A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen
Witchuda Prasatkaew , Phochit Nanthanawat , Nanthika Khongchareonporn , Sutin Kingtong
DOI:10.1016/j.jes.2018.05.001
Received November 30, 2017,Revised January 01, 1900, Accepted May 02, 2018, Available online May 12, 2018
Volume 31,2019,Pages 325-333
A monoclonal antibody specific to sea bass (Lates calcarifer) vitellogenin (VTG) was developed, for use as a tool for monitoring endocrine disrupting chemicals (EDCs). VTG was induced in sea bass by intramuscular injection of 17β-estradiol (E2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry (LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41 (MAb-sea bass VTG 41) was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay (ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E2-treated sea bass and others EDCs (Nonylphenol, Benzo[a]pyrene and CdCl2). Moreover, cross-reactivity was also found in E2-treated coral grouper (Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.
