摘要
应用草鱼生长激素（ｇｃＧＨ）单克隆抗体及多价兔抗血清建立了草鱼ＧＨ非竞争式酶’联免疫吸附测定ＥＬＩＳＡ系统。用正辛酸法对腹水单抗进行了分离纯化，获得了高纯度的单抗制备物。聚丙烯酸胺凝胶电泳表明纯化的单抗由分子量分别为５５ｋＤ和２５ｋＤ的两条蛋白带组成。用纯化单抗铺底，用兔抗血清作后续抗体建立了一种测定草鱼ＧＨ的非竞争式双抗夹心ＥＬＩＳＡ方法。交叉试验表明该测定系统只与草鱼ＧＨ和基因重组鲤生长激素（ｒｃＧＨ）具有剂量依存的结合反应，而与大马哈鱼生长激素（ｓＧＨ）、牛生长激素（ｂＧＨ）、大马哈鱼促性腺激素（ｓＧｔＨ）、及黑鲢促性腺激素（ｂｓｃＧｔＨ）等均无交叉反应。该 ＥＬＩＳＡ方法的灵敏度可达０．８ｎｇ／ｍｌ，组内变异系数为 ５．９ ％，组间变异系数为７．６％，回收率达９０％以上。初步应用表明，鲤和团头鲂垂体抽提液、草鱼血清、鲤血清及鲫血清在该测定系统中有剂量依存的反应曲线，而大口鲶、黄颡鱼、中华鲟及黄鳝鱼垂体抽提液及大口鲶、胡子鲶和罗非鱼血清在该测定系统中没有交叉反应。
A highly sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) for grass carp. (Ctenopharyngodon idellus) growth hormone (gcGH) was developed using monoclonal anti- bodies (MCAs) against gcGH as coating antibody and gcGH rabbit antiserum as linking antibody. The MCAs were purified from ascites fluid using the caprylic acid method and consisted of two bands with molecular weights of 55kD and 25kD in SDS-PAGE. The ELISA recognized gcGH and recombinant common carp (Cyprinus carpio) GH (rcGH), but did not recognize salmon (Oncorhynchus tshawytscha) GH.bovine GH.salmon gonadotropin (GtH) and black silver carp (Aristichythys nobolis) GtH. The ELISA recognize GH in common carp and bluntnose black bream (Megalobrama ambly- cephala) pituitary extracts, but not in nor-them sheatfish (Silurus soldatovi), yellow catfish (Pelteoba- grus fulvidraco), Chinese sturgeon (Acipenser sinensis ) and rice field cell (Monopterus albus ) extracts; and recognized GH in common carp, grass carp and goldfish plasma, but not in northern sheatfish, walking catfish (Clarias batrachus) and tilapia (Tilapia nilotica) plasma. The sensitivity of the ELISA was about 0.8ng/ml. Inter-and intraassay coefficients of variance of the ELISA were about 7.6% and 5. 9%, resistively. Recovery test data indicate>90% recovery.
出处
《动物学报》
SCIE
CAS
CSCD
1996年第4期386-393,共8页
ACTA ZOOLOGICA SINICA
基金
国家自然科学基金资助项目,批文号:39300102&&
关键词
草鱼
ELISA
非竞争式
生长激素
Grass carp,Growth hormone, Monoclonal antibody, Noncompetitive ELISA.
