基于光谱自编码微球的多样品免疫荧光混合筛选和荧光成像

研究了微球粒度、温度、pH和包被液浓度对聚苯乙烯微球免疫吸附的影响。将20种编码微球与20个样品对映包被,进行免疫荧光混合筛选,分析荧光信号最强微球的光谱编码,确定了阳性样品的编号。其异硫氰酸荧光素(FITC)荧光标记抗体筛选灵敏度可达体积稀释比1∶2000。采用氩离子激光扫描荧光成像技术,成功实现了基于光谱自编码微球的多样品免疫荧光混合筛选和荧光成像。     

应用荧光微球技术进行免疫检测研究

以聚苯乙烯羧基荧光微球为固相载体,将人免疫球蛋白G(IgG)和猪血红蛋白(Hb)分别与微球偶联,应用Luminex-100TM荧光微球检测系统检测抗体IgG和抗原Hb与荧光微球的偶联效率.结果表明,采用100μg·mL-1抗体或抗原浓度可以获得较高的偶联效率,且检测快速灵敏.应用荧光微球进行抗原抗体免疫检测可行.     

高通量药物筛选中的荧光微球

简要介绍了邻近闪烁分析技术及由其发展而来的Alphascreen分析技术 ,对其中使用的荧光微球的要求、特点作了介绍 ,并按荧光物质在微球中所处的位置对荧光微球的制备方法作了较为详细的讨论。     

功能化碳微球构建自组装膜

采用浓HNO3和H2O2对原始CMSs进行表面修饰,使其团聚现象得到改善;以氧化后CMSs作为结构基团,采用垂直沉积法实现CMSs的自组装,得到CMSs薄膜.考察了溶剂、悬浮液pH值、悬浮液浓度和反应温度对自组装的影响.采用场发射扫描电子显微镜和原子力显微镜对产物进行表征分析,结果表明,以NaOH溶液为溶剂配制悬浮液、...     

溶胀法制备高强度聚苯乙烯荧光及编码微球

通过对荧光微球制备的传统溶胀法的改进,以二氯甲烷作溶胀剂,0.25%SDS水溶液作分散体系(促进疏水性染料溶胀进入种子微球以增加微球荧光强度),制备出了荧光强度高,单分散性好(平均粒径3.6μm,变异系数3.7%),光学性质稳定,定量染色的绿色、橙色、红色三种荧光微球。将发生荧光共振能量转移的两种染料同时溶胀进种子微球中,制备了能产生双荧光信号的荧光编码微球。     

磁性高分子微球固定化酶的制备及应用

利用磁性高分子微球通过化学反应固定化酶,可以借助外部磁场方便地分离回收固定化酶,将固定化酶放入磁场稳定的流动床反应器中还可以减少持续反应体系中的操作.简要地介绍了磁微球的制备方法,包括包埋法、分散聚合、乳液聚合和悬浮聚合,对磁性微球固定化酶的制备方法和原理进行了探讨,论述了磁性高分子微球固定化酶的特点及应用.     

自乳化制备新型含醛基纳米微球

采用Witting等一系列化学反应合成了一种既含醛基又含糖基的乙烯基单体1,2:3,4-Di-O-异亚丙基-6-O-(2'-甲醛-4'-苯乙烯基)-D-半乳糖(IVDG),该单体为白色粉末状,熔点为97～98℃,用元素分析、质谱和核磁分析进行了结构表征。在AIBN的引发下,60℃四氢呋喃中、24h单体转化率可达到68%,聚合物相对分子质量为130000,表明该单体具有较高的聚合活性。经88%甲酸水解,脱去保护基团异亚丙基后,所得的聚合物具有两亲性,不需要外加乳化剂,在水中能够自组装形成含有大量醛基的纳米微球,动态光散射数据表明粒径为450nm,其形貌可在透射电镜下直接观察。该聚合物在生物医药领域有潜在的应用前景。     

逐层自组装法制备纳米二氧化钛空心微球

本文采用溶胶-凝胶法与自组装技术相结合的工艺,以聚乙二醇为模板制备了多层组装的TiO2空心微球。采用TEM对产物进行了表征。以甲基橙溶液的脱色降解率,考察了不同煅烧温度和组装层数对TiO2空心微球光催化性能的影响。结果表明：500℃煅烧下组装四层的TiO2空心微球光催化效果最佳,2h内对甲基橙的催化效果可达到97.25%。     

交联聚苯乙烯微球的制备

以聚乙烯醇为分散剂、水为反应介质、过氧化二苯甲酰为引发剂,异戊醇为致孔剂,采用苯乙烯——二乙烯基苯悬浮共聚悬浮聚合的方法,通过优化反应条件,成功制得了平均粒径为0．8mm的多孔微球。研究了引发剂浓度,制孔剂浓度,分散剂浓度和搅拌速度对微球粒径的影响。并用扫描电镜（SEM）对微球进行了表征。     

喹噁啉-2-羧酸残留的酶联免疫检测方法及试剂盒

该发明属于免疫化学分析技术领域。公开了一种用于喹噁啉-2-羧酸残留检测的酶联免疫方法及试剂盒,该方法主要包括药物改造,免疫原、包被原、抗体的制备以及样品前处理和ELISA检测方法的建立。试剂盒主要由喹噁啉-2-羧酸（QCA）特异性抗体、QCA标准品、包被有QCA与）γ-氨基丁酸反应后与卵清蛋白偶联物的酶标板组成。     

