白血病细胞酶联免疫酶催化银沉积于插指电极阵列电化学免疫分析新方法

建立了一种检测白血病细胞表面抗原的细胞酶联免疫电化学分析新方法. 该方法兼有细胞酶联免疫分析抗原、抗体结合的特异性和插指电极阵列酶催化银沉积电化学分析的灵敏性. 在聚苯乙烯微孔板中包被白血病细胞, 先后加入鼠抗人抗体及碱性磷酸酶(ALP)标记的马抗鼠抗体, ALP催化抗坏血酸磷酸酯(AAP)水解成抗坏血酸(AA), AA使银离子还原成银单质并沉积到插指电极阵列表面, 导致插指电极阵列上相邻两个梳齿导通. 通过对电导率的测定, 可实现对细胞表面抗原的高灵敏分析. 此分析方法灵敏度高(可检测出50个左右的HL-60细胞)、特异性好, 且可用于大量样品的分析, 为白血病等肿瘤疾病的早期诊断和免疫分型提供了新技术. 此外, 该方法也可用于细胞表面分子基因工程抗体活性的检测.     

免疫化学方法测定动物组织中呋喃唑酮残留标示物

以自制特异性抗体为核心试剂,建立了以苯甲醛为衍生试剂的呋喃唑酮残留标示物间接竞争ELISA方法.通过方阵滴定法和间接竞争法确定ELISA方法最佳反应条件:抗原最佳包被浓度200 μg/L,抗体最佳稀释倍数1:2.5×105,抗体与药物最佳工作配比40 μL: 60 μL,最佳竞争时间1 h.检出限为0.1 μg/L,检测范围0.1～25.6 μg/L,线性关系良好.在空白组织中(猪肌肉、猪肝脏、鸡肌肉、鸡肝脏、鱼肉)添加0.4、1.0和5.0 μg/kg AOZ,回收率55.8%～96.6%,相对标准偏差均小于20%.测定20份不同组织的空白样品,方法的检出限为0.4～0.5 μg/kg.以100 mg/kg呋喃唑酮饲喂动物,其组织样品同时经HPLC方法和本方法测定,数据表明本方法具有实际检出能力,测定结果与仪器方法的测定结果相近.通过与德国同类试剂盒比较实验表明,本方法的灵敏度、精密度、准确度接近于进口试剂盒水平,可用于动物可食性组织肝脏和肌肉中AOZ筛选.     

Determination of oxytocin in milk of cows administered oxytocin

To address people's concerns of exogenous oxytocin (OT) administration to lactating bovines, a study was undertaken to (a) establish an enzyme immunoassay (EIA) for OT determination in milk, (b) quantify OT in milk of cows administered OT, and (c) study influence of pasteurization on OT stability in milk. A sensitive EIA validated according to the criteria of European Union—Decision 2002/657/EC was developed for OT in skim milk in an analytical range of 10-250 pg mL⁻¹ with a decision limit (CCα) of 30 pg mL⁻¹ and detection capability (CCβ) of 41.5 pg mL⁻¹. Milk samples collected from cows (n = 38) administered either 25 or 50 IU OT prior to milking were investigated for the presence of OT. There was no significant difference among both groups with the mean concentrations of OT being 15.8 and 14.9 pg mL⁻¹ for cows subjected to 25 and 50 IU OT administration, respectively. The OT levels in skim milk of control cows (n = 30; untreated) were basal (around 10 pg mL⁻¹). All the analyzed milk samples were below the CCα value of 30 pg mL⁻¹. Pasteurization of OT spiked milk samples at different temperature and sample holding conditions reduced the immunological activity of OT to 43% at 110 °C. However, no further decline occurred in the immunological activity with increased pasteurization temperature and time. It was concluded that the milk OT concentrations after OT administrations were minimal and below the assay decision limit. However, OT was quite stable to pasteurization in OT spiked milk.     

Chemiluminometric enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor based on cross-flow chromatography

A chemiluminometric biosensor system for point-of-care testing has been developed using an immuno-chromatographic assay combined with an enzyme (e.g., horseradish peroxidase) tracer that produces a light signal measurable on a simple detector. Cross-flow chromatography, a method previously investigated by our laboratory, was utilized in order to accomplish sequential antigen-antibody binding and signal generation. This enzyme-linked immunosorbent assay (ELISA) was effectively carried out on a plastic chip that was redesigned to simplify the fabrication process. To enhance the sensitivity, biotin-streptavidin capture technology was employed in preparing an immuno-strip that was then incorporated onto the chip in order to generate the ELISA-on-a-chip (EOC) biosensor. Samples containing cardiac troponin I (cTnI) were analyzed using the EOC. A chemiluminescent signal proportional to the analyte concentration was produced by adding a luminogenic substrate to the tracer enzyme complexed with the analyte on the chip. The luminescent signal was detected in a dark chamber mounted with a cooled charge-coupled device and the signal was converted to optical density for quantification. This EOC biosensor system was capable of detecting cTnI present in serum at concentrations as low as 0.027 ng mL⁻¹, 30 times lower than those measured using the conventional rapid test kit with colloidal gold as the tracer. In addition, the final data was acquired within 30 s after the addition of the enzyme substrate, which was faster than the detection time required when using a colorimetric substrate with the same tracer enzyme.     

Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay

We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y = 456.35x + 80,382 (R = 0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.     

Organic-inorganic matrix for electrochemical immunoassay: Detection of human IgG based on ZnO/chitosan composite

A new strategy to construct amperometric immunosensor for human IgG assay based on ZnO/chitosan composite as sensing platform has been described. This material, which combined the advantages of inorganic species, ZnO and organic polymer, chitosan, can maintain biological activity well. A sequential sandwich immunoassay format was performed on the ZnO/chitosan composite supported by glass carbon electrode (GCE) using goat-anti-human IgG antibody (IgG Ab) and human IgG as a model system. Amperometry was used to determine the amount of horse-radish peroxidase (HRP) fixed on the sensor surface, which was related to the content of the desired human IgG. Assay conditions that were optimized included the amount of labeled antibody, the incubation time and temperature, the pH of the substrate solution, etc. Using hydroquinone as a mediator, amperometric detection at −150 mV (versus SCE) resulted in a detection range 2.5-500 ng mL⁻¹, with a detection limit of 1.2 ng mL⁻¹. The simple manipulations of the construction of ZnO/chitosan composite, as well as low-cost and broad linear range, are the main features of the proposed immunosensing method.     

Investigation of voltammetric enzyme-linked immunoassay based on new system of ODA-H_2O_2-HRP

A voltammetric enzyme-linked immunoassay based on a new system of ODA-H2O2-HRP has first been developed and used in the detection of HRP and labelled HRP. By this method, the enzyme-catalyzing reaction of H2O2 oxidizing odianisidine (ODA) couples the electrode-reduction reaction of the oxidizing product of odianisidine, which produces a sensitive polarographic wave at potential of -0.56V (SCE) in Britton-Robinson buffer solution. In using this polarographic wave, a detection limit to HRP is 3.7×10-12g/mL and a linear range 1.0×10-11-2.0×10-9g/mL. And the mechanisms of the coupling reaction and the process of electro-reduction in the ODA-H2O2-HRP voltammetric enzyme-linked immunoassay system have also been carefully studied.     

增强型酶催化发光免疫分析法高灵敏测定血清中心肌肌钙蛋白Ⅰ

基于辣根过氧化物酶（HRP）催化 H₂O₂-Luminol系统,对一种新型酚类衍生物 4-（1,2,4-三氮唑-1-基）苯酚对化学发光的增强作用进行了研究。用HRP标记急性心肌梗死标志物心肌肌钙蛋白Ⅰ（cTnⅠ）单克隆抗体,通过cTnⅠ的双抗体夹心免疫反应,建立了简单、灵敏和快速检测人血清中的cTnⅠ含量的增强型酶发光免疫分析方法。实验结果表明,cTnⅠ在1.2 ~ 24 ng/mL浓度范围内与增强型发光强度具有良好的线性关系（R=0.99）,变异系数（n= 8）为 4.7%。最后,使用该方法对人血清样品中的cTnⅠ含量进行测定,测试结果具有较好的稳定性和精度,能够满足临床检测要求。     

Electrochemical Immunosensor for α‐Fetoprotein Based on Gold Nanoparticles/Graphene‐Prussian Blue

The electrochemical immunosensor for α‐fetoprotein (AFP) was fabricated based on the platform of gold nanoparticles (GNP)/graphene (Gr)‐prussian blue (PB). By electrodeposition, GNP were modified on the surface of the prepared Gr‐PB. The anti‐AFP‐1,1′‐ferrocenedicarboxylic acid (FcDA) as label was directly immobilized on the platform of GNP/Gr‐PB. And after the immunoreactions, the formed complex inhibited the electron transfer and decreased the catalytic current of FcDA toward the reduction of H₂O₂. And in the range of 10–3200 pg·mL^?1, the decreased current is linear with the concentration of AFP, with a detection limit of 3 pg·mL^?1. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme linked immunosorbent assay (ELISA) method.     

普鲁士蓝增敏压电均相免疫分析法测定免疫球蛋白G

建立了普鲁士蓝增敏的均相压电免疫分析法,用于人尿液中免疫球蛋白G(IgG)的检测.本方法以蛋白质为种子,形成蛋白质-普鲁士蓝粒子,该粒子不断“滚雪球”增大,使得压电检测信号明显放大.在pH 4.0、盐浓度0.1 mol/L,5 mmol/L FeC13,2.5 mmol/L K4Fe(CN)6(K4Fe(CN)6与蛋白浓度比≥5000),FeCl3加样速度为0.5 μL/s的优化条件下,IgG的检测范围为0～625 nmol/L;检出限为2.4 nmol/L.α1-微球蛋白,β2-微球蛋白、尿微量白蛋白均无明显干扰.本方法用于临床样本的IgG测定,并与免疫比浊法进行对比,两者结果一致,表明本方法可行.