猪瘟病毒抗原胶体金快速检测试纸的研究

应用胶体金标记技术(Immunogold Labelling Technique)与免疫层析技术相结合,在玻璃纤维膜、硝酸纤维膜的检测线(T)和对照线(C)上分别喷上胶体金与猪瘟(CSF)单克隆抗体(Ab2)的偶联物、CSF单克隆抗体(Ab2)和羊抗鼠IgG多克隆抗体,制成检测CSF抗原的"猪瘟抗原胶体金快速检测试纸".用该试纸检测"猪瘟弱毒活疫苗"和猪瘟脾淋弱毒苗均显阳性,而对猪源性的其它病毒、细菌抗原均显阴性.经临床测试,该试纸具有微量、特异、快速、简便和结果容易判定的优点,可作为检测猪瘟抗原的一种新试剂.  

直接竞争ELISA法快速检测动物源性食品中磺胺嘧啶残留

采用重氮化-偶联法将磺胺嘧啶（SD）与人血清白蛋白（HSA）相连制备了免疫抗原SD—HSA，与卵清白蛋白（OVA）相连制备了包被抗原SD—OVA，用过碘酸钠法将SD—OVA与辣根过氧化物酶（HRP）连接制备了酶标抗原（SD—OVA—HRP）。用SD—HSA免疫BALB／c小鼠，经融合、筛选和克隆化制备了单克隆抗体，利用单抗建立了直接竞争ELISA方法。该方法具有好的特异性，在1—729ng／mL浓度范围标准曲线方程Y=12．05x＋2．2538，R^2=0．995，IC50为41．7ng／mL，在猪肉、鸡肉、鸡肝、鸡蛋、牛奶中的检测限分别为20、20、20、5、5μg/kg。在20μg／kg和100μg／kg水平猪肉、鸡肉、鸡肝、牛奶、鸡蛋样品中添加，回收率为82．78％-104．6％。与高效液相色谱方法比较，二者的阳性符合率为81．3％，阴性符合率为83．3％，总符合率为88．9％；与同类英国RANDOX试剂盒比较，二者符合率为100％。  

抗狂犬病毒单克隆抗体的纯化及标记

对一株稳定、特异、高效的单克隆抗体（mAb）进行了纯化和荧光素（FITC）的标记,利用标记产物与已有的抗狂犬病毒核蛋白单克隆抗体标记物相比较,效果良好,二者可以结合（或单独）使用;应用标记好的荧光抗体与FITC-抗狂犬病毒免疫球蛋白比较进行血清样品效价测定实验,来确定纯化、标记后的单克隆抗体的灵敏度,结果表明本实验制备的单克隆抗体与FITC-抗狂犬病毒免疫球蛋白具有相同的灵敏度,该单克隆抗体测得的血清样品效价结果与FITC-抗狂犬病毒免疫球蛋白测得的结果相同。因此,此荧光抗体可应用于狂犬病病毒检测（FAT）、病毒毒力测定（TCID50）、中和抗体实验（FAVN）等方面的检测实验,此荧光抗体可为狂犬病的相关研究提供有力的保障。  

<em>Mycoplasma hyopneumoniae</em>-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage <em>via</em> increasing nitric oxide production,oxidative stress,and caspase-3 activation

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.  

用荧光标记细菌法研究丝兰提取物对瘤胃原虫对细菌吞噬的影响

采用荧光染色标记细菌(fluorescence labeled bacteria,FLB)技术,研究山羊瘤胃中原虫对细菌的吞噬速率.试验选用3头装有永久瘘管的徐淮山羊作为瘤胃液供体,采用体外培养法研究在精粗比为1:9的情况下,探讨不同丝兰提取物水平(A组0 mg/L、B组35 mg/L、C组70 mg/L)对瘤胃原虫吞噬细菌速率的影响.荧光镜检与回归分析结果表明:原虫吞噬细菌的速率:A组:339.9 cells/(cell·h);B组:314.7 cells/(cell·h);C组:339.9 cells/(cell·h).结果表明荧光标记细菌技术能够应用于瘤胃原虫吞噬细菌速率的研究.[动物营养学报,2009,21(3):417-422][中文全文见<动物营养学报>网站(www.ChinaJAN.com)中文版2009年21卷3期]  

Simultaneous double labelling of routinely processed paraffin tissue sections using combined immunoperoxidase, immunofluorescence, and digital image editing

An innovative image editing system based on a sequential immunoperoxidase-immunofluorescence technique on routine histological sections is described. With this technique it is possible to identify different antigens in different cells, as well as co-localised antigens in the same cell. The method uses digital image editing to mix two independently captured images into one merged image. The technique was performed with indirect immunoperoxidase, followed by sequential indirect immunofluorescence, digital image acquisition and image editing. Multiple staining examples using anti-cytokeratin, anti-vimentin and anti-calbindin antibodies on canine skin and cerebellum, and feline pleural mesothelioma sections were performed in order to investigate the capabilities of the proposed technique. Our data demonstrated that this method can be easily used to assess multiple protein staining studies with minimum laboratory equipment, and that it allows a better structural visualisation of the tissue morphology compared to double immunofluorescence. Moreover, in contrast to double-immunoperoxidase, with this method it is possible to easily co-localise two different antigens in the same cell compartment.  

胶体金标记抗圆弧青霉菌毒素—青霉酸单克隆抗体的研究

本研究探讨了胶体金标记抗圆弧青霉菌毒素—青霉酸单克隆抗体探针的制备方法，摸索其最佳反应条件并对其活性进行检测。结果表明，胶体金标记抗青霉酸单克隆抗体的最佳pH为8.2，1 mL胶体金标记的最小蛋白量为25 μg。金标抗体与二抗发生牢固结合，即金标探针具有活性。该研究为青霉酸的胶体金免疫层析检测技术奠定了一定的基础。  

食源性动物组织中CSFV和PRRSV双重双色荧光定量RT-PCR检测方法的建立及应用

为建立同时检检测食源性动物组织中猪瘟病毒(CSFV)和猪蓝耳病病毒(PRRSV)的双色荧光定量RT-PCR方法。本研究根据SCFV和PRRSV基因序列设计特异性的引物和不同荧光标记的TaqMan荧光探针,通过优化反应的体系和扩增条件,建立了能够检测食源性动物组织中SCFV和PRRSV的双重双色荧光定量PCR的方法。其检测下限为1×102拷贝/μL,而且与其他一些猪病病毒无交叉反应,具有良好的特异性。该方法重复性和稳定性试验表明其组内和组间的变异系数最高分别为4.2和4.5。对比试验表明,该方法对CSFV和PRRSV检验的敏感性为常规RT-PCR方法的200倍;该方法的建立为食源性动物组织中CSFV和PRRSV提供了有效手段,该方法特异性和敏感性较好,能够应用于临床检测。  

胶体金免疫技术在猪瘟诊断上的应用进展

胶体金免疫技术是一种新型免疫学检测技术.它具有简便、快速、特异性强、敏感性高、结果判断直观等优点.在猪瘟的快速诊断中得到了广泛应用,显示出了良好的应用前景.论文就胶体金免疫技术及其在猪瘟快速诊断上的应用现状及发展前景作一简要综述.