酶联免疫吸附分析法测定食品中的苏丹红I号

本研究建立了测定食品中苏丹红I号含量的间接竞争酶联免疫分析法。首先对苏丹红I号分子作了的修饰，再与载体蛋白交联制得免疫原和包被抗原，经动物免疫制得抗苏丹红I号的抗体。在包被抗原为100μg／L，抗体为1：100，000倍稀释，标记二抗为1：15000倍稀释的优化条件下，测得检出限为0．12μg／L，IC50值（标准曲线中吸光度抑制至最大吸光度值的50％时所对应的待测物浓度）为0．74μg／L。苏丹红I号在番茄酱和辣椒面中的回收率分别为106％和110％。样品仅需甲醇萃取再用缓冲液简单稀释就可以直接进行ELISA测定。     

Quantitative immunofluorescent assay of full-length,recombinant CD4 in solution and mapping of its epitopes

A specific, rapid, and sensitive method for the detection of CD4 in solution was developed using pairs of fluorescently stained monoclonal antibodies which do not cross-compete. The assay is quantitated by flow cytometry using Simply Cellular microbeads (SC beads) as the primary support for the first anti-CD4 mAb. This method uses the standard conditions for anti-CD4 monoclonal antibody binding, washing, detection, and quantitation by flow cytometry of the CD4 antigen either bound to the SC beads or expressed on the cell surface. The monoclonal antibody used (Leu 3a PE) is the standard reference used to evaluate the CD4 concentration. This method differs from ELISA techniques, which need an antigen standard curve and thus can be influenced by the quality and source of the antigen. This type of assay is also a procedure which enables determination of the level of oligomerization of the bound antigen. It can be used for any antigen to which monoclonal antibodies recognizing at least two distinct epitopes are available. The use of soluble or full-length CD4 derivatives as potential therapeutic agents against AIDS, would benefit from a precise quantitation of the CD4 molecules which still have their proper tertiary structure.     

Simultaneous flow-injection assay of creatinine and creatine in serum by the combined use of a 16-way switching valve, some specific enzyme reactors and a highly selective hydrogen peroxide electrode

Creatine and creatinine in serum were assayed simultaneously in a noble flow-injection system made up by a 16-way switching valve with two sample loops, three enzyme reactors positioned in a serial way, and a delay coil needed to separate two peaks corresponding to two sample portions injected simultaneously. A Nafion/poly(1,2-diaminobenzene) bilayer modified electrode was used to selectively detect the hydrogen peroxide generated as one of the end products in the last enzyme reactor, without any interferences from electroactive species (such as l-ascorbate and urate) and proteins present in the serum. Because two sample portions passed through the flow line with different residence time, two peaks were obtained. The first peak corresponded to creatine and the second peak to the total of creatine and creatinine. The maximum currents of both peaks were linearly related to the concentration of creatine and total of creatine and creatinine in the range of 1-100 μM, respectively; 20 samples h⁻¹ could be processed with an R.S.D. <1.6%.     

甜叶醇衍生物的合成及其生物活性

本文对甜叶醇的结构进行修饰合成14个新化合物。其中以化合物4对棉花种子的发芽及根促进作用最佳。     

Citrate and calcium determination in flavored vodkas using artificial neural networks

The development of multianalyte sensing schemes by combining indicator-displacement assays with artificial neural network analysis (ANN) for the evaluation of calcium and citrate concentrations in flavored vodkas is presented. This work follows a previous report where an array-less approach was used for the analysis of unknown solutions containing the structurally similar analytes, tartrate and malate. Herein, a two component sensor suite consisting of a synthetic host and the commercially available complexometric dye, xylenol orange, was created. Differential UV-Visible spectral responses result for solutions containing various concentrations of calcium and citrate. The quantitation of the relative calcium and citrate concentrations in unknown mixtures of flavored vodka samples was determined through ANN analysis. The calcium and citrate concentrations in the flavored vodka samples provided by the sensor suite and the ANN methodology described here are compared to values reported by NMR of the same flavored vodkas. We expect that this multianalyte sensing scheme may have potential applications for the analysis of other complex fluids.     

