预制备抗体—DNA偶联物的免疫聚合酶链反应检测HBsAg

目的：探讨一种实用敏感的乙型肝炎表面抗原（HBsAg)检测方法。方法：利用亲和素作为桥梁连接生物素化DNA和抗体，形成抗体－亲和素－DNA偶联物，在抗原抗体特异性反应的基础上，聚合酶链反应（PCR）扩增抗体所连的DNA片段，通过检测DNA来判断相应抗原存在与否。结果：免疫PCR检测敏感度为常规酶联免疫试剂盒的300倍，结论：预制备抗体＿DNA偶联物的免疫PCR是一种检测HBsAg的敏感方法。     

免疫聚合酶链反应检测方法初探

为了提高抗原抗体检测的灵敏度，利用生物素亲和素系统对免疫聚合酶链反应（免疫－ＰＣＲ）检测方法的建立做了初步探索，结果表明，免疫－ＰＣＲ可检测到少至１００ｆｇ的抗原，比酶联免疫吸附试验平行对照高１０００倍，该法仅限于实验模型的建立，其实际应用价值还有待于深入研究。     

免疫聚合酶链反应检测方法初探

为了提高抗原抗体检测的灵敏度，利用生物素亲和素系统对免疫聚合酶链反应（免疫－ＰＣＲ）检测方法的建立做了初步探索。结果表明，免疫－ＰＣＲ可检测到少至１００ｆｇ的抗原，比酶联免疫吸附试验平行对照高１０００倍。该法仅限于实验模型的建立，其实际应用价值还有侍于深入研究。     

酶免疫法检测丙型肝炎病毒RNA的聚合酶链反应产物

建立一灵敏，快速的检测丙型肝炎病毒逆转录－套式聚合酶链反应产物的酶免疫法。方法，对第２次ＰＣＲ所用的引物进行双标记，使扩增产物同时被修饰有生物一不和荧光素成分，再经过固相的链亲合素进行捕获，辣根过氧化酶记的抗－ｆｌｕｏｒｅｓ－ｃｅｉｎ反应后显色测定     

聚合酶链反应检测霍乱弧菌

为快速、准确地检测霍乱弧菌，控制霍乱流行，研究建立了一种聚合酶链反应（ＰＣＲ）检测方法。根据霍乱弧菌肠毒素ＣＴ基因序列，自行设计一对特异引物，扩增片断为２９６ｂｐ，同时采用一种高效率、快速、简便的方法提取并纯化ＤＮＡ，该法能成功地检测各种样品中的产ＣＴ肠毒素Ｏ１群霍乱弧菌。应用ＰＣＲ技术和常规分离培养方法对９０９份腹泻患者粪便标本和１８份环境水样标本进行平行检测。结果表明，两种方法的检测结果一致，粪便标本阳性率为４．２％（３８／９０９）；水样标本为２２．２％（４／１８），其中１份粪便标本和１份水样标本，是经过第２次增菌培养后，检出用性，而ＰＣＲ方法未见假阳性及假阴性结果。表明ＰＣＲ方法准确、快速、灵敏，并可在２、３小时内检测出含３个菌细胞的标本。因此，该方法可广泛应用于检测各种临床、环境标本中的产ＣＴ肠毒素Ｏ１群霍乱弧菌。     

聚合酶链反应检测乙型肝炎血清HBV—DNA的临床意义

我们对112例乙型病毒性肝炎用聚合酶链反应(PCR)检测血清HBV-DNA,并与血清乙肝病毒标志(HBVM)检测对照,以观察其临床意义,现将结果报告如下。 1 对象与方法 1.1 对象 112例乙型病毒性肝炎为我院传染科1995年6月～1996年6月的住院患者,男86例,女26例;年龄8～70岁,其中20～30岁65例,占58％。依据1990年(上海)全国病毒性肝炎学术会议修订的诊断标准分     

斑点金免疫法检测HBsAg

斑点金免疫法检测ＨＢｓＡｇ吴海裘，潘明宽（宁海县人民医院，浙江宁海３１５６００）关键词斑点金免疫法，乙型肝炎表面抗原快速斑点免疫渗滤试验（ＤＩＧＦＡ）是近年来发展起来的一种新技术［１］），１９８９年有人在此技术中用胶体金代替酶制备标记物［２］。国内已...     

