乙肝表面抗原压电免疫传感器阵列的研制

采用戊二醛交联法,将乙肝表面抗体固定于石英晶体表面,研制成HBsAg压电免疫传感器阵列.根据频率改变值考察了传感器的固定化过程和传感器对乙肝表面抗原的响应特性.该传感器对乙肝表面抗原进行定量分析,线性范围为1～25 μg/ml,线性相关系数为0.9977.取临床血清进行检测,将结果与临床常用的ELISA法比较,探索了该传感器应用于临床检测的可行性.     

电化学免疫传感器的研究进展

近年来，随着电化学免疫传感器在各方面的广泛应用，关于提高它的性能、优化其检测方法的研究也越来越多。根据检测信号的不同，将其分为电位型、电流型、电导型、电容型免疫传感器。并逐一介绍了它们有关国内外研究进展。     

DNA电化学传感器中电极修饰条件的探讨

目的 探讨ssDNA单层膜的制备的最佳条件.方法利用自组装单分子膜技术,将人工合成并修饰的含22个碱基的特异性单链DNA序列固定到金电极上制备成DNA修饰的金电极,通过控制DNA溶液的浓度、作用时间,以及改变修饰基团探讨最佳的修饰条件,并利用循环伏安法初步研究了该修饰电极的电化学行为.结果 通过对不同标记探针、不同探针浓度及不同标记时间的表征,结果显示巯基化ssDNA和二硫键修饰的ssDNA探针浓度为80 μmol/L,自组装固定时间为12 h时对DNA探针的固定效率最佳.结论 巯基化ssDNA和二硫键修饰的ssDNA通过自组装方法可在金电极表面形成稳定、有序的自组装单分子膜(self-assembled monolayer,SAM),可应用于SAM为基底的DNA传感器的设计与应用.     

用于禽流感同步检测的压电免疫传感器阵列的研制

目的 制作用于禽流感检测的四通道压电蛋白芯片.方法 采用聚乙烯亚胺(PEI)粘附、戊二醛(Glu)交联法,将禽流感H5、H7、H9、HI抗原分别固定于石英晶体的银电极表面.结果 得到了抗原的最佳固定条件和压电蛋白芯片对阳性、阴性标准血清的响应情况.结论 该传感器对阳性标准血清的响应值与阴性标准血清的噪声值之比大于2,可用于定性定量检测.     

电化学发光定量检测乙肝血清标志物的观察

近年来,非放射免疫分析技术迅速应用于临床。特别是电化学发光技术（ECLIA）以其自动、快速,特异性强、灵敏度高等优点展示出良好的应用前景。ECLIA技术定量检测血清HBVM,灵敏度为1pmol,达到和超过了放射免疫分析（Radioimmunoassay,RIA）水平,用于定量检测乙肝血清标志物,改变了目前国内常用酶联免疫吸附试验（ELISA）,只能定性检测的现状。     

用于禽流感H5、H9亚型同步检测的压电免疫传感器阵列的研制

目的：研制禽流感H5、H9亚型同步检测的压电免疫蛋白芯片。方法：采用聚乙烯亚胺（PEI）粘附、戊二醛（Glu）交联的方法，将禽流感H5、H9抗原固定于石英晶体的金电极表面。探讨了抗原的最佳固定条件，研究了压电蛋白芯片对阳性、阴性标准血清的响应情况。结果：该芯片用于检测浓度范围分别为0．123mg／mL-0．615mg／mL、0．123mg／mL-1．23mg／mL的禽流感H5、H9标准血清时，呈较好的线性关系，相关系数分别为0．9585、0．9854。结论：阳性标准血清的响应值与阴性标准血清的噪声值之比大于2，可用于定性定量检测。     

用于输血四项指标检测的压电免疫传感器阵列的研制

采用聚乙烯亚胺粘附，戊二醛交联，固定相应的敏感材料研制了四通道压电免疫传感器阵列。该阵列可快速同步检测乙肝表面抗原（HBsAg）、丙肝病毒抗体（HCV）、梅毒螺旋体抗体（TP）、爱滋病毒（HIVI＋2）抗体，考察了该传感器阵列的灵敏度、特异性、稳定性，并应用于临床样品的检测，同时与EUSA法进行比较。结果表明阳性血清的响应值与阴性血清的噪声值之比均大于4，可用于输血四项指标的同步检测,其检测灵敏度略高于ELISA法。     