热挤压铸造新型镁合金微观组织及细胞生物活性分析

采用热挤压铸造工艺制造新型镁合金, Mg-Nd-Zn-Zr-Mn (平衡-3-0.2-0.4-0.2%)基, 研究了新型镁合金表面特征、力学性能及细胞生物活性. 选择NZ30K添加Mn元素制成新镁合金, 挤压前通过均匀化热处理, 减少挤压过程中铸态合金中的粗大析出相以及树枝晶形成的带状组织. 光谱测试分析合金成分; 显微观察合金铸态、横纵截面; 扫描电镜扫描; X射线衍射分析. 将合金制成金属棒、螺钉、接骨板等形状, 测试力学性能. 进行体外细胞培养, 利用DMEM完全培养基制作镁合金浸提液, 滤膜过滤后4℃保存; 大鼠骨髓间充质干细胞提取培养, 待细胞生长至70%~80%传代于24孔板种板, 添加浸提液培养12、24、36h, 利用线粒体膜电位检测试剂盒检测细胞凋亡活性. 以纯镁作为对照组, 进行力学性能测试及细胞活性凋亡测试. 热挤压后合金的组织由细小的再结晶晶粒与变形晶粒组成, 与铸态合金相比, 其组织明显细化. 经挤压加工后, Mg-3Nd-0.2Zn-0.4Zr-0.2Mn合金横截面为细小的等轴晶组织, 组织均匀性好; 纵截面出现了晶粒尺寸相对较大的长条状组织, 组织均匀性稍差. 扫描电镜图显示Mg-3Nd-0.2Zn-0.4Zr-0.2Mn合金中第二相颗粒沿挤压方向被碾碎成更细小的颗粒, 只有非常少量的弥散分布的颗粒状析出相, 而在该合金中有较多被碾碎的第二相, 发生了明显的动态再结晶, 挤压后获得的组织不均匀, 大晶粒被发生再结晶的小晶粒包围. 从X射线衍射图中可以看出该合金铸态组织主要由α-Mg、Mn、Mg12Nd和Y相等这几相组成. 力学性能测试表明, 新型镁合金综合力学性能明显优于纯镁金属. 短期细胞培养中, 新型镁合金无明显细胞毒性, 对细胞生长有积极促进作用. 新型镁合金热挤压后的横截面为细小等轴晶组织, 组织明显细化且均匀性好, 力学性能有极大提升; 在短期细胞培养过程中新型镁合金与纯镁都表现出无细胞毒性, 新型镁合金对细胞活性的提升优于纯镁组.     

Development of direct competitive enzyme-linked aptamer assay for determination of dopamine in serum

A rapid and cost-effective screening method based on a competitive enzyme-linked aptamer assay (ELAA) for dopamine (DA) in serum has been optimized and validated. In this paper, we report advantageous sensitivity and specificity of aptamer assays as compared to the existing antibody based-immunoassays. The RNA aptamer (67 mer) was immobilized via site-directed immobilization with biotin both at the 3′-end on aptamer and at neutravidin plate. Various factors such as incubation temperature, divalent ion – Mg²⁺ ion and treatment of serum solution were evaluated for the performance of ELAA. The aptamer was incubated for 1 h at 4 °C in the assay buffer containing 5 mM Mg²⁺ ion, and serum was diluted (1:9, serum:assay buffer) and filtrated through a 3 kDa dialysis membrane to extract the proteins present in the serum. Assay was performed with 0.01 μg mL⁻¹ of aptamer and 1.205 × 10⁻⁷ M DA-HRP conjugate using the optimized method. A dose–response curve was constructed, and the limit of detection and a dynamic range for the DA were determined as 1.0 × 10⁻¹² M and four orders (1.0 × 10⁻⁷ M to 5.0 × 10⁻¹¹ M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown a good agreement with the correlation coefficient (R² = 0.9872): Abs. (in serum) = 0.9612 × Abs. (in buffer) − 0.0556. The cross-reactivity evaluation demonstrated that norepinephrine showed some cross-reactivity (3.68%) whereas 3-methoxytyramine, epinephrine, homovanillic acid and 3,4-dihydroxyphenylacetic acid showed almost no cross-reactivity (<1%). Percent recoveries of DA in serum were quite satisfactory (∼95%). This paper describes usefulness of the aptamer assay in monitoring DA in human serum.     

A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M₁ (AFM₁) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM₁ surrogate, was generated by immunizing rabbits with F(ab′)₂ fragments from the anti-AFM₁ monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM₁ mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM₁ and the anti-Id antibody (y = 31.91x − 8.47, r = 0.9997). The assay was applied to analyze AFM₁ in spiked milk samples. The IC₅₀ value of the surrogate calibrator curve was 2.4 μg mL⁻¹, and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y = 0.81x + 9.82, r = 0.9922).     

Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals

Availability of highly sensitive assays for metal ions can help monitor and manage the environmental and food contamination. In the present study, a monoclonal antibody against Copper(II)–ethylenediaminetetraacetic acid was used to develop two sensitive ELISAs for Cu(II) analysis. Cobalt(II)–EDTA–BSA was the coating antigen in a heterologous indirect competitive ELISA (hicELISA), whereas Co(II)–EDTA–BSA–horseradish peroxidase (HRP) was the enzyme tracer in a heterologous direct competitive ELISA (hdcELISA). Both ELISAs were validated for detecting the content of Cu(II) in environmental waters. The ELISA data agreed well with those from graphite furnace atomic absorption spectroscopy. The methods of developing the Cu(II) hicELISA and hdcELISA are potentially applicable for developing ELISAs for other metals. The chelator–protein complexes such as EDTA–BSA and EDTA–BSA–HRP can form a suite of metal complexes having the consistent hapten density, location and orientation on the conjugates except the difference of the metal core, which can be used as ideal reagents to investigate the relationship between assay sensitivity and antibody affinities for the haptens and the analytes. The strategy of conjugating a haptenated protein directly with HRP can reduce the loss of HRP activity during the conjugation reaction and thus can be applicable for the development of ELISAs for small molecules.