Development of specific enzyme-linked immunosorbent assay for determining LH and FSH levels in tilapia, using recombinant gonadotropins

We recently produced Oreochromis niloticus recombinant LH and FSH as single-chain polypeptides in the methylotrophic yeast Pichia pastoris. Glycoprotein subunit a was joined with tilapia (t) LH beta or tFSH beta mature protein-coding sequences to form afusiongenethat encodes a "tethered" polypeptide, in which the gonadotropin beta-subunit forms the N-terminal part and the alpha-subunit forms the C-terminal part. Recombinant (r) gonadotropins were used to develop specific and homologous competitive ELISAs for measurements of FSH and LH in the plasma and pituitary of tilapia, using primary antibodies against rtLH beta or rtFSH beta, respectively, and rtLH beta alpha or rtFSH beta alpha for the standard Curves. The wells were coated with either rtLH beta (2 ng/ml) or rtFSH beta (0.5 ng/well), and the filial concentrations of the antisera were 1:5000 (for tLH) or 1:50,000 (for tFSH). The sensitivity of the assay was 15.84 pg/ml for tLH and 0.24 pg/ml for tFSH measurements in the plasma, whereas for the measurements in the pituitary, the sensitivity was 2.43 ng/ml and 1.52 ng/ml for tLH and tFSH, respectively. The standard curves for tFSH and tLH paralleled those of serially diluted pituitary extracts of other cichlids, as well as of serially diluted pituitary extract of seabream, European seabass and hybrid bass. We examined plasma tFSH and tLH levels in the course of one reproductive cycle, between two successive spawnings, it three individual tilapia females. Plasma levels of both FSH and LH increased during the second day after the eggs had been removed, probably related to the vitellogenic phase. LH levels increased toward spawning, which occurred oil the 11th day. FSH levels also increased on day of cycle, probably due to recruitment of a new generation of follicles for the successive spawning. The development of specific ELISAs using recombinant gonadotropins is expected to advance the Study of the distinct functions of each of these important hormones. (c) 2007 Published by Elsevier Inc.