硒对儿童桥本甲状腺炎的影响

为探讨硒对儿童桥木甲状腺炎的干预效果,采用双盲前瞻性随机对照试验观察了亚硒酸钠对桥本甲状腺炎患儿甲状腺球蛋白抗体(TGA) 和甲状腺微粒体抗体(TMA)的影响。结果表明,亚硒酸钠治疗组治疗后,TMA显著下降(P<0.01),而TGA没有差异(P>0.05)。提示硒可降低儿童桥本甲状腺炎TMA的质量分数。     

Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody

Although Staphylococcus Protein A (SpA) affinity chromatography is the state of the art capture step for antibody purification, non-affinity methods are more economical. We used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to evaluate the purification of a recombinant IgG₁ antibody from cultured cells, with two different processes: (1) SpA capture followed by cation-exchange chromatography (CEX); and (2) CEX capture, followed by anion exchanger, then hydrophobic interaction chromatography. Efficiencies were similar in sodium dodecylsulphate polyacrylamide gel electrophoresis and size-exclusion chromatography; however, 2-D DIGE revealed higher efficiency with SpA than with CEX capture. Thus, 2-D DIGE is a valuable tool for downstream process development.     

Picogram-detection of estradiol at an electrochemical immunosensor with a gold nanoparticle|Protein G-(LC-SPDP)-scaffold

Low picograms of the hormone 17β-estradiol were detected at an electrochemical immunosensor. This immunosensor features a gold nanoparticle|Protein G-(LC-SPDP)¹-scaffold, to which a monoclonal anti-estradiol capture antibody was immobilised to facilitate a competitive immunoassay between sample 17β-estradiol and a horseradish peroxidase-labelled 17β-estradiol conjugate. Upon constructing this molecular architecture on a disposable gold electrode in a flow cell, amperometry was conducted to monitor the reduction current of benzoquinone produced from a catalytic reaction of horseradish peroxidase. This current was then quantitatively related to 17β-estradiol present in a sample. Calibration of immunosensors in blood serum samples spiked with 17β-estradiol yielded a linear response up to ∼1200 pg mL⁻¹, a sensitivity of 0.61 μA/pg mL⁻¹ and a detection limit of 6 pg mL⁻¹. We attribute these favourable characteristics of the immunosensors to the gold nanoparticle|Protein G-(LC-SPDP) scaffold, where the gold nanoparticles provided a large electrochemically active surface area that permits immobilisation of an enhanced quantity of all components of the molecular architecture, while the Protein G-(LC-SPDP) component aided in not only reducing steric hindrance when Protein G binds to the capture antibody, but also providing an orientation-controlled immobilisation of the capture antibody. Coupled with amperometric detection in a flow system, the immunosensor exhibited excellent reproducibility.     

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 μg l⁻¹ for carbaryl and TCP, respectively. The sensitivity attained (I₅₀ value) was around 30 μg l⁻¹ for both compounds. Linear working ranges were 15-53 μg l⁻¹ for carbaryl and 13-83 μg l⁻¹ for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.     

Development of an ICP-MS immunoassay for the detection of anti-erythropoietin antibodies

A sandwich-type immunoassay linked with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the detection of anti-erythropoietin antibodies (anti-EPO Abs). Recombinant human erythropoietin (rhEPO) was immobilized on the solid phase to capture anti-rhEPO Abs specifically. After the immunoreactions with Au-labeled goat-anti-rabbit IgG, a diluted HNO₃ (2%) was used to dissociate Au nanoparticles which was then introduced to the ICP-MS for measurements. Under the optimized conditions, the calibration graph for anti-EPO Abs was linear in the range of 35.6-500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹ (3σ, n = 9). The relative standard deviation (R.S.D.) for three replicate measurements of 30.9 ng mL⁻¹ of anti-EPO Abs was 8.43%. The recoveries of anti-EPO Abs in sera at the spiking level of 50, 100, 150, 200 and 400 ng mL⁻¹ were 99.2%, 101.5%, 95.0%, 94.0% and 102.9%, respectively. For the real sample analysis, 26 samples from healthy people and 53 samples from patients with rhEPO treatments were studied. One sample from patients showed significantly higher anti-EPO Abs from other samples, indicating a possibility of immune response of this patient.     

