犬瘟热病毒抗原胶体金免疫层析检测方法的建立

发布时间：2018-04-27 15:42

  本文选题：犬瘟热病毒 + 抗原检测　； 参考：《西北农林科技大学》2015年硕士论文

【摘要】：犬瘟热是由犬瘟热病毒引起犬的一种高度接触性、致死性传染病。犬瘟热病毒属于副粘病毒科麻疹病毒属,同属成员还有麻疹病毒、牛瘟病毒、小反刍兽疫病毒和鲸豚麻疹病毒。感染犬瘟热病毒犬临床症状为精神沉郁、体温升高、厌食、拉稀,后期出现神经症状,表现为共济失调、四肢瘫痪等。最终,口吐白沫、尖叫而死亡。犬瘟热病毒易感动物主要是幼犬和貂,成年动物发病但不死亡。我国养犬业、皮毛经济动物业及野生动物安全受到了该病严重威胁。现有的犬瘟热病毒检测方法主要包括血清中和试验、免疫荧光试验、ELISA、RT-PCR、病毒分离及包涵体检查。但是这些方法都需要专业人员在特殊仪器下进行操作,并且耗时几小时到几天不等。因此建立一种在临床上操作简易、快速、可靠并且廉价的犬瘟热病毒检测方法非常重要。本研究使用了两株可以与重组CDV-N蛋白和犬瘟热病毒结合的抗CDV-N蛋白单克隆抗体,研发了一个可以检测犬瘟热病毒抗原的免疫层析试纸条。本研究的内容及结果如下:将两株抗CDV-N蛋白单克隆抗体1B7和2F11,分别腹腔注射Balb/c小鼠制备单抗腹水,纯化并鉴定。以胶体金标记纯化的1B7单抗作为示踪剂,将纯化的单克隆抗体2F11和商品化的羊抗鼠IgG二抗分别作检测线(T线)和质控线(C线)喷点于硝酸纤维素膜上,建立了检测动物排出物和排泄物中犬瘟热病毒抗原的胶体金免疫层析方法。判定标准为:①待检的样品中含有CDV,可与金标单抗形成CDV-1B7复合物,经层析膜流经T线和C线时,CDV-1B7复合物就会和T线上的2F11单抗以及C线上的羊抗鼠IgG二抗结合,形成两条红色的线,判定为阳性结果;②若待检血清中不含CDV,则只有质控线出现;③若质控线未形成红色线条时,则该方法检测无效。经过探索该胶体金的最佳反应条件,我们最终确定T线包被浓度为600μg/mL、C线浓度为800μg/mL、金标单抗最佳标记pH为8.6、浓度为14.26μg/mL。本研究中制备的胶体金免疫试纸条特异性良好,只在检测犬瘟热病毒时出现阳性结果,对其他病毒如犬细小病毒(CPV)、犬冠状病毒(CCV)、犬副流感病毒(CPIV)、伪狂犬病毒(PRV)、新城疫病毒(NDV)无交叉反应;在敏感性试验中,该方法检测细胞毒最低浓度为102.5 TCID50/mL;对同一样品不同批次重复试验,该方法检验均为同一结果,并且在4℃可稳定储存6个月不改变特性;与商品化CDV检测试剂盒对比,两者有良好的符合性。综上所述,本试验建立了犬瘟热病毒抗原胶体金免疫层析检测方法,为犬瘟热病毒检测提供了一种快速、简易的方法。
[Abstract]:Canine distemper is a highly contagious and fatal disease caused by canine distemper virus. Canine distemper virus belongs to the measles virus genus of paramyxovirus family, and its members include measles virus, rinderpest virus, small ruminant zoonotic virus and whale dolphin measles virus. The clinical symptoms of canine distemper virus infection were depression, elevated body temperature, anorexia, dilation, and later neurological symptoms, such as ataxia, quadriplegia and so on. Finally, foaming at the mouth, screaming and dying. Canine distemper virus susceptible animals are mainly puppies and minks, adult animals develop but do not die. China's canine industry, fur and economic animal industry and wildlife safety has been seriously threatened by the disease. The existing detection methods of canine distemper virus mainly include serum neutralization test, immunofluorescence test ELISART-PCR, virus isolation and inclusion body examination. But these methods require professionals to operate under special instruments and take hours to days. Therefore, it is very important to establish a simple, rapid, reliable and inexpensive method for detection of canine distemper virus. In this study, two monoclonal antibodies against CDV-N protein and canine distemper virus were used to detect the antigen of canine distemper virus, and an immunochromatographic test strip was developed to detect the antigen of canine distemper virus. The contents and results of this study are as follows: two McAbs, 1B7 and 2F11, were injected intraperitoneally into Balb/c mice to prepare ascites, purified and identified. The purified 1B7 McAb labeled with colloidal gold was used as tracer. The purified monoclonal antibody (2F11) and the commercial goat anti mouse IgG (second antibody) were sprayed on the nitrocellulose membrane. A colloidal gold immunochromatographic method for the detection of canine distemper virus antigen in animal excreta and excreta was established. It was determined that the sample to be tested by the standard of: 1 contained CDV, which could form CDV-1B7 complex with gold labeled McAb. When the membrane was flowing through T and C lines, the CDV-1B7 complex would bind to 2F11 monoclonal antibody on T line and sheep anti-rat IgG second antibody on C line. If there is no CDV in the serum, only the quality control line appears if the red line is not formed, then the method is ineffective. After exploring the optimum reaction conditions of the colloidal gold, we finally determined that the concentration of T line coating was 600 渭 g / mL, the best pH of gold labeled McAb was 8.6, and the concentration was 14.26 渭 g / mL. The colloidal gold immunoassay strip prepared in this study has a good specificity and only positive results in the detection of canine distemper virus. There was no cross reaction for other viruses such as CPV, CCV, CPIVV, PRV, NDV, and so on, and no cross reaction was found for other viruses, such as canine parainfluenza virus (CPV), pseudorabies virus (PRV) and Newcastle disease virus (NDV). The lowest concentration of cytotoxicity detected by this method was 102.5 TCID 50% mL.The results of repeated tests for different batches of the same sample were the same, and it could be stably stored at 4 鈩，

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