纳米硒标记单抗快速检测新技术

Lateral flow immunochromatography was employed widely in disease detection field, especially lateral flow kit or test strip based on colloidal gold. Colloidal gold has superior stability, sensitivity, but colloidal gold synthesis is relatively complicated and expensive. In contrast with colloidal gold, the selenium nanoparticle was more stable, sensitive, prepared. Moreover, it is more economical in raw material cost. The effect in test strip of selenium nanoparticle was confirmed on melamine rapid test kit. However, present investigations have not been studied systematically with the aim of the preparation of selenium nanoparticle to identify the effects for test strips. This study represented a template method for the preparation of selenium nanoparticle by reducing seleninic acid with ascorbic acid at room temperature in the presence of different templates, screening the best templates and its working concentration by biocompatibility, stability, precision and morphological features. Technology of selenium nanoparticle conjugated-McAb was established by conjugated with anti-HIV McAb and anti-hepatitis virus McAb, proving selenium nanoparticles’ generality. Meanwhile, the stability and sensitivity were checked based on clinical samples by corresponding colloidal gold kit and ELISA kit. Preparation of kit to detecting different diseases about HIV, hepatitis virus and others was final target of this research. The penetration of rapid test kit and on-site, in-home testing was enhanced as more economic costs of selenium nanoparticles.

免疫层析技术在疾病快速检测中的应用很广泛，尤其是胶体金试剂盒；虽然胶体金具有良好的稳定性、灵敏等优点，但其存在制备相对复杂、成本昂贵等问题。我们通过制备三聚氰胺纳米硒快速检测试剂盒发现纳米硒具有①制备更加简便、②性能更加稳定、③更加灵敏以及④更加经济等优点；本课题拟通过①纳米硒的研制：抗坏血酸还原亚硒酸，采用不同稳定剂并筛选其出最适宜的工作浓度，并对纳米硒的稳定性、敏感性、生物相容性、形态学特性进行控制，②硒标技术的建立：用纳米硒标记抗艾滋病毒抗体、抗肝炎病毒抗体等抗体，验证本研究制备的纳米硒的通用性，③临床标本的佐证：利用胶体金法试剂盒对比纳米硒试剂盒效果，利用ELISA等方法校验试剂盒稳定性和灵敏性。最终旨在研究出适宜于筛检艾滋病、肝炎等传染性等疾病的试剂盒；由于纳米硒制备原料成本更为经济，因此运用纳米硒制备试剂盒可以提高试剂盒的普及度以及家庭自检使用率。

免疫层析技术在疾病快速检测中的应用很广泛，尤其是胶体金试剂盒；虽然胶体金具有良好的稳定性、灵敏等优点，但其存在制备相对复杂、成本昂贵等问题。我们前期通过制备三聚氰胺纳米硒快速检测试剂盒发现纳米硒具有制备更加简便、性能更加稳定、更加灵敏以及更加经济等优点；本课题通过.（1）纳米硒的研制：利用抗坏血酸还原亚硒酸，分别采用阿拉伯树胶粉（GA）、明胶（Gelatin）、十二烷基磺酸钠（SDS）、聚乙二醇（PEG）、SDS＋PEG作为稳定剂，并对纳米硒的稳定性、敏感性、生物相容性、形态学特性进行控制，最终筛选SDS＋PEG为稳定剂，且其浓度为5 mM SDS+0.1M PEG，最终获得澄清透明、分散均匀、粒径均一、性能稳定的纳米硒溶液，纳米硒的粒径为36.63nm；.（2）硒标技术的建立：用上述纳米硒成功标记抗盐酸克伦特罗抗体，标记浓度为2 μg/mL，制备的试剂盒可以有效检出3ng/mL的盐酸克伦特罗，该结果用酶联免疫吸附实验（ELISA）验证；成功标记抗人绒毛膜促性腺激素抗体，标记浓度为6 μg/mL，制备的试剂盒灵敏度可达10 mIU/mL，该结果通过经过ELISA验证；.（3）实际标本的佐证：从某猪场收集18份猪尿液样本，纳米硒试剂盒与ELISA检测结果一致，符合率100%；从医院收集20例孕妇血清与7例正常人血清进行实际样本检测。实验过程中采用双盲实验，将血清样本滴加到自制的纳米硒侧向层析检测试剂盒中进行检测，阳性样本与阴性样本均检出，与ELISA结果符合率为100%。分别与相应的胶体金试剂盒对比，以检测CLE为例，将纳米硒用做竞争法试纸条的标记物时，与胶体金相比，其制备成本降低了30%，灵敏度提高了25%；以检测HCG为例用于夹心法试纸条的制备中，纳米硒与胶体金的制备成本与灵敏度一致。