多聚螯合物酶联抗体交联磁性纳米乳胶的脂联素免疫透射比浊方法研究

采用多聚螯合物酶联抗体交联磁性纳米乳胶的脂联素免疫透射比浊法检测了血清中脂联素. 发现多聚螯合物酶上含有大量HRP酶, 可以极大地放大检测信号; 在优化条件下, 免疫比浊信号强度在脂联素0.005~0.2ng?mL-1范围内变化时, 并随着ADPN浓度的增加呈线性关系(R2=0.998), 检出限为2 pg?mL-1. 该方法能成功用于血清样本中脂联素的检测.     

光纤荧光免疫分析系统的研制及应用

将光纤传感技术、荧光分析与免疫分析技术结合,自行设计了Y型分叉光纤、光强可调的光纤固定架及性能优良的荧光测定池,建立了新型光纤荧光免疫系统。系统性能可靠,操作简便,既可进行普通的荧光分析,又可进行光纤荧光免疫分析。将系统用于临床血清标本中肺炎支原体抗体的测试,结果与荧光显微镜法和ELISA法结果一致。该新型光纤荧光免疫系统在临床、环保等领域有广泛的应用前景。     

单克隆抗体-白介素2融合蛋白一级结构的确证

建立单克隆抗体-白介素2融合蛋白一级结构确证的方法.用基质辅助激光解吸飞行时间质谱测定单克隆抗体-白介素2融合蛋白的精确相对分子质量,毛细管等电聚焦方法测定其等电点,通过N-末端氨基酸序列的测定以及肽图分析,证实了抗体-白介素2融合蛋白一级结构表达的正确性,同时为单克隆抗体-白介素2融合蛋白的质量标准研究奠定了基础.     

三唑磷的免疫分析技术研究

将合成的三唑磷半抗原采用活性酯法分别与牛血清白蛋白和卵清蛋白共价偶联制备突出三唑磷分子结构特征的人工抗原与包被抗原。以人工抗原免疫新西兰白兔获得抗血清,采用硫酸铵分步盐析和DEAE纤维素反相吸附法从抗血清中分离纯化对三唑磷具特异性亲合力的抗体,以辣根过氧化物酶采用混合酸酐法标记半抗原。在此基础上,首次成功建立了对三唑磷具高特异性的间接竞争、包被抗体直接竞争酶联免疫吸附分析（ELISA）技术。在优化条件下,三唑磷测定的线性浓度范围为0．001～1．0mg／L,检出限0．11μg/L,其他类似结构的常用有机磷酸酯类杀虫剂和苯唑醇不干扰三唑磷的测定。     

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。     

Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

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.