实时荧光定量聚合酶链反应检测乙型肝炎病毒感染的临床意义

我们采用实时荧光定量聚合酶链反应(FQ-PCR)方法对HBV感染760例血清中HBVDNA进行定量检测，并与常规PCR检测结果进行比较分析，取得了较好的效果，现总结如下。     

逆转录聚合酶链反应检测风疹病毒

为了早期诊断风疹感染，应用逆转录。聚合酶链反应（ＲＴ－ＰＣＲ）方法对８０例临床怀疑风疹感染患者的咽拭物检测风疹病毒。ＰＣＲ阳性者４３例，占５３％；同时用酶联免疫吸附法对每份血清作了ＩｇＭ检测，阳性者１２例，占１５％。统计学处理（Ｐ＜０．０１），表明两种检测方法差异有显著性。扩增产物的特异性通过地高辛标记探针的点杂交分析得到证实。该ＲＴ－ＰＣＲ方法具有快速、早期、灵敏度高和特异性强的特点，有希望发展成为临床诊断胎儿风疹感染的一种重要手段，并为产前咨询提供可靠的依据。     

Radioimmunoassay for detecting hepatitis B core antigen in serum from patients with chronic hepatitis B infection

The core antigen component of hepatitis B virus (HBV), HBcAg, may be used as a marker of viral replication. We describe a radioimmunoassay for HBcAg in serum, and compare results with those by other similar methods. Samples are centrifuged over a sucrose gradient containing 2-mercaptoethanol, and the resulting pellets are resuspended in a nonionic detergent solution. After a bead coated with anti-HBcAg is incubated with this suspension, 125I-labeled anti-HBcAg is added, and is also bound by the antigen. The specificity of the method was verified by blocking with purified IgG antibodies to HBcAg. When we tested by this method for HBcAg in sera from 60 patients with chronic HBV infection, all those with circulating HBV-DNA polymerase tested positive for HBcAg. All sera from HBV-negative controls showed no detectable HBcAg. The correlation between the presence of HBcAg and HBV-DNA polymerase was significant (r = 0.715, p less than 0.001). Samples can be tested more quickly and easily with this method, and its sensitivity compares well with that of other similar methods.     

胶体金免疫层析法检测乙型肝炎病毒表面抗原

目的建立一种简易快速、自测式胶体金免疫层析法（ＧＩＣＡ）用于检测血清中乙型肝炎病毒表面抗原（ＨＢｓＡｇ）。方法采用柠檬酸三钠还原法制备胶体金颗粒、标记乙型肝炎病毒表面抗体（抗ＨＢｓ），制成免疫层析检测试条。血清中ＨＢｓＡｇ与测试条上金标记抗体结合后沿着硝酸纤维素膜移动，与膜上的固相抗体结合形成肉眼可见的红色线条。结果ＧＩＣＡ试条只与ＨＢｓＡｇ有特异性反应，与乙型肝炎病毒核心抗原、ｅ抗原等无交叉反应。检测中国药品生物制品检定所ＨＢｓＡｇ标准品，灵敏度可达１ｎｇ／ｍｌ。用ＧＩＣＡ与酶免疫法比较检测了３９５份不同血清标本中ＨＢｓＡｇ，两法符合率为９９．０％。结论ＧＩＣＡ检测血清中的ＨＢｓＡｇ特异性强、灵敏度高、简便快速，无需特殊仪器设备，有广泛应用价值。     

Solid-phase enzyme immunoassay for hepatitis B surface antigen.

