Interference of sugars with the binding of biotin to streptavidin and avidin

Streptavidin and avidin have found widespread use as detection reagents in immunology, biochemistry and cell biology due to their high affinity binding to biotin, but the cellular functions of these proteins are not known. We have found that various sugars interfere with the binding of streptavidin and avidin to biotin. Mannose was most effective in inhibiting the binding to biotin followed by other saccharides. The inhibitory effect is most probably due to interactions of the sugars with residues in the binding pocket of streptavidin and avidin for biotin. These results show that great caution has to be exercised in the evaluation of experiments conducted with these detection reagents in the presence of sugars.     

链霉亲和素-自噬相关基因融合蛋白的原核表达及纯化

目的 构建链霉亲和素（streptavidin,SA）和自噬相关基因（Beclin 1）重组质粒,并对融合蛋白SA-Beclin 1进行表达和纯化.方法 利用基因重组技术将核心SA与Beclin 1序列连接,克隆人pQE80形成pQE80-SA-Beclin 1重组载体,IPTG诱导融合蛋白原核表达,镍亲和凝胶层析柱纯化融合蛋白,Western blot鉴定.结果 PCR成功扩增核心SA活性中心,酶切鉴定和测序均证实重组载体pQE80-SA-Beclin 1构建成功;IPTG诱导后SA-Beclin 1融合蛋白（含His标签）在大肠杆菌中高效表达,SDS-PAGE分析表达的融合蛋白以包涵体为主,经镍亲和凝胶层析柱纯化得到融合蛋白,Western blot鉴定其相对分子量约为72000kD,与预期相符.结论 本文成功构建了重组质粒并表达纯化了SA-Beclin 1融合蛋白,为进一步研究SA-Beclin 1的功能及临床应用奠定了基础.     

链霉亲和素化载紫杉醇相变型PLGA纳米粒的制备及体外超声显影

目的制备链霉亲和素化载紫杉醇相变型PLGA纳米粒(PTX-PLGA-SA/PFPs),并观察其体外低强度聚焦超声(LIFU)致相变后超声增强显影特性。方法采用单乳化法(O/W)制备载紫杉醇相变型PLGA纳米粒,高效液相色谱法检测紫杉醇包封率;碳二亚胺法连接链霉亲和素(SA),共聚焦激光显微镜观察二者连接情况,流式细胞术检测二者链接率;体外LIFU致相变观察超声增强显影情况。结果制备的纳米粒粒径为(322.2±85.6)nm,表面电位(-5.66±3.46)mV。紫杉醇的包封率及载药量分别为(71.56±6.51)%、(6.57±0.61)%,与链霉亲和素的连接率为(97.16±1.20)%。LIFU功率7.5 W作用3min时可明显增强该纳米粒在体外的B-mode及造影模式下的超声显影。结论成功制备了PTX-PLGA-SA/PFPs纳米粒,其紫杉醇包封率高、链霉亲和素连接率高,体外声致相变后可显著增强超声显影。     

A simple binding assay for the direct determination of biotin in urine

BACKGROUND: We have devised a simple assay to detect adequate biotin intake, which uses an alternative configuration from most existing assays. METHODS: The assay depends on the competition of streptavidin peroxidase for immobilized biotin or soluble biotin in standards or samples. Immobilized streptavidin peroxidase is detected using tetramethylbenzidine, and the plates are read at 450 nm. The assay was normalised by determining the biotin/creatinine ratio in the urine of healthy adults. Urinary biotin excretion was measured in unsupplemented pregnant women. The half-life of biotin excretion after a single oral supplement was determined for healthy volunteers. RESULTS: Urinary biotin excretion in unsupplemented pregnant women was 2.9+/-1.9 micromol/mol creatinine (mean+/-S.D.) and was significantly lower (p<0.001) than those of healthy males and females, which were 9.0+/-5.4 and 7.0+/-2.1 micromol/mol creatinine (mean+/-S.D.), respectively. The half-life of a single oral biotin supplement was 30-40 h, with excretion returning to basal levels at 70 h. CONCLUSION: We have devised a novel binding assay for the direct determination of total biotin excretion in urine, which is suitable for routine clinical laboratory. The assay is inexpensive, simple, rapid, and could be fully automated.     

鼻咽癌血清学检测方法的改进

目的 改进现有鼻咽癌血清学早期诊断方法,提高检测灵敏度.方法 用链霉亲和素.生物素放大免疫酶法与既有常规免疫酶法对鼻咽癌防治示范基地所取294份普查血清IgA/VCA,IgA/EA抗体进行血清学检测,并用SPSS统计软件对检测结果进行χ2检验和t检验.结果 共检测30岁以上294份普查人群血清,其中鼻咽癌患者血清106份,健康人血清188份.改进的链霉亲和素一生物素放大免疫酶法与既往已有的免疫酶法相比,血清IgA/VCA、IgA/EA抗体的检测阳性率增加;检测血清抗体的几何平均数明显提高.结论 改进方法可以提高血清学检测的灵敏度,同时保证了检测结果的特异性,提高了鼻咽癌的检出率,可以应用于鼻咽癌早期筛查工作.     

