Submitted to: General and Comparative Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/7/2003
Publication Date: 10/1/2003
Citation: Drennon, K., Moriyama, S., Kawauchi, H., Small, B.C., Silverstein, J., Parhar, I., Shepherd, B. 2003. Development of an enzyme-linked immunosorbent assay (elisa) for the measurement of plasma growth harmone (gh) levels in channel catfish (ictalurus punctatus): assessment of environmental salinity and gh-secretagogues on plasm. General and Comparative Endocrinology. 133:314-322.
Interpretive Summary: A technique and assay to measure blood levels of an important hormone in the growth axis, growth hormone (GH), was developed for channel catfish. This is an entirely new assay and should be a tremendous tool for working on growth and many other traits in channel catfish. In addition, two treatments expected to cause a rise in blood GH levels were conducted and found to lead to the expected rise in GH levels. These experiments served to validate the technique of GH measurement and demonstrate that channel catfish respond to both salinity increase and to injection of other stimulatory hormones by releasing more GH.
Technical Abstract: We report the development of a sensitive, and specific, competitive, antigen-capture enzyme-linked immunosorbent assay (ELISA) for the measurement of channel catfish (Ictalurus punctatus) growth hormone (cfGH). The detection limit of the assay (90% binding) was 2.0 ng/ml and the ED50 value was 67.3 ng/ml. Recovery assays showed that recovery of spiked plasma samples to be 102%. Dose-response inhibition curves using serially-diluted pituitary homogenates and plasma samples consistently showed parallelism with our standard curves using purified catfish GH. The GH antibody (rabbit anti-catfish GH) specificity was demonstrated in competitive binding curves employing heterologous hormones as well as purified channel catfish prolactin (PRL). These studies show that there was no significant binding of cfPRL (competitive inhibition of cfGH binding), or heterologous hormones, within the working range of the assay. To physiologically validate the assay, fresh water-adapted (FW) catfish were injected (100 mg/g body weight, 3 injections every 5 days) with either bovine GHRH1-29-amide or the synthetic hexapeptide GHRP-2 (KP-102: D-Ala-D-b-Nal-Ala-Trp-D-Phe-Lys-NH2) suspended in corn oil. Following the last injection, ½ of the animals were sampled for plasma and the remaining transferred 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 secretagogues, KP-102 or bGHRH. Our findings demonstrate that two mechanisms of GH elevation, one which is seen in euryhaline telesots (salinity-induced GH levels) and another, which has been recently described in teleosts (GHRP-induced GH levels), are present in the channel catfish which is a stenohaline teleost