【作者】 王鹤佳；

【导师】 沈建忠；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 本研究采用混合酸酐法制备了玉米赤霉醇（Zeranol,ZER）免疫原，免疫BALB／c小鼠，用间接酶联免疫吸附法（ELISA）对抗体效价进行了检测，并建立了ELISA检测方法。ZER免疫原（ZER-7-半琥珀酸酯-BSA，以下简称Z7BSA）中半抗原与载体蛋白的偶联比为10∶1，包被原（ZER-7-半琥珀酸酯-OVA，以下简称Z70VA）中半抗原与载体蛋白的偶联比为8∶1。抗血清效价为1∶8000。利用杂交瘤技术获得了两株稳定分泌ZER单克隆抗体（以下简称单抗）的杂交瘤细胞株IC12和9B4。经鉴定两株单抗亚型均为IgG₁，杂交瘤细胞染色体数目为95～102条，间接ELISA测定腹水效价为1∶3.2×10⁵，单抗分子量为151.8KD。该单抗与ZER同系物β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、玉米赤霉烯酮的交叉反应率分别为35.5％，47.4％，7.1％，73.0％，56.3％；与己烯雌酚、雌二醇、睾酮、克伦特罗的交叉反应率均小于0.1％。该细胞株体外传代和冻存复苏后抗体分泌稳定。 用方阵滴定法确定包被原最适工作浓度为0.5μg／ml，单抗最适稀释倍数为1∶5000，辣根过氧化物酶标记羊抗鼠IgG（以下简称酶标二抗）最适稀释倍数为1∶5000。采用间接竞争ELISA方法建立检测ZER的标准曲线，在0.5ng／ml～100ng／ml范围内呈线性相关，回归方程为y=-66.844x+424.15，相关系数R²=0.996，50％抑制浓度(IC₅₀)为3.0ng／ml，以10％抑制率对应的浓度计算，则该方法对ZER的检测限为0.6ng／ml。 以该单抗为基础，进行牛尿中玉米赤霉醇酶联免疫检测试剂盒的研制。确定了试剂盒中各种溶液的配方。试剂盒最低检测限为0.60ng／ml，试剂盒对空白牛尿的最低检测限为7.74ng／ml。以10ng／ml，21ng／ml，35ng／ml 3个浓度添加空白牛尿，回收率在70.0％～116.0％之间，板内变异系数在6.0％～15.9％之间，批内变异系数为15.3％，批间变异系数为14.3％，均低于20％。试剂盒于2～8℃下可保存6个月。更多还原

【Abstract】 In this study, Zeranol(ZER) was coupled to carrier protein bovine serumalbumin(BSA) and ovalbumin(OVA) using the mixed anhydride method. The ZER-BSA was used as immunogen and the ZER-OVA used as coating antigen. The coupling ratio of ZER to BSA was 10:1 and to OVA was 8:1. BALB/c mice were immuned by the immunogen and the titer of anti-serum was 1:8000 by two-dimensional titration method.Two hybridomas producing anti-ZER monoclonal antibodies were obtained by limited dilution, named 1C 12 and 9B4. The characteristics of monoclonal antibody was performed by EL1SA test, chromosomal counting and SDS-PAGE electrophoresis. The results indicated that the subclass of the McAb was IgGl, the titer of ascitic fluid was 1: 3.2×105, the molecular weight was 151.8KD .the chromosomal numbers were 95-102. The cross-reactivities of McAb to zeranol, β-zearalanol,α-zearalenol, β-zearalenol, zearalanone, zearalenone were 100%, 35.5%, 47.4%, 7.1%, 73.0%, 56.3% respectively, and to diethylstilbestrol, 17 β-estradiol, testosterone, clenbuterol were all below 0.1% . The indirect competitive Enzyme Linked Immunosorbent Assay(ELISA) for ZER was developed. The best concentration of coating antigen was 0.5ug/ml, the optional working dilution of the McAb and enzyme -conjugate were all 1:5000 , The linear detecting range of calibration curves to detect ZER was 0.5ng/ml~100ng/ml , The formular was y = -66.844x+424.15, R2 = 0.996, the 50% inhibition concentration(IC50) was 3.0ng/ml. The limit of detection was 0.6ng/ml.The enzyme immunoassay kit for the quantitative analysis of zeranol was developed with McAb against ZER. The lower detection limit of ZER test was 0.60ng/ml. According to the sample preparation protocol, the detection limit was 7.74ng/ml in bovine urine. When the spiked concentration of ZER were 10ng/ml, 21ng/ml and 35ng/ml, the recovery range was all from 70.0% to 116.0%, the coefficient range of variation was from 6.0% to 15.9%. The ELISA kit could be reserved for 6 months at 2~8℃.更多还原