TY - JOUR
T1 - Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins
AU - Burow, Susann
AU - Fontaine, Romain
AU - von Krogh, Kristine
AU - Mayer, Ian
AU - Nourizadeh-Lillabadi, Rasoul
AU - Hollander-Cohen, Lian
AU - Cohen, Yaron
AU - Shpilman, Michal
AU - Levavi-Sivan, Berta
AU - Weltzien, Finn Arne
N1 - Publisher Copyright:
© 2019 The Author(s)
PY - 2019
Y1 - 2019
N2 - The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs. The advantages of this protocol include: • The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%). • The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity. • Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.
AB - The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs. The advantages of this protocol include: • The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%). • The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity. • Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.
KW - Competitive enzyme-linked immunosorbent assay
KW - Follicle-stimulating hormone
KW - Gonadotropin quantification
KW - Luteinizing hormone
KW - Oryzias latipes
KW - Specific antibodies
KW - Specific enzyme-linked immunosorbent assay (ELISA) for determining Fsh and Lh levels in medaka
UR - http://www.scopus.com/inward/record.url?scp=85067572429&partnerID=8YFLogxK
U2 - 10.1016/j.mex.2019.06.011
DO - 10.1016/j.mex.2019.06.011
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AN - SCOPUS:85067572429
SN - 2215-0161
VL - 6
SP - 1473
EP - 1479
JO - MethodsX
JF - MethodsX
ER -