Immunochromatographic flow-injection method for detection of human serum immunoglobulin G antibodies to Helicobacter pylori

This study reports on the development of an immunochromatographic flow-injection (FI) method for the quantitation of human serum IgG antibodies to Helicobacter pylori. Patients’ sera were injected into the carrier stream of a FI manifold incorporating an H. pylori immuno-adsorbent reactor. The immuno-adsorbent was prepared by covalently linking H. pylori antigens to periodate oxidized Sepharose CL-4B. Any H. pylori antibody present in the serum is bound to the immuno-sorbent. The bound antibody was quantified by injecting into the carrier stream anti-human IgG peroxidase conjugate followed by TMB/H₂O₂ as the enzyme substrate. The FI system was optimized with respect to antigen loading (1.0 mg/ml gel), bioreactor length (3.0 cm), enzyme conjugate (0.2 mg/ml) and sample loop size (250 μl). One hundred clinical samples were analyzed for their H. pylori antibody concentrations and the results evaluated with respect to their receiver-operator characteristics (ROC). When a cut-off absorbance of between 0.7 and 0.75 was used for positive and negative samples a specificity (>95%), a sensitivity (>90%)and an overall accuracy (>94%) were obtained.     

胶体金免疫层析法快速检测腹泻性贝毒软海绵酸的研究

腹泻性贝毒是一类分布较广的赤潮毒素,严重威胁到人类的健康和安全。本文用胶体金标记利用细胞融合技术制备的抗软海绵酸单克隆抗体,使用卵清蛋白合成高偶联比的包被抗原,以硝酸纤维素膜为载体,利用免疫层析技术原理,建立了快速检测软海绵酸的免疫层析试纸条方法。方法检出限12 ng/mL(0.96纳克/条)。     

CdTe/ZnSe核壳量子点免疫层析试纸条检测克伦特罗的研究

采用巯基丁二酸作为表面修饰剂,水相法合成水溶性的CdTe/ZnSe核壳量子点,然后在N-羟基琥珀酰亚胺(NHS)的作用下,将CdTe/ZnSe核壳量子点与抗克伦特罗多克隆抗体(Anti-CLE pAb)连接。通过凝胶电泳和斑点杂交实验,验证CdTe/ZnSe核壳量子点与Anti-CLE pAb连接成功,并且CdTe/ZnSe-Anti-CLE pAb偶联物能识别克伦特罗-BSA抗原(CLE-BSA)。光谱分析表明,量子点与抗体连接后荧光增强,荧光峰位从628nm红移至635nm。将合成的CdTe/ZnSe-Anti-CLE pAb偶联物作为指示克伦特罗(CLE)分子的荧光标记物,制备出一种用于检测CLE的免疫层析试纸条,其最低检测量可达1μg/L。与ELISA法的对比实验表明,此试纸条能应用于CLE残留的快速检测。     

Electrophoresis-Assisted Multilayer Assembly of Nanoparticles for Sensitive Lateral Flow Immunoassay**

Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive point-of-need method. A major limitation of LFIA is a high limit of detection (LOD), which impacts its diagnostic sensitivity. To overcome this limitation, we introduce a signal-enhancement procedure that is performed after completing LFIA and involves controllably moving biotin- and streptavidin-functionalized gold nanoparticles by electrophoresis. The nanoparticles link to immunocomplexes forming multilayer aggregates on the test strip, thus, enhancing the signal. Here, we demonstrate lowering the LOD of hepatitis B surface antigen from approximately 8 to 0.12 ng mL⁻¹, making it clinically acceptable. Testing 118 clinical samples for hepatitis B showed that signal enhancement increased the diagnostic sensitivity of LFIA from 73 % to 98 % while not affecting its 95 % specificity. Electrophoresis-driven enhancement of LFIA is universal (antigen-independent), takes two minutes, and can be performed by an untrained person.     