串联亲和纯化标记的HMGB1细胞因子诱导片段1融合表达载体构建和蛋白纯化及其功能初步探讨

目的：构建高迁移率族蛋白B1（HMGB1）中细胞因子诱导片段1（CIF1）与串联亲和纯化（TAP）系统中纯化标签链霉亲和素结合肽（SBP）和钙调蛋白结合肽（CBP）序列相融合的原核表达载体,观察重组蛋白对小鼠单核／巨噬细胞白血病细胞释放肿瘤坏死因子-α（TNF-α）的影响,为HMGB1参与炎症反应作用机制的研究以及CIF1结构域细胞膜表面受体蛋白的获得和鉴定奠定基础。方法：采用PCR方法分别扩增出cBP-SBP和CIF1的编码序列,构建表达质粒pET-14b／CBPSBP—CIF。利用Ni—NTA亲和树脂纯化融合蛋白,纯度达85％以上。采用纯化后的蛋白诱导小鼠单核／巨噬细胞白血病细胞RAW264．7,利用LiquiChip液相蛋白芯片系统检测RAW264．7细胞释放TNF-a的水平变化。结果：表达载体构建正确,CIF1重组蛋白表达量高,纯度好。当蛋白浓度大于0．5ng／μl时,CIF1重组蛋白诱导细胞TNF-α的分泌量与对照组相比差异具有显著性（P〈0．05）,并且在CIF1蛋白浓度O～2．5ng／μl范围内,TNF-α的分泌量与CIF1重组蛋白的浓度呈显著的剂量依赖关系。结论：原核表达纯化了His—CBP—SBP—CIF1融合蛋白,该融合蛋白可以有效地作用于巨噬样细胞内的信号转导过程,介导了炎症因子TNF—α的分泌表达。     

Liquid-phase hybridization and capture of hepatitis B virus DNA with magnetic beads and fluorescence detection of PCR product

The polymerase chain reaction (PCR) exceeds all hitherto known detection limits. This sensitivity could lead to false positive results. Every manipulation increases the risk of contamination via, for example, aerosols. Most protocols for the extraction of template nucleic acids are complicated and possible centrifugation steps do not reduce the risk of aerosols. In addition, most of the methods for analysis are time-consuming and cannot be applied to different template materials. An alternative extraction method has been developed. The fast chemical denaturation of template by guanidine thiocyanate was followed by liquid hybridization to biotinylated oligonucleotides. The template nucleic acid could be washed after binding to streptavidin-coated paramagnetic beads to reduce influence on the enzymatic amplification steps. PCR of hepatitis B virus deoxyribonucleic acid was used to demonstrate how easy, versatile, and time-saving this method is without centrifugation. The level of extracted nucleic acids was quantitated and the properties for sensitive extraction were evaluated. After PCR an additional step was developed which used fluorescent staining to detect positive amplifications. This is useful to identify positive results in predominantly negative samples.     

链霉亲和素修饰的CdSe–ZnS量子点对聚合酶链反应的影响作用初步研究

目的:探讨链霉亲和素修饰的CdSe–ZnS量子点对聚合酶链反应的影响。方法:将链酶亲和素修饰的量子点浓度进行稀释观察其对于聚合酶链反应体系的浓度效应;反应体系中含有一定浓度的链酶亲和素修饰的量子点,在不同的退火温度下进行扩增,观察链酶亲和素修饰的量子点对退火温度的影响;将不同浓度的BSA加入到含有一定浓度的链酶亲和素修饰的量子点的反应体系中,观察BSA的影响作用;将不同公司的Taq酶与不同浓度链酶亲和素修饰的量子点组合,观察对扩增体系的影响。结果:一定浓度链霉亲和素修饰的量子点可以促进聚合酶链反应的扩增效率,拓宽PCR的退火温度范围;BSA可以逆转链霉亲和素修饰的量子点对于PCR的优化作用;量子点可能是与Taq酶相互作用,来影响PCR的扩增效能。结论:一定浓度的链霉亲和素修饰的量子点可以优化PCR的反应体系。     

The use of an electroporation apparatus for the production of murine hybridomas

Antigen-directed electrofusion was carried out using biotin-streptavidin to bridge antigen-specific splenocytes to myeloma cells. Electrofusion was performed using a commercial electroporation apparatus. Electrofusion conditions were optimized by measuring the survival of myeloma cells after a range of electrical pulse conditions. The procedure was tested with antigens of high and low immunogenicity. The yields of hybridomas secreting antibodies specific for both antigens were considerably increased by the use of the electrofusion procedure.     

A solid-phase technique for preparation of no-carrier-added technetium-99m radiopharmaceuticals: application to the streptavidin/biotin system

A high effective specific activity (HESA) formulation of a biotin-containing ^99mTc ligand [RP488: dimethyl-Gly-Ser-Cys(Acm)-Lys(Biotin)-Gly] conveniently prepared from solid phase was compared to a typical low effective specific activity (LESA) solution formulation to demonstrate improved targeting to streptavidin in an in vitro assay and in an in vivo rat model. RP488 was coupled to a maleimide-functionalized polyethylene glycol resin via a thiol ether linkage and labeled with ^99mTc-gluconate at room temperature, followed by elution of the HESA ^99mTc-RP488 in saline (minimum specific activity 1000 TBq/mmol by amino acid analysis). Both HESA and LESA ^99mTc-RP488 labeled at > 90% purity. In vitro, HESA ^99mTc-RP488 incubated with streptavidin-agarose was bound quantitatively, but there was competition from addition of increasing amounts of cold RP488. In rats, radiotracer uptake was evident at the site of implantation of streptavidin-agarose beads for the HESA dose, less uptake of low effective specific activity (LESA) material, and no appreciable uptake in the control rats of the LESA or HESA dose. The target-to-background ratio for HESA ^99mTc-RP488 was 5.4 times that of the control. The solid-phase technology offers a convenient way to prepare high specific activity receptor-targeting ^99mTc radiopharmaceuticals.