Drennon Katherine, Moriyama Shunsuke, Kawauchi Hiroshi, Small Brian, Silverstein Jeffrey, Parhar Ishwar, Shepherd Brian
Department of Biology, University of Kentucky, T.H. Morgan School of Biological Sciences, 101 T.H. Morgan Building, Lexington, KY 40506-0225, USA.
Gen Comp Endocrinol. 2003 Oct 1;133(3):314-22. doi: 10.1016/s0016-6480(03)00194-1.
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.
我们报道了一种用于测定斑点叉尾鮰（Ictalurus punctatus）生长激素（cfGH）的灵敏、特异、竞争性抗原捕获酶联免疫吸附测定法的开发。该测定法的检测限（90%结合率）为2.0 ng/ml，ED(50)值（标准曲线范围150 - 0.59 ng/ml）为67.3 ng/ml。添加cfGH的血浆样本回收率测定为102%。使用系列稀释的垂体匀浆和血浆样本绘制的剂量 - 反应抑制曲线与使用纯化cfGH绘制的标准曲线始终显示出平行关系。在使用异源激素和纯化的斑点叉尾鮰催乳素（cfPRL）的竞争结合曲线中证明了生长激素抗体（兔抗斑点叉尾鮰生长激素）的特异性。这些研究表明，在该测定法的工作范围内，cfPRL（对cfGH结合的竞争性抑制）或异源激素没有显著（0.006%）结合。为了从生理角度验证该测定法，给斑点叉尾鮰注射（100 μg/g体重，每5天注射3次）溶解于玉米油中的牛生长激素释放激素（1 - 29）酰胺或合成六肽生长激素释放肽 - 2（KP - 102：D - Ala - D - β - Nal - Ala - Trp - D - Phe - Lys - NH(2)）。最后一次注射后，一半动物采集血浆样本，其余动物从淡水（FW）转移至12 ppt的海水（BW：半咸水）中。转移至BW后24小时，再次采集动物血浆样本。与淡水（FW）组相比，所有BW组（对照组、KP - 1０2组和bGHRH组）的血浆生长激素水平均显著升高（p < 0.001）。此外，用生长激素促分泌素KP - 102或bGHRH处理后，血浆生长激素水平也显著升高（p < 0.0０1）。我们的研究结果表明，狭盐性斑点叉尾鮰存在两种生长激素升高的调节机制，一种见于广盐性硬骨鱼（盐度诱导的生长激素水平），另一种是最近在硬骨鱼中描述的（生长激素释放肽诱导的生长激素水平）。
