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Data on the Characterization of Follicle-Stimulating Hormone Monoclonal Antibodies and Localization in Japanese Eel Pituitary

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Data on the Characterization of Follicle-Stimulating Hormone Monoclonal Antibodies and Localization in Japanese Eel Pituitary

Dae-Jung Kim et al. Data Brief.

Abstract

Monoclonal antibodies were generated against recombinant follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica; rec-FSH was produced in Escherichia coli and purified using Ni-NTA Sepharose column chromatography. In support of our recent publication, "Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica" [1], it was important to characterize the specificity of eel follicle-stimulating hormone antibodies. Here, the production and ELISA system of these monoclonal antibodies are presented. The affinity-purified monoclonal antibodies specifically detected eel rec-FSH in ELISA and on western blots of rec-FSH produced from CHO cells. Immunohistochemical analysis revealed that FSH staining was specifically localized in the eel pituitary.

Keywords: FSH; Japanese eel; Monoclonal Antibody.

Figures

Fig. 1.
Fig. 1
Construction of eel FSHβ/α expression vector and expression of rec-FSHβ/α in E. coli. (A) Construction of rec-tethered eel FSHβ/α cDNA by using overlapping PCR. The expression vector was subcloned into pRSET under the T7 promoter (designated as pRSET-eel FSHβ/α). (B) The pRSET vector was transformed into E. coli. After culture, whole-cell, soluble, and inclusion-body fractions were subjected to SDS-PAGE. One strain was cultivated in large-volume cultures and the protein was purified using Ni-NTA Sepharose column chromatography.
Fig. 2.
Fig. 2
Indirect ELISA for testing the reactivity of antibodies. The reactivity of culture supernatants from selected hybridoma cells was checked by performing indirect ELISA according to the protocol described in Section 2.5. Six positive clones were selected based on testing the supernatants of the hybridoma cells.
Fig. 3.
Fig. 3
Quantification of rec-FSHβ/α and LHβ/α amounts by using sandwich-ELISA system. (A) Proteins (rec-FSHβ/α and LHβ/α) produced from CHO cells and baculovirus-infected cells were analyzed by performing sandwich ELISA. (B) The HRP-conjugated anti-eel FSH monoclonal antibody eFA-C11, diluted 100–3200 times, was tested; this dilution range of the HRP-labeled antibody efficiently described the standard curve. (C) Quantities of rec-FSHβ/α produced from stable cell lines were also determined. (D) Western blotting performed using eFA-C5 antibody is shown here. The 2nd antibody used was goat anti-mouse (1:3000). The monoclonal antibody detected a single ~34-kDa FSHβ/α band in the medium (M) and cell lysates (C).
Fig. 4.
Fig. 4
Localization of FSHβ-subunit expression in the eel pituitary during oocyte-maturation. Adjacent sections of the pituitary were stained with eFA-C14, an antiboy that specifically binds to eel FSHβ-subunit, and detection was performed using swine anti-mouse IgG secondary antibodies (1:1000). Representative IHC analyses are shown. The staining revealed the specific site of FSHβ-subunit in the pituitary. (A) GSI>0.5%; (B) 5.7%; (C) 33.8% (immediately before ovulation); (D) immediately after ovulation; (E) on Day 7 after ovulation.

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References

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