A solid-phase enzyme immunoassay is described for measuring hepatitis B surface antigen in human serum or plasma. Immunologically purified antibody labeled with horseradish peroxidase was used as the indicator. In the assay system, antibody-coated controlled-pore glass is used as a solid support and there are three sequential incubations, totaling 2 h, at room temperature. Results for serially diluted positive and reference sera compare favorably to radioimmunoassay in sensitivity and specificity.     

A topological model for hepatitis B surface antigen.

A model of hepatitis B surface antigen has been derived, based on extensive sequence analysis and biochemical data. The surface antigen sequences of the human, woodchuck, ground squirrel and duck hepadnaviruses were examined using hydrophobicity, hydrophobic moments, flexibility and secondary structure prediction. The helix phase diagram, which is a modified version of Eisenberg's hydrophobic moment plots and which specifically addresses the problem of transmembrane helices, was used to examine the predicted helices. In this model four transmembrane helices are predicted. The N and C termini and the second hydrophilic region, which bears the major B-cell antigenic determinants, are external. It is suggested that the transmembrane helices may pack to form a channel through the membrane and may also be involved in the mechanisms of cell entry. A significant difference between the duck hepadnavirus and the mammalian HBsAg sequences was found, hence care must be taken when extrapolating data between the duck and the human surface antigen.     

A novel hepatitis B virus surface antigen immunoassay as sensitive as hepatitis B virus nucleic acid testing in detecting early infection

BACKGROUND: The aim was to considerably enhance the sensitivity of hepatitis B virus (HBV) surface antigen (HBsAg) detection and investigate whether the window period for HBV detection could be reduced.
STUDY DESIGN AND METHODS: A high-sensitivity chemiluminescent enzyme immunoassay (CLEIA) was developed for quantitative HBsAg detection by a combination of monoclonal antibodies, each one for a specific epitope of HBsAg, and by improving the conjugation technique. The sensitivity of the assay was compared with that of the existing chemiluminescent immunoassay (CLIA). Commercially available seroconversion panels and samples of HBV-infected chimpanzees were tested with the developed prototype to assess whether the window period for HBsAg detection could be reduced to that for DNA detection.
RESULTS: Compared to the existing CLIA, the CLEIA prototype detected HBsAg with approximately 230-fold higher sensitivity and showed a reduced window period. HBsAg detection by the CLEIA prototype and HBV DNA detection by polymerase chain reaction (PCR) occurred simultaneously. The mean time for the CLEIA prototype to first detect HBsAg was approximately 17.4 days less than that for the existing systems. Further, CLEIA prototype enabled HBsAg detection even in anti-HBs–positive seroconversion samples. In the inoculated chimpanzees the HBsAg and HBV DNA became detectable simultaneously and concentrations increased in parallel, whereas HBsAg remained detectable longer than HBV DNA in the declining phase of viremia.
CONCLUSION: The CLEIA prototype yielded results comparable with those of HBV DNA PCR. This novel high-sensitivity assay may be useful for early detection of HBV infection and monitoring patients with a history of infection.     

Chronic hepatitis B and hepatitis B surface antigen carriers in university students

A study was made of latent hepatic diseases in university students. 1.4% to 1.6% of the students were positive for hepatitis B surface antigen (HBsAg), and 10.3% for anti-HBs. Of 28 students with HBs-antigenemia, 2 had chronic persistent hepatitis, and 3 minimal hepatitis, 23 being healthy carriers. Hepatitis B e-antigen (HBeAg) was detected in 44% of the students with HBsAg, and anti-HBe in 13%. Anti-HBe was significantly more frequently found in female students with HBsAg than in male students. Though most of the students with HBsAg had high titer of antibody to hepatitis B core antigen (anti-HBc), there were a small number of cases showing low titer. HBsAg and anti-HBs was detected in the same serum specimens of 2 carrier students. Liver damage was also found in 3 students without HBs-antigenemia.