title: Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica.
abstract: We prepared monoclonal antibodies (mAbs) against a recombinant tethered follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica that was produced in Escherichia coli. Positive hybridomas (clones eFA-C5, eFA-C10, eFA-C11, eFA-C12, eFA-C13, and eFB-C14) were selected by using the eel FSH antigen in ELISA, and anti-eel FSH mAbs were purified from culture supernatants by performing affinity chromatography. Three of the 6mAbs were characterized and their isotypes were identified as IgG2b (eFA-C5 and eFA-C11) and IgG1 (eFB-C14). In western blotting assays, the mAbs recognized the antigen as a 24.3-kDa band, and further detected bands of 34 and 32kDa in the supernatants of CHO cells transfected with cDNA encoding tethered eel FSHβ/α and LHβ/α, respectively. PNase F-mediated deglycosylation of the recombinant proteins resulted in a drastic reduction in their molecular weight, to 7-9kDa. The mAbs eFA-C5 and eFA-C11 recognized the eel FSHα-subunit that is commonly encoded among glycoprotein hormones, whereas eFB-C14 recognized the eel FSHβ-subunit, and immunohistochemical analysis revealed that the staining by these mAbs was specifically localized in the eel pituitary. We also established an ELISA system for detecting rec-tethered FSHβ/α and LHβ/α produced from CHO cell lines. Measurement of biological activities in vitro revealed that only weak activity of rec-FSHβ/α was detected. The activity of rec-LHβ/α was found to be increased in a dose-dependent manner for eel oocyte maturation.
authors.affiliation: Aquaculture Research Division, National Institute of Fisheries Science (NIFS), Busan 46083, Republic of Korea.
authors.forename: Dae-Jung
authors.initials: DJ
authors.lastname: Kim
authors.affiliation: Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong 17579, Republic of Korea.
authors.forename: Chae-Won
authors.initials: CW
authors.lastname: Park
authors.affiliation: Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong 17579, Republic of Korea.
authors.forename: Dong-Wan
authors.initials: DW
authors.lastname: Kim
authors.affiliation: Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong 17579, Republic of Korea.
authors.forename: Hong-Kyu
authors.initials: HK
authors.lastname: Park
authors.affiliation: Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong 17579, Republic of Korea.
authors.forename: Munkhzaya
authors.initials: M
authors.lastname: Byambaragchaa
authors.affiliation: Aquaculture Research Division, National Institute of Fisheries Science (NIFS), Busan 46083, Republic of Korea.
authors.forename: Nam-Sil
authors.initials: NS
authors.lastname: Lee
authors.affiliation: Dept. of Research and Development, Institute of Gyeongbuk Marine Bioindustry, Ulgin 36315, Republic of Korea.
authors.forename: Sun-Mee
authors.initials: SM
authors.lastname: Hong
authors.affiliation: Aquaculture Research Division, National Institute of Fisheries Science (NIFS), Busan 46083, Republic of Korea.
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Seo
authors.affiliation: Department of Food Science & Nutrition, Hoseo University, Asan 31499, Republic of Korea.
authors.forename: Myung-Hwa
authors.initials: MH
authors.lastname: Kang
authors.affiliation: Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Anseong 17579, Republic of Korea. Electronic address: ksmin@hknu.ac.kr.
authors.forename: Kwan-Sik
authors.initials: KS
authors.lastname: Min
mesh_terms: D000811:Anguilla / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005640:Follicle Stimulating Hormone / Q000276:immunology* / Q000378:metabolism; D037201:Follicle Stimulating Hormone, beta Subunit / Q000378:metabolism; D037101:Luteinizing Hormone, beta Subunit / Q000378:metabolism; D009866:Oogenesis; D010902:Pituitary Gland / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D037201:Follicle Stimulating Hormone, beta Subunit; D037101:Luteinizing Hormone, beta Subunit; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone
keywords: Follicle-stimulating hormone; Japanese eel; Monoclonal antibody; Recombinant