蝰蛇毒压电免疫传感器固定方法的研究

目的：为研制蝰蛇毒压电免疫传感器，研究抗蛇毒抗体固定于石英晶体银电极表面的固定技术。方法：采用马抗蝰蛇毒血清抗体和抗蝰蛇毒鸡卵黄抗体作为生物敏感材料，对比研究了胱胺自组装-PSS反相吸附法和PEI粘附-戊二醛交联法：比较了采用两种固定方法所制的压电免疫传感器的性能。结果：鸡卵黄抗体采用PEI粘附-戊二醛交联法效果较好，其制备的IgY压电免疫传感器检测蝰蛇毒灵敏度为0．5ug／mL；而马血清抗体用胱胺自组装-PSS反相吸附法较好，其制备的IgG’免疫传感器检测蝰蛇毒灵敏度为10ug／mL。结论：以PEI粘附-戊二醛交联法固定抗蝰蛇毒鸡卵黄抗体所制备的蝰蛇毒压电免疫传感器的性能稳定，特异性好，可实现蛇毒的快速检测。     

电沉积纳米金修饰的16通道电流型PSA免疫传感器的制备

近年来有关电化学免疫传感器的研究报道虽然很多,但普遍存在如预处理操作繁琐、灵敏度低、特异性差和成本昂贵等不足。本研究通过电沉积纳米金修饰16通道丝网印刷电极,以提高免疫传感器的性能。采用恒电位沉积法将氯金酸(HAu Cl4)直接还原成纳米金,并修饰于16通道丝网印刷碳电极(16-SPCE)表面;以浓度为0.05mg/m L的HAu Cl4溶液和150 s电沉积时间为电沉积纳米金修饰16-SPCE的最优条件。利用静电吸附原理将前列腺抗原(PSA)抗体固定在电极表面,结合双抗夹心法制备免疫电极。通过循环伏安法和稳态时间电流曲线法表征免疫电极的性能。对30份前列腺癌患者和3份正常人血清进行PSA检测,与标准ELISA方法对照。结果表明:获得的免疫电极线性检测范围为0.2~100 ng/m L,其线性回归方程为Y=134.558X+72.705,线性相关系数为1,检测限为0.14 ng/m L。采用免疫传感器检测到的前列腺癌患者血清PSA水平与标准ELISA方法检测结果具有良好的线性相关性(R=0.944,P0.001),正常人的检测值符合正常人血清中PSA的含量。所研制的免疫传感器具有灵敏度高、特异性强、成本低和反应时间短等特点,具有应用前景。     

血清中丙肝病毒的压电免疫传感器检测

构建一种新型的丙肝压电免疫传感器.采用AT切型、基频为10MHz石英晶体作为换能器,在清洗后的晶体金电极表面固定蛋白A之后固定丙肝的单克隆抗体,最后检测血清样品中的丙肝病毒.研究了不同固定方法的固定效果,并采用最佳方法做了阴阳对照实验、清洗实验、封阻实验、温度实验等,得到了初步的结果.此传感器具有特异性好、不需标记、操作简单、能实时在线检测和重复使用等优点,在临床实验诊断应用中具有潜在前途.     

乙型重型肝炎的内科治疗

我国有大量HBV感染患者,且有相当一部分患者病情进展成肝衰竭,又称乙型重型肝炎.HBV相关肝衰竭预后差,易引起严重并发症,比如败血症、肝性脑病、肝肾综合征等.肝移植被认为是目前治疗乙肝相关肝衰竭的有效方法,但其昂贵的花费以及器官来源的缺乏、较高的移植后排斥反应率使得该项治疗的应用受到限制.因此有效的内科治疗手段正受到关注.在此将目前治疗乙型重型肝炎的内科治疗方法综述如下.     