临床诊断试剂及其诊断方法研究进展

本文结合作者工作,综述了现代临床化学所用诊断试剂,以及诊断方法学研究的某些进展与发展趋向。     

Rapid pretreatment-free immunochromatographic assay of chloramphenicol in milk

A pretreatment-free immunochromatographic assay for detection of chloramphenicol (CAP) in milk was developed. The assay is based on competition between CAP molecules in the sample and immobilized CAP-protein conjugate for binding to monoclonal anti-CAP antibodies conjugated with colloidal gold particles (average diameter 30 nm). The assay is carried out in the course of sample flowing along test strip with immobilized reactants, and its results can be detected by the naked eye or by a photometric device. Effect of the concentration of immunoreactants on assay characteristics was studied. The assay protocol with maximal sensitivity and reliability was optimized using measured values of brightness of lines. Detection limit for CAP is 10 ng mL⁻¹. Assay duration is 10 min, and it can be carried out at room temperature without any additional devices and reactants. The developed test strip has been applied to CAP detection in dairy products.     

Immune fluorescence test strips based on quantum dots for rapid and quantitative detection of carcino-embryonic antigen

A QDs-based immune fl uorescence test strips was built up for carcino-embryonic antigen detection to realize cancers POCT diagnostic, with a sensitivity of 0.72 ng/mL in 25 min.     

A competitive immunochromatographic assay for testosterone based on electrochemical detection

An immunochromatographic assay using nitrocellulose membrane was combined with electrochemical detection using an electrode chip in order to quantitatively detect testosterone as a model analyte. The electrode chip consisted of a gold working electrode, a counter electrode and a pseudo-reference electrode, all fabricated on the bottom of a 3.2 mm × 3.2 mm well. Competitive immunoreactions on the membrane were initiated by flowing a solution containing testosterone and horseradish peroxidase (HRP)-labeled testosterone (a competitor) over the membrane. Prepared membrane was placed in a solution containing ferrocenemethanol (FcOH) and H₂O₂ in the well of the electrode chip, and the enzyme reaction was detected by amperometry. Labeled HRP captured on the membrane catalyzed the oxidation of FcOH to the oxidized form FcOH⁺, which was reduced electrochemically by the electrode chip. The electrochemical response of the reduction current decreased with increasing concentration of testosterone over the range 1–625 ng/ml.     

Development of a competitive lateral flow immunoassay for progesterone: influence of coating conjugates and buffer components

Several aspects of the development of competitive lateral flow immunoassays (LFIAs) are described. The quantitation of progesterone is taken as an example. The LFIA format consisted of a nitrocellulose membrane spotted with various progesterone conjugates as the test line. A mixture of primary antibody and secondary antibody adsorbed to colloidal carbon was used for signal generation. A digital scanner and dedicated software were used to quantitate the response. A reappraisal of the checkerboard titration, often used in the optimisation of immunoassays, is discussed. Surprisingly, the highest sensitivity of the LFIA format (IC₅₀ of 0.6 μg L⁻¹ progesterone in buffer) was achieved by using a high coating concentration of the analyte–protein conjugate and a high dilution of the antibody solution. Immediate addition of all reagents in LFIA was superior to premixing the components and allowing prereaction. Of several blocking agents tested bovine serum albumin was superior in performance, whereas the combination of ovalbumin and progesterone substantially influenced test results.     

量子点标记免疫层析技术检测血液中的降钙素原

为了快速定量检测血液中降钙素原( Procalcitonin,PCT)含量,首先制备CdSe/ZnS量子点,再将量子点标记抗抗钙素单抗,标记单抗后量子点荧光发射峰从620 nm红移至625 nm,峰形保持良好。以制作的量子点标记免疫层析试纸条及其相应荧光测定系统。因减少单抗用量可降低试纸条成本,本研究采用结合垫上喷涂适量量子点,标记单抗并仅有检测线的结构。通过检测信噪比,优化此试纸条的参数。本研究的量子点荧光标记试纸条及其配套测定系统可在20 min内快速检测PCT,定量检测线性范围0.2~100μg/L,灵敏度达到0.1μg/L。22份血液样本检测结果与目前商品设备检测结果的Pearson相关系数为0.9995,K-S检验的Sig为1.0,说明两种检测方法的一致性。快速定量检测PCT具有定量测量细菌性感染、临床指导抗生素合理应用的重要意义。