| In recently years, the potential adverse effects of environmental estrogens on the reproducetive system of aquatic wildlife have become a major research topic. To evaluate the estrogenic activity of chemicals, the Organization for Economic Co-operation and Development (OECD) and US Environmental Protection Agency (US EPA) recommended a fish screening assay protocol that uses vitellogenin (Vtg) as a core endpoint. Zebrafish (Danio rerio) is a recommended test species in the guidelines and widely used in the detection of environmental estrogens, and the measurement of Vtg was normally performed using enzyme-linked immunosorbent assays (ELISA). The precise quantification of Vtg induction in zebrafish is the key for investigating estrogenic chemicals, while the precise quantification of Vtg was disturbed by the currently used detection methods and and experimental organisems. In order to establish a high robust and sensitive ELISA protocol for zebrafish Vtg, the antigens and antibodies used in the assay along with the formats and operating steps were compared and optimized in the present study. Besides, a biosensor for detection of zebrafish Vtg using Octet molecular interaction system was developed, which would provide a rapid screening method for environmental estrogens. Firstly, zebrafish Vtg and lipovitellin (Lv) was purified, characterized, and then used to prepare rabbit anti-Vtg polyclonal antibody, rabbit anti-Lv polyclonal antibody, and anti-Lv monoclonal antibody. Following determination of titers and specificity of these antibodies, and immunological similarity between Vtg and Lv, several ELISA methods and a biosensor for quantification of zebrafish Vtg were developed based on the purified Vtg, purified Lv, and prepared antibodies. Moreover, the Vtg-blank period of zebrafish was determined. These findings would be helpful in providing novel methodology improvements for precise quantification of zebrafish Vtg. The results revealed that:(1) Two Vtgs with molecular weights of approximately 483 kDa and 458 kDa were purified by gel filtration, ion-exchange chromatography followed by ultrafiltration from the whole body homogenate (WBH) of male zebrafish exposed to 100 ng/L 17β-estradiol (E2) for 7 days. The results of specific staining revealed that two Vtgs were both phospholipoglycoproteins. Two Vtgs were encoded by different Vtg genes and showed high homology with each other as identified by MALDI-TOF/TOF MS. Using the purified Vtg and the prepared anti-Vtg polyclonal antibody, competitive and sandwich ELISA methods for zebrafish Vtg were developed and compared. The sandwich ELISA was found to be not only conducted more easily, but also had higher precision and sensitivity, with a detection limit of 2.2 ng/mL and a working range from 3.9 to 250 ng/mL. Thus, this ELISA format was recommended for Vtg detection. Furthermore, Western blot and ELISA were used to detect the changes in Vtg-derived yolk proteins and newly produced Vtg during zebrafish developmental process, and 9 to 56 days post-hatching (dph) was confirmed to be the Vtg-blank period. Finally, Vtg induction of the 21 dph fish exposed to E2 were measured by the optimized sandwich ELISA to verify the usage of juveniles during Vtg-blank period in the screening for environmental estrogens.(2) Zebrafish Lv was purified form the ovary extracts through a two-step chromatographic method. Lv was also characterized as a phospholipoglycoprotein with molecular weight of approximately 445 kDa and reduced to two polypeptides of approximately 117 and 102 kDa in SDS-PAGE. Additionally, the amino acid composition of zebrafish Lv was comparable to Lvs from other fish species. Compared to Vtg, Lv showed higher stability after -80℃ storage, multiple freeze/thaw cycles, and heat treatment. Additionally, the use of treated Lv in the ELISA development did not change the slopes of standard curves, which could dramatically promote the precision of the ELISA assay. The Lv-based sandwich ELISA had a detection limit of 4.3 ng/mL and a working range from 7.8 to 250 ng/mL. The intra- and inter-assay coefficients of variations were below 10%. Finally, the usefulness of the ELISA was verified by detecting the Vtg induction in juvenile zebrafish exposed to monocrotophos, a potential exogenous estrogen has been proved in our laboratory.(3) Two monoclonal antibodies recognized different antigen epitopes of zebrafish Lv were prepared and applied to develop two sandwich ELISA assays for the quantification of zebrafish Vtg. Compared with polyclonal antibodies, the monoclonal antibody-based ELISA showed higher sensitiviy, with a detection limit of 0.78 ng/mL and a working range from 1.95 to 250 ng/mL. Parallelism between the Lv standard curve and WHB from E2-exposed male zebrafish confirmed the validity of the assay for quantifying Vtg in the WHB. The monoclonal-based Vtg ELISA was further applied to quantify Vtg induction in juvenile zebrafish exposed to E2 for 14 days.(4) A biosensor for quantification of zebrafish Vtg using purified Lv and anti-Lv IgG was developed on the Octet molecular interaction system. This method was simple, fast, label-free, and automated, which making the detection of Vtg accomplished within 15 min. The assay had a working range from 78 to 5000 ng/mL, and the inter-assay coefficient of variations was 1.13%. To verify the usage of the biosensor for detection of estrogenic effects, it was used to quantify Vtg concentration in zebrafish exposed to E2.In summary, this study indicated that the sandwich ELISA was more suitable for accurate quantification of Vtg due to its high sensitivity and precision. Besides, the Vtg-blank period during the development of zebrafish was determined, which provide a basis for the selection of experimental fish. Zebrafish Lv was found to have high stability, and the use of Lv instead of Vtg as the standard would promote the robustness of the ELISA. The monoclonal antibodies against zebrafish Lv were firstly prepared and used to develop zebrafish Vtg sandwich ELISA which showed higher sensitivity than the polyclonal antibodies-based ELISA. Besides, the high homogenous monoclonal antibody would help to establish a standardized test system for zebrafish Vtg. In addition, a biosensor for rapid detection of zebrafish Vtg was developed using the Octet molecular interaction system, offering an alternative method in estrogenic chemical screening. These findings will provide important methodological basis for improvements of the current test guidelines. |
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