PMC

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 . Six positive clones were selected based on testing the supernatants of the hybridoma cells.

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.

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.

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).