A novel enzyme-linked immunosorbent assay based on anti-lipovitellin monoclonal antibodies for quantification of zebrafish (Danio rerio) vitellogenin.
Vitellogenin (Vtg) in zebrafish (Danio rerio) is a recommended biomarker endpoint for detecting estrogenic activity of chemicals under the OECD test guidelines. The present paper reports the development of a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the quantification of zebrafish Vtg based on monoclonal antibodies (MAbs) against lipovitellin (Lv), the major yolk protein derived from Vtg. The purified Lv was used to immunize mice and the spleen cells of mice were fused with myeloma cells. Two high-affinity MAbs (H3A8 and H4D9) were screened from hybridoma cells. Western blot analysis revealed that two MAbs were highly specific to zebrafish Vtg and recognized different antigenic epitopes because MAb H3A8 detected a main band of 143kDa in purified Vtg, while MAb H4D9 reacted with two clear bands of purified Vtg at 117 and 102kDa. Using MAb H3A8 as the coating antibody, HRP-labeled MAb H4D9 or HRP-labeled PAbs as the detecting antibody, two sandwich ELISAs for Vtg quantification were developed. The sandwich ELISA developed using HRP-labeled MAb H4D9 had a working range of 1.95-250ng/mL, with a detection limit of 0.78ng/mL, which was lower than that of the assay based on HRP-labeled PAbs. Parallelism between Lv standard curves and dilution curves of whole-body homogenates (WBH) from E2-treated male zebrafish confirmed the validity of the ELISAs for quantifying zebrafish Vtg. Finally, the usefulness of two assays for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to 17β-estradiol.
Marine Life Science College, Ocean University of China, Qingdao 266003, China.
Jun
J
Wang
Marine Life Science College, Ocean University of China, Qingdao 266003, China.
Wei
W
Wang
Marine Life Science College, Ocean University of China, Qingdao 266003, China.
Hua
H
Tian
Marine Life Science College, Ocean University of China, Qingdao 266003, China.
Xiaona
X
Zhang
Marine Life Science College, Ocean University of China, Qingdao 266003, China. Electronic address: rusg@ouc.edu.cn.
Shaoguo
S
Ru
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western; D004527:Egg Proteins / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity; D014819:Vitellogenins / Q000276:immunology*; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D015027:Zebrafish / Q000097:blood / Q000276:immunology*; D029961:Zebrafish Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D004527:Egg Proteins; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D029961:Zebrafish Proteins; C486450:vtg1 protein, zebrafish; D004958:Estradiol; C001549:lipovitellin
Lipovitellin; Monoclonal antibodies; Sandwich ELISA; Vitellogenin; Zebrafish