to the Vtg standard curve, and paralleled serial whole-body homogenate dilutions of male zebrafish exposed to 17β-estradiol, demonstrating that the Lv-based ELISA could be used for quantification of zebrafish Vtg. Zebrafish Lv showed high stability during purification process, heat treatment, − 80 °C storage, and repeated freeze/thaw cycles. Additionally, the standard curve of Lv stored at − 80 °C for 3 months exhibited higher robustness than that of Vtg stored under the same conditions. Finally, the usefulness of the ELISA for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to monocrotophos.