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.     

稀土上转换发光材料标记抗体的制备及在免疫组化中的应用

采用微波辐射法合成了具有上转换发光特性的六方相纳米粒子NaGdF₄: Yb³⁺,Er³⁺(UCNPs), 其晶粒大小约为65 nm, 且粒子在980 nm的激发光下显示绿光(550 nm). 进一步在NaGdF₄: Yb³⁺,Er³⁺纳米晶的表面包覆了一层二氧化硅层, 进行氨基功能化后获得了表面共价结合氨基基团的粒径为70 nm的上转换发光纳米微球NaGdF₄: Yb³⁺,Er³⁺@SiO₂-NH₂(UCNPs@SiO₂-NH₂). 通过共价键将UCNPs@SiO₂-NH₂与多克隆抗体免疫球蛋白联接, 将标记后的多克隆抗体应用于传统的免疫组化检测子宫内膜腺细胞中基质金属蛋白酶组织抑制剂-4(TIMP-4)蛋白的表达. 结果表明, 微波合成的稀土上转换发光纳米材料形貌规则且粒径均一, 包覆硅壳后材料具有良好的分散性和水溶性, 荧光强度高且稳定, 在980 nm激发光下对生物组织无背景荧光, 可以很好地检测组织中蛋白质的表达.     

基于CSA-SQP的预测控制器参数整定研究

针对预测控制器参数整定困难, 提出了一种基于克隆选择和序列二次规划的预测控制器参数整定算法, 建立了一个基于免疫原理和序列二次规划算法进行控制器参数整定的机制, 并给出了参数整定问题中的抗原、抗体及亲和力的定义. 在此基础上, 针对系统的不确定性干扰, 构造了基于事件触发的参数调整框架. 最后, 将算法应用于仿真实验, 通过与设定值控制结果的对比, 证明所提出的预测控制器参数整定方法是有效的.     

Preparation of Functionalized Fe3O4@SiO2 Magnetic Nanoparticles for Monoclonal Antibody Purification

Magnetic Fe₃O₄@SiO₂ nanoparticles with superparamagnetic properties were prepared via a reverse mi-croemulsion method at room temperature. The as-prepared samples were characterized by transmission electron mi-croscopy(TEM), X-ray diffractometry(XRD), and vibrating sample magnetometry(VSM). The Fe₃O₄@SiO₂ nanoparticles were modified by (3-aminopropyl)triethoxysilane(APTES) and subsequently activated by glutaraldehyde(Glu). Protein A was successfully immobilized covalently onto the Glu activated Fe₃O₄@SiO₂ nanoparticles. The adsorption capacity of the nanoparticles was determined on an ultraviolet spectrophotometer(UV) and approximately up to 203 mg/g of protein A could be uniformly immobilized onto the modified Fe₃O₄@SiO₂ magnetic beads. The core-shell of the Fe₃O₄@SiO₂ magnetic beads decorated with protein A showed a good binding capacity for the chime-ric anti-EGFR monoclonal antibody(anti-EGFR mAb). The purity of the anti-EGFR mAb was analyzed by virtue of HPLC. The protein A immobilized affinity beads provided a purity of about 95.4%.     

超大孔聚合物亲和色谱层析介质的制备及评价

探索了用席夫碱法在超大孔聚合物微球表面偶联Protein A配基制备亲和层析介质的方法,以亲水化后的聚丙烯酸酯类超大孔微球为基质,考察了氧化剂浓度(H5IO6)对配基偶联量的影响,以扫描电子显微镜表征微球表面形貌,结果发现其偶联Protein A后微球仍能够保持其大孔结构。考察了该类亲和介质在不同操作流速(361~3 600 cm/h)下的动态载量,在3 600 cm/h操作流速下,人抗体载量与361 cm/h相比仅下降10%左右,表明该类介质适合抗体大分子的快速传质,能够满足快速、高效、高通量分离纯化的需求。