鸡新城疫病毒免疫金标试纸条的研制及其应用
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摘要
本实验采用柠檬酸三钠还原氯金酸，制备胶体金颗粒。根据不同粒径的胶体金颗粒在检测时所呈现的颜色鲜艳(容易分辨)程度，选择20nm的胶体金颗粒作为标记物。新城疫病毒用低温超速离心法进行提纯，然后免疫动物；对制备的血清采用辛酸-硫酸铵盐析法进行纯化；抗体经常规PAGE和琼脂扩散试验，结果显示辛酸-硫酸铵法纯化的抗体，纯度和活性均较高。胶体金标记最低稳定量和最适的pH的试验结果为30μg/ml及pH为9.0；对标记后的抗体用低温超迷离心法进行纯化。经过试验胶体金标记抗体最适稀释倍数为1：2，兔抗新城疫抗体和羊抗兔抗体的合适点样量分别为500μg/ml和300μg/ml。制作的试纸条能够在5min左右显示清晰的条带。同时加入的稳定剂，能够使抗体保持较长时间的活性和使金标抗体释放较为完全，在4℃干燥器中保存8个月后效果仍然很好。用本试纸条和ELISA对157个样品进行检测。结果显示：新城疫胶体金免疫层析法是一种快速、灵敏度高、特异性强的检测NDV的方法，可以用于流行病学调查，及SPV鸡场的净化。
本试纸条的研制为进一步研究和开发一系列畜禽疾病快速诊断金免疫层析试纸条奠定了实验基础。
The different diameter colloidal gold particles were synthesized by reducing hydrogen tetrachloroaurate with sodium citrate using the microwave oven which was a heating up installation.At the same time, we gained the referenced data which prepared colloidal gold particles form 100 to 500ml. The 20nm colloidal gold particles were selected.according to the different diameter colloidal gold particles showing different color(easy to judge). The NDV was purified on the zonal ultracentrifuging at 4 ,then use the method of colloidal gold immunoelectronmicroscopy to detect NDV. The result showed that the NDV had been purified. Using the NDV immunized rabbit and chicken, the received serum was purified by caprilic acid/ammonium sulfamate precipitation. The purified antibodies were analyzed by the conventional PAGE and agar diffusion test.And the result showed that the IgG in high purity and good activities.The gold sol was adjusted to pH 9.0 then at 4癈, the labeled rabbit anti-NDV IgG was purify on zonal ul
tracentrifuging. We diluted the labeled rabbit anti-NDV IgG in PBS(containing 1% BSA) at the rate of 1: 2. The labeled antibodies was immobilized to nitrocellulose membrane(NC). The two kind of antibody concentration was optimized by the square matrix test. We selected the HF135 to prepare the test strip.Meanwhile, a optimized and stable formula was obtained. The test strip was placed in 4"C desiccator.after 8 month its effect of detection and releasing still hold the line. 157 clinical samples were detected by the strip comparing with HA, ELISA, The test strip show is easy to be justified;time-saving,sensitive,and suitable for at grassroot level and epidemiology investigate.
These key techniques and the date will help us research and develop a series of GICA strip to detect the domestic animals and poultry.
本试纸条的研制为进一步研究和开发一系列畜禽疾病快速诊断金免疫层析试纸条奠定了实验基础。
The different diameter colloidal gold particles were synthesized by reducing hydrogen tetrachloroaurate with sodium citrate using the microwave oven which was a heating up installation.At the same time, we gained the referenced data which prepared colloidal gold particles form 100 to 500ml. The 20nm colloidal gold particles were selected.according to the different diameter colloidal gold particles showing different color(easy to judge). The NDV was purified on the zonal ultracentrifuging at 4 ,then use the method of colloidal gold immunoelectronmicroscopy to detect NDV. The result showed that the NDV had been purified. Using the NDV immunized rabbit and chicken, the received serum was purified by caprilic acid/ammonium sulfamate precipitation. The purified antibodies were analyzed by the conventional PAGE and agar diffusion test.And the result showed that the IgG in high purity and good activities.The gold sol was adjusted to pH 9.0 then at 4癈, the labeled rabbit anti-NDV IgG was purify on zonal ul
tracentrifuging. We diluted the labeled rabbit anti-NDV IgG in PBS(containing 1% BSA) at the rate of 1: 2. The labeled antibodies was immobilized to nitrocellulose membrane(NC). The two kind of antibody concentration was optimized by the square matrix test. We selected the HF135 to prepare the test strip.Meanwhile, a optimized and stable formula was obtained. The test strip was placed in 4"C desiccator.after 8 month its effect of detection and releasing still hold the line. 157 clinical samples were detected by the strip comparing with HA, ELISA, The test strip show is easy to be justified;time-saving,sensitive,and suitable for at grassroot level and epidemiology investigate.
These key techniques and the date will help us research and develop a series of GICA strip to detect the domestic animals and poultry.
引文
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