梭鱼卵黄蛋白原的化学发光免疫检测法

Development of chemiluminescent immunoassay for vitellogenin in red lip mullet,Liza haematocheila

运用建立化学发光免疫(chemiluminescence immunoassay,CLIA)检测法,对梭鱼血清中主要卵黄蛋白原(Vg)类型(B类型;VgB)进行定量测定。梭鱼VgBCLIA法按两步法操作进行,使用纯化的梭鱼VgB制备特异型抗血清(a-VgB)。优化抗体浓度和培育时间等检验条件后,检验范围设定为3.91～500ng/mL。卵黄发生期的梭鱼雌鱼血清和雌激素处理后的梭鱼稚鱼血清稀释曲线与纯化的梭鱼Vg曲线平行,而梭鱼雄鱼稀释血清中几乎不发生相应的免疫学反应。对天津原种场养殖的10尾梭鱼血清VgB水平的定量检测结果表明,在雌鱼(9尾)血清中检测到VgB但个体间差异较大,变化范围为3.0～2700.1μg/mL,并表现出随着卵巢发育而增长的变化趋势。相对而言,成熟梭鱼雄鱼血清VgB水平则极低(2.7μg/mL),表明在其生长环境中不存在雌激素活性。另外,所有个体的组织学观察也未呈现出明显的性腺异常情况。研究为梭鱼主要雌激素诱导生物标志物(VgB)的量化提供了一种新的手段,较以往的检验方法具有更高的灵敏度,有助于建立多元化的水生环境中雌激素活性的检测体系。

A specific and sensitive chemiluminescent immunoassay（CLIA） was developed for quantification of a major vitellogenin（Vg） subtype（B-type Vg;VgB） in the serum of red lip mullet（Liza haematocheila）.The mullet VgB CLIA was performed using two-site method,with subtype-specific antiserum developed for purified red lip mullet VgB（a-VgB）.Assay conditions were optimized with regard to antibody concentration,as well as incubation time,resulting in the typical assay range from 3.91 to 500 ng/mL.Dilution of vitellogenic female mullet serum and the serum from estrogen-treated juvenile mullet appeared to be parallel to purified mullet Vg,while dilution of male mullet serum hardly revealed any positive immunoreactivity in the developed CLIA.Serum levels of VgB were quantified in 10 individuals of red lip mullet reared in an aquaculture center in Tianjin City,China.In females（n=9）,production of VgB was evident but varied in their serum,the levels were ranged from 3.0 to 2 700.1 μg/mL,showing a typical trend of increase during the ovarian growth.In contract,serum level of VgB in a matured male mullet was extremely low（2.7 μg/ mL）,indicating no sign of estrogenic activities in this environment.In addition,no trace of gonadal abnormality was evident when histological observation was performed for all individuals.The present study provided a new tool for the quantification of the major estrogen-inducible biomarker（i.e.,VgB） in red lip mullet,which appeared to be better in the sensitivity in comparison with our previous assay and thus enabled us to evaluate a variety level of estrogenic activities in aquatic environment.Basic information such as typical,albeit preliminary,reproductive changes in circulating VgB levels were also provided and will endow to set a ＂normal＂baseline,which is necessary to be established prior to interpreting＂abnormal＂inductions of this biomarker.