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance

The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 10² copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 10⁴ copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms.Antiviral therapy in patients with chronic hepatitis B is associated with improved outcomes. Several oral agents have been approved by the U.S. Food and Drug Administration for the therapy of chronic hepatitis B, including lamivudine (LMV), adefovir (ADV), entecavir (ETV), telbivudine (LdT), and tenofovir (TFV). However, hepatitis B virus (HBV) mutations associated with antiviral resistance are a major problem in the treatment of chronic hepatitis B. Antiviral resistance and poor adherence are the most important factors in the treatment failure of nucleoside and nucleotide analogue therapies for hepatitis B (8, 12, 16, 20, 25). The sensitive and early detection of emerging resistance is important for monitoring the viral dynamics associated with treatment and to improve therapeutic decision making. Consequently, the method for the detection of antiviral-resistant HBV must be rapid, sensitive, and accurate for reliable diagnosis (13, 21).Various methods have been proposed for the detection of antiviral-resistant HBV mutants. Direct sequencing can identify known and potential new resistance mutations, but it is a time-consuming and laborious method. Furthermore, sequencing can detect minor populations comprising more than 20% of the total virus population (8, 13, 26). In particular, the sequencing method is able to correctly determine the nucleotide mixture at position 1 (R = A or G) and position 3 (K = T or G, R = A or G, and S = C or G) of codon 204 (11, 23). Thus, the mutant mixture (M204V/M204I) of codon 204 is difficult to correctly determine as the mixture of mutants and the wild type (M204V/M204I/M204) by direct sequencing. Other methods have been used to overcome some of the limitations of sequencing, including PCR restriction fragment length polymorphism (RFLP) analysis and reverse hybridization (line probe assay and oligonucleotide array). However, these methods have disadvantages in terms of specificity, sensitivity, operational complexity, and interpretation of results (1, 8, 13, 17). Most hybridization methods usually make use of a DNA probe, which can be a synthetic oligonucleotide or longer enzymatically generated DNA. Although DNA probes work well, they have problems with selectivity, sensitivity, and stability under various conditions (24). Recently, however, several nucleic acid analogs, such as peptide nucleic acids (PNAs), have been developed to overcome the limitations of natural nucleic acids for specificity, sensitivity, hybridization kinetics, thermodynamic properties, and stability (2, 15).PNAs are a nucleic acid analog in which the negatively charged ribose-phosphate backbone is replaced by uncharged N-(2-aminoethyl)-glycine units. The uncharged nature of PNA is responsible for the better thermal stability of the PNA:DNA duplex compared to that of the DNA:DNA duplex. The stability of the PNA:DNA duplex is greatly affected by the presence of a single-base mismatch. A PNA:DNA mismatch is more destabilizing than a mismatch in a DNA:DNA duplex, and they therefore are highly sequence selective. The stronger binding properties and biological stability of PNA give more specific, more sensitive, and more accurate results in the detection of target sequences. These properties of PNA are widely used as a molecular tool in molecular biology and biotechnology. An increasing number of applications for PNA technology have been described, detecting specific gene sequences in advanced diagnostic methods such as PCR clamping, real-time PCR, FISH (fluorescence in situ hybridization), and microarrays (3, 4, 10, 24). The PNA array (for research use only, not for use in diagnostic procedures) (Panagene Inc., Daejeon, South Korea) is an in vitro microarray used to detect the presence of different genetic variants of HBV in human blood samples (PANArray DR HBV).The primary aim of this study was to develop a PNA array for the detection of HBV mutations associated with antiviral resistance. The secondary aim was to evaluate the accuracy of the PANArray DR HBV by comparing the results of this assay to those of direct sequencing for concordance.     

Hepatitis B vaccination

As hepatitis B virus does not replicate in tissue culture systems, it was impossible to prepare a vaccine in the conventional way. However, the surface-antigen of the virus is present in abundance in the blood of certain virus carriers. This phenomenon has been used to develop plasma-derived hepatitis B vaccines and these vaccines are now available. In low incidence countries vaccination should be restricted to high-risk groups after initial screening for antibodies to hepatitis B virus. In areas where hepatitis B virus infection is highly endemic immunisation of the whole population, immediately after birth, is probably the most effective strategy. The high cost of the currently available plasma-derived vaccine is a constraint on the implementation of public health programmes for the prevention of hepatitis B. A second generation of vaccines — made by DNA recombinant technology — is expected to be available in the near future and could be the answer to these problems.     

Hepatitis B Virus X Protein: The X Factor in Chronic Hepatitis B Virus Disease Progression

Introduction: Hepatitis B virus (HBV) is the most common aetiological factor causing hepatocellular carcinoma (HCC). HBx gene plays an enigmatic role in HBV-related HCC. In this study we have analysed amino acid substitutions in HBx from HBV-infected individuals of different clinical stages. Materials and Methods: HBV-infected individuals (n = 93) were recruited in the study. DNA was extracted from plasma, amplified, and DNA sequencing was performed using specific primers targeting HBx gene (540 bp). Results: Among the study participants, 57% had chronic HBV infection, 30% had chronic liver disease (CLD) and 13% had HBV related HCC. Genotypes such as D1, D2, D3, A1, C2 and B2 were identified of which genotype D2 was predominant (78%). HBxC-terminal deletion was observed in four hepatitis B e antigen (HBeAg) negative participants with CLD. The frequency of aminoacid substitution in proapoptotic domain was higher in HBeAg negative participants including I127V (34%), K130M (34%), V131I (40%). The frequency of double mutation (K130M+V131I) and triple mutation (I127V+K130M+V131I) were found to be higher (32% and 36%) in HBeAg negative participants. Also, we identified L5M substitution (4.3%) in HBeAg positive participants with advanced liver disease. Conclusion: In HBx gene, aminoacid substitutions at positions 127, 130, 131 are associated with poor expression of HBeAg. We suggest screening for HBx aminoacid substitutions especially in patients with HBeAg negative chronic HBV infection to predict the clinical outcome and enable early treatment to prevent disease progression.