Development of an enzyme-linked immunosorbent assay for the measurement of plasma growth hormone (GH) levels in channel catfish (Ictalurus punctatus): assessment of environmental salinity and GH secretogogues on plasma GH levels.
We report the development of a sensitive, and specific, competitive, antigen-capture enzyme-linked immunosorbent assay for the measurement of channel catfish (Ictalurus punctatus) growth hormone (cfGH). The detection limit of the assay (90% binding) was 2.0ng/ml and the ED(50) value (standard curve range 150-0.59 ng/ml) was 67.3 ng/ml. Recovery of cfGH-spiked plasma samples was determined to be 102%. Dose-response inhibition curves using serially diluted pituitary homogenates and plasma samples consistently showed parallelism with the standard curves using purified cfGH. The GH antibody (rabbit anti-catfish GH) specificity was demonstrated in competitive binding curves employing heterologous hormones and purified channel catfish prolactin (cfPRL). These studies show that there was no significant (0.006%) binding of cfPRL (competitive inhibition of cfGH binding), or heterologous hormones, within the working range of the assay. To physiologically validate the assay, catfish were injected (100 microg/g body weight, 3 injections every 5 days) with either bovine GHRH(1-29)-amide or the synthetic hexapeptide GHRP-2 (KP-102: D-Ala-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH(2)) suspended in corn oil. Following the last injection, half of the animals were sampled for plasma and the remaining transferred from fresh water (FW) to 12 ppt seawater (BW: brackish water). Twenty-four hours after transfer to BW, animals were again sampled for plasma. Plasma GH levels were significantly (p<0.001) elevated in all the BW groups (control, KP-102, and bGHRH), compared with the FW (fresh water) groups. In addition, plasma GH levels were significantly (p<0.001) elevated by treatment with either of the GH secretogogues, KP-102 or bGHRH. Our findings demonstrate that two regulatory mechanisms of GH elevation, one which is seen in euryhaline teleosts (salinity-induced GH levels) and another, which has been recently described in teleosts (GHRP-induced GH levels), are present in the stenohaline channel catfish.
Drennon
Katherine
K
Department of Biology, University of Kentucky, T.H. Morgan School of Biological Sciences, 101 T.H. Morgan Building, Lexington, KY 40506-0225, USA.
Moriyama
Shunsuke
S
Kawauchi
Hiroshi
H
Small
Brian
B
Silverstein
Jeffrey
J
Parhar
Ishwar
I
Shepherd
Brian
B
D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005618:Fresh Water / Q000737:chemistry; D013006:Growth Hormone / Q000097:blood* / Q000187:drug effects / Q000276:immunology; D013007:Growth Hormone-Releasing Hormone / Q000819:agonists* / Q000097:blood / Q000494:pharmacology*; D007059:Ictaluridae / Q000097:blood*; D009842:Oligopeptides; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D011388:Prolactin / Q000097:blood; D011817:Rabbits; D012623:Seawater / Q000737:chemistry*
D009842:Oligopeptides; D011388:Prolactin; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; C091874:growth hormone-releasing peptide-2