脱二氧喹烯酮多克隆抗体的制备及其直接竞争ELISA检测方法的建立

利用脱二氧喹烯酮（DQCT）的酮基为活性基团,分别采用混合酸酐法和碳二亚胺法合成了其免疫原（DQCT-BSA）和包被原（DQCT-OVA）,用过碘酸钠氧化法将DQCT-OVA与辣根过氧化物酶（HRP）偶联,制备了酶标抗原（DQCT-OVA-HRP）。用DQCT-BSA免疫Balb／C小鼠,获得了效价1：128000的抗DQCT的多克隆抗体。分别用间接竞争ELISA法和直接竞争ELISA法检测DQCT并比较,最终建立了直接竞争ELISA检测方法,其IC50为39．8μg·mL^-1,检测范围为1．6-1258．9μg·mL^-1。为后续实际样品中脱二氧喹烯酮的检测奠定了基础。     

Evaluation of Pseudoenantiomeric Mixed Carbonates as Efficient Fluorogenic Probes for Enantioselectivity Screening

We report mixed carbonates as enzyme substrates and demonstrate their application as fluorogenic probes for lipase and esterase enantiopreference screening. By the application of pseudoenantiomers with distinct fluorescence behaviors, it is possible to evaluate the activity and enantiopreference of hydrolytic enzymes. In order to validate our method, enantioselectivities calculated from fluorometric measurements were compared with the results obtained from larger‐scale kinetic resolution.     

咸鸭蛋清抗生物素蛋白的分离纯化及快速测定方法的建立

采用2-亚氨基生物素琼脂糖凝胶4B从酸热变性后咸鸭蛋清中亲和分离了抗生物素蛋白,并利用生物素化的过氧化物酶偶联起显色反应,建立了一种简便的抗生物素蛋白含量测定新方法．进而对咸鸭蛋清抗生物素蛋白进行了普查。实验结果表明：咸鸭蛋清抗生物索蛋白回收率达60．1％±5．0％,纯化倍数为222．5,纯化蛋白经SDS—PAGE电泳均显示单一蛋白染色带,其对应的分子量约为67．8K,咸鸭蛋清抗生物素蛋白质量分数约为0．05％,是抗生物索蛋白潜在的资源,本研究为咸鸭蛋清的回收利用打下了基础。     

High-Throughput Screening of Signal Peptide Library with Novel Fluorescent Probe

Nitrile hydratase (NHase) is an excellent biocatalyst for the synthesis of amide compounds and is composed of two heterologous subunits. However, the secretory expression of NHase has been difficult to achieve because of its complex expression mechanism. In this work, a novel fluorescent probe Rho-IDA-CoII was synthesized by a one-pot method. Rho-IDA-CoII could specifically label His-tagged proteins in vitro, such as for staining in-gel, Western blot, and ELISA analysis. Furthermore, Rho-IDA-CoII combined with dot blots could quantitatively detect His-tagged proteins at between 1–10 pmol and perform high-throughput screening for the NHase signal peptide library. Recombinant Bacillus subtilis WB800/phoB-HBA with the extracellular expression of NHase was screened (ca. 6500 clones). After optimization of fermentation conditions, the NHase activity in the culture supernatant reached 17.34±0.16 U/mL. This is the first time that secretory NHase has been expressed in B. subtilis successfully.     

脑膜炎球菌W135群多糖含量和分子大小双抗体夹心ELISA检测方法的建立及初步应用

目的建立检测A、C、Y、W135群脑膜炎球菌(Neisseria meningitidis,Nm)多糖疫苗(Groups A,C,Y,W135 menig-nococcal polysaccharide vaccine,MPV4)中W135群多糖含量和分子大小的双抗体夹心ELISA法,并进行初步应用。方法以抗W135群Nm多克隆抗体作为包被抗体,建立双抗体夹心ELISA法,采用棋盘滴定法筛选包被抗体与酶标抗体的最佳工作浓度,对W135群Nm多糖进行特异性定量测定,并验证线性关系的重复性;对建立的ELISA法进行特异性、准确度、精密度及定量限的验证;采用建立的ELISA法检测10批W135群Nm多糖样品和10批无关流脑多糖样品,进行W135群Nm多糖的鉴别试验;采用建立的ELISA法测定MPV4多糖含量、多糖分子大小和回收率。结果经棋盘滴定法确定双抗体夹心ELISA法的最佳包被抗体工作浓度为10μg/ml,最佳酶标抗体工作浓度为1∶15 000稀释,W135群Nm多糖在2.5~20 ng/ml浓度范围内剂量反应曲线线性关系良好,相关系数大于0.99。采用建立的双抗体夹心ELISA法检测W135群Nm多糖为强阳性,检测其余样品的结果均为阴性;试验内及试验间测定16、8、4 ng/ml W135群Nm多糖含量的变异系数在1.1%~9.0%之间,回收率在87.5%~105.0%之间,定量限为4 ng/ml;检测W135群Nm多糖的阳性符合率和无关多糖的阴性符合率均为100%。采用该法测定3批MPV4中W135群多糖含量、分子大小及回收率均符合申报MPV4疫苗暂行规程关于W135群Nm多糖的质量标准。结论建立的双抗体夹心ELISA法可用于MPV4中W135群多糖含量和分子大小的测定。     

High-Throughput NanoBiT-Based Screening for Inhibitors of HIV-1 Vpu and Host BST-2 Protein Interaction

Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.     

A High‐Throughput Mass‐Spectrometry‐Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

The most commonly employed glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position in potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a qualitative mass spectrometry–based glycosidase assay amenable to high‐throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high‐throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved through the observation of an appropriate decrease in mass between a potential sugar substrate and its corresponding product by electrospray ionization mass spectrometry (ESI‐MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.