鸡白细胞介素-6原核表达及双抗夹心ELISA方法的建立

应用分子生物学技术原核表达ChIL-6，以ChIL-6重组蛋白为免疫原，按免疫程序分别制备兔抗和鼠抗IL-6重组蛋白的多克隆抗体。应用此抗体建立双抗夹心EL-ISA方法后，为了优化此方法本试验对该方法的最佳试验条件、标准曲线、重复性和初步应用进行确定。结果显示，经SDS-PAGE和Western-blot分析，表明ChIL-6在大肠杆菌中正确表达，为了建立检测ChIL6的双抗夹心ELISA方法，本试验应用表达的重组蛋白制备兔抗和鼠抗多克隆抗体。建立的双抗夹心ELISA方法最佳反应条件为包被抗体的质量浓度为50mg／L，4℃过夜；酶标二抗稀释度为1：400，37。C1h；应用该方法检测感染金黄色葡萄球菌后的ChIL-6，其结果与本实验室之前应用人的ELISA试剂盒检测的结果相似。结果表明，本试验建立的检测ChlL-6的双抗夹心ELISA方法可用于临床。     

Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA

【Objective】 This study was intend to obtain cathepsin L1(rFgCat L1) specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】 Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened, 1×10⁶ cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method, antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody, and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control, and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】 After immunization, the antibody titers in serum of 4 mice were all more than 10⁴.After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures, the antibodies secreted in the cell supernatant were stable, with titers of 2⁹ and 2¹⁰ respectively, with ascites titers of 10⁷ and 10⁸.Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type, both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies, the antigen titer of the two monoclonal antibodies were comparied, 7G6 was used as the coating antibody, and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.     

Seismic Load Tests on Reinforced Concrete Beam-Column Sandwich Joints with High Shear Stress

Seven reinforced concrete Beam-Column sandwich joint specimens were tested under cyclic load. The performances of these specimens consisting of loads resistance, deflection, ductility, displacement, energy dissipation could satisfy the requirement of structures for seismic design. The phenomena that concrete in joint region was crushed by axial load or eccentric load were never observed,and final failure for a multitude of specimens occurred by shear force. Though a few of performances such as beam bar anchorage capacity were not as well as those of traditional joints ,it is feasible for sandwich joints used while establishing structures.     

A cusp catastrophe model study of destabilization of soft sandwich rock slope

A slope geomechanical model was proposed based on the destabilization characteristics of soft sandwich rock slopes. Based on an analysis of the effects of internal external factors on slope stability, the cusp catastrophe model of slopes can be employed to analyze and predict the necessary conditions leading to slope catastrophes. During the processing of the mass and energy exchange with the external, this may lead to the change of controlling variables. Slides or quick or slow creeping thus may take place due to environmental complexity. This work helps deepen understanding of the formation of landslides and makes feasible the applcation of the catastrophe model to slope destabilization investigations.     

应用A蛋白夹心酶联免疫吸附法鉴定黄瓜花叶病毒血清组

根据Ａ蛋白夹心酶联免疫吸附法（ＰＡＳ－ＥＬＩＳＡ）试验结果可以将我国分离的１６个黄瓜花叶病毒（ＣＭＶ）分离物及４个ＣＭＶ标准毒株（Ｆｎｙ、Ｌｎｙ、Ｍ、ＷＬ）区分为２个血清组．１４个分离物及标准毒株Ｆｎｙ、Ｍ属ＤＴＬ血清组，２个分离物及标准毒株Ｌｎｙ、ＷＬ属ＴｏＲＳ血清组     

应用ELISA双抗体夹心法检测鸡病毒性关节炎病毒的研究

本试验建立了检测鸡病毒性关节炎病毒的ELISA双抗体夹心法.取代反应、阻断试验均为阴性,与NDV、MDV和IBDV无交叉反应,对已知阳性标本的检测均为阳性;其敏感性比琼扩试验高80倍以上,这表明ELISA夹心法具有较高的特异性和敏感性.在人工感染后2～27d,关节滑膜、腱鞘和脾脏中病毒检出率为100％;还从肝脏、法氏囊和脑组织中检测到病毒.     

应用双抗体夹心法ELISA检测IBDV的研究

从抗ＩＢＤＶ高免蛋黄液中提取ＩｇＧ，用作包被抗体和酶标记，建立了检测ＩＢＤＶ的双抗体夹心法ＥＬＩＳＡ。经抗原阻断，无关病毒对照、取代、验证等项试验，并与常规ＡＧＰ法比较，对来源不同的１００多个样品进行检测，结果表明：该法对患鸡腔上囊、脾脏的检出率均为１００％，比ＡＧＰ法敏感１００倍以上，用肉眼观察阳性与阴性之间颜色差异显著，不存在非特异性反应。试验证明该法具有满意的待异性、敏感性、快速性和稳定性，是ＩＢＤ早期病原学诊断和流行病学调查的有效手段。     

柞蚕微孢子虫胶体金免疫层析检测法

利用柞蚕微孢子虫孢子液直接免疫家兔获得柞蚕微孢子虫多克隆抗体后,分别采用双抗夹心法和竞争法制作胶体金免疫层析试纸条,建立能简便、快捷、准确诊断柞蚕微粒子病的柞蚕微孢子虫胶体金免疫层析检测法。双抗夹心法和竞争法分别是将柞蚕微孢子虫多克隆抗体与25 nm和17 nm的胶体金颗粒结合并固定在金标垫上,然后均将羊抗兔二抗包被在硝酸纤维素膜上作为质控点(C),但是2种方法制作的胶体金免疫层析试纸条的反应模式不同:前者是以柞蚕微孢子虫多克隆抗体作为检测点(T),而后者以柞蚕微孢子虫孢壁蛋白作为检测点(T)。2种方法制作的胶体金免疫层析试纸条的检测时间均为10 min,检测灵敏度为0.8×107个/mL,与柞蚕血淋巴无交叉反应,特异性强,操作简单,其中,以双抗夹心法制作的试纸条显色更清晰,结果更可靠,更具实用价值。     

3种ELISA法检测Bt杀虫蛋白的比较研究

运用3种ELISA法对提纯的Bt杀虫蛋白进行检测,结果表明:直接法ELISA和夹心法ELISA的灵敏度相同,都为0.94 ng/孔,但直接法ELISA中Bt蛋白量与OD值间呈极显著的线性关系,夹心法ELISA中Bt蛋白量与OD值间呈显著的线性关系;间接法ELISA的灵敏度较前两者低,使用HRP-IgG的灵敏度为15 ng/孔,Bt蛋白量与OD值间呈显著的线性关系,使用AKP-IgG的灵敏度为3.75ng/孔,Bt蛋白量与OD值间呈极显著的线性关系。