HBsAg diagnostic kits in the detection of hepatitis B virus mutation within "a" determinant

The performance of currently available hepatitis B surface antigen (HBsAg) commercial kits was analyzed by using a panel of 212 well-characterized plasma donors all over the country and a panel of nine recombinant HBsAg mutants containing single point or combinations of mutations between amino acid residues 124 and 147 of the "a" determinant. HBsAg commercial kits in this study were machine-based immunoassays with a one-step sandwich ELISA method using either an automatic closed system or manual system. The sensitivity of all machine-based assays evaluated with 105 HBsAg plasma panels was 100% (95% CL = 95.6-99.9%), whereas the specificity with 107 HBsAg negative plasma ranged from 99.07% to 100% (95% CL = 94.2-99.9%). The relative performance of these kits to detect the hepatitis B virus (HBV) mutant panel members of the "a" determinant was found to differ. Interestingly, any commercial kits with monoclonal antibody capture and polyclonal antibody detection (mono/poly), but not mono/mono Ab capture and detection, could pick up the common HBsAg Gly145Arg mutant either solely or in combination with other mutations within the "a" determinant. New versions of HBsAg test kits should recognize multiple HBsAg epitopes in order to detect mutant HBsAg, together with providing good analytical sensitivity and specificity, because of the importance of these assays in HBV diagnosis and in protecting the safety of the blood supply.     

G145R突变后HBsAg"a"决定簇合成肽的免疫学特性分析

目的研究G145R突变后乙肝表面抗原(HBsAg)"a"决定簇合成肽的免疫学特性改变情况. 方法首先合成2条短肽P1-wt和P2-145R,分别代表野毒株和G145R突变后HBsAg"a"决定簇合成肽.然后用β-巯基乙醇(2-ME)变性试验以及用乙肝疫苗标准品制备的小鼠多抗血清研究2条合成肽的空间构象以及抗原性异同.最后用等量合成肽免疫小鼠,用酶免疫法、竞争抑制试验和Western blot试验等研究G145R突变后"a"决定簇合成肽的免疫原性改变.结果合成肽P1-wt和P2-145R用2-ME温和变性后,PAGE结果表现为相对分子质量(Mr)为4×103左右的单一条带,而变性前2条合成肽均表现为Mr是从4×103到30×103的弥漫条带,主带位置在5×103和10×103,分别相当于二聚体和四聚体位置;用HBsAg的多克隆抗体(anti-HBs)检测合成肽的抗原性,结果在固定抗原量的前提下,在抗体1∶32 000稀释时仍可检测到P1-wt的阳性结果,而当同一抗体稀释到1∶8000时,P2-145R检测结果即为阴性.合成肽P2-145R免疫小鼠产生的抗体与P1-wt合成肽的反应滴度比与P2-145R合成肽本身的反应低4～8倍. 结论针对HBsAg"a"决定簇合成肽可自发形成一定的空间构象,G145R突变株的HBsAg"a"决定簇的抗原性和免疫原性与野毒株相比发生了明显改变,为正确评价G145R变异株的流行危害以及现行疫苗的保护效果提供了实验依据.     

四种乙肝病毒表面抗原ELISA诊断试剂的评价

目的 对四种国内乙肝病毒表面抗原诊断试剂进行评价。方法 用四种试剂分别对标准品和未知血清进行检测，用几种常用诊断试验评价方法对检测结果进行分析和比较。结果 四种试剂对标准血清的检测结果较好，A、B两种试剂的总符合率为100％，另两种的总符合率也在90％以上，其中B试剂有较好的精密性。δ值比较显示A和B两种试剂有较好的诊断特性。四种试剂在一定的抗原浓度范围内都有良好的线性关系。针对于未知样本的检测中，四种试剂与采用电发光法的罗氏诊断试剂有较好的一致性。结论 国内主要厂家的HBsAg ELISA诊断试剂具有较高的检测效能。在实际工作中，可以通过常用的诊断试剂筛选实验，经统计分析后筛选出较高效能诊断试剂用于检测。     

Impact of changes in the amino acid sequence of the "a" determinant on the diagnostic capacities of test systems for the detection of HBsAg

The diagnostic capacities of 4 commercial test systems were comparatively estimated for the detection of HBsAg, by applying a panel of samples with the established amino acid sequence of the "a" determinant of HBsAg. The Roche Elecsys HBsAg test system demonstrated the highest sensitivity - the maximum HBsAg concentrations were found in 19 of 31 cases. Escape mutations in the major hydrophilic region (MHR) of HBsAG (P120S, M133T) were responsible for differences in the sensitivity of 4 test systems by 10- to 40-fold. There were also samples that showed differences in the diagnostic capacities of the test systems to detect HBsAg, but without amino acid replacements in the area of the "a" determinant, which seems to be associated with amino acid replacements in other regions of HBsAg.     

用现行国产HBsAg EIA检测乙肝S基因突变体问题的研究

目的 探讨乙型肝炎表面抗原（ＨＢｓＡｇ）突变体对抗体结合能力的影响，寻找鉴别变异株的办法和规律，并为改进试剂质量进行基础研究。方法 用２３家国产ＨＢｓＡｇ ＥＩＡ检测１０株重组表达ＨＢｓＡｇ突变体和１株原型ＨＢｓＡｇ。结果 ２３家国产试剂均能检测出原型、重组表达ＨＢｓＡｇ。表达的１０株突变体，１号试剂可全部检出，其它试剂的检出率分别为：对突变体抗原１４５精氨酸、１３３亮氨酸、１２６天冬酰胺的检出率     

A hepatitis B virus variant found in the sera of immunised children induces a conformational change in the HBsAg "a" determinant.

The emergence of variants in the outer envelope proteins of hepatitis B virus (HBV) are found among individuals vaccinated against HBV and asymptomatic carriers of the infection. For example, children in The Gambia vaccinated against hepatitis B may show serological evidence of breakthrough infections, particularly if anti-HBs antibodies induced by the vaccine are low in titre. A single-point mutation at nucleotide 421 of the S gene is associated with such breakthrough infections. In the present study, the antigenicity of variant HBV S protein expressed as HBsAg particles in a vaccinia virus expression system has been characterised using a panel of monoclonal antibodies directed against linear and conformational determinations of the S protein. A cellular ELISA procedure using expressed antigen in Vero cells revealed differences in reactivity using four of the six antibodies that had been raised against the adw subtype of HBV and recognise conformational epitopes in the a determinant. In two instances, an enhanced reactivity for the variant antigen was found, confirming that point mutations in the a determinant of the S protein between residues 139 and 147 may result in significant changes in conformation. These findings also demonstrate that there are distinct antigenic differences between the vaccine strains of HBsAg/ adw subtype and the predominant HBsAg subtype circulating in West Africa. The implications of this work are that serodiagnosis of HBV infections may be unreliable in populations where there is a possibility of variant HBV infections emerging in the face of increasing herd immunity to HBV as a result of vaccination, particularly using monoclonal antibody-based diagnostic tests. Such variants may play a role in the maintenance of HBV infections in endemic regions.     

Synthesis of diagnostic test kits for syphilis with polymeric particles

This investigation aimed to derive alternative guidelines for syphilis. In the first step of this study monodisperse PGMA particles were synthesised by dispersion polymerization of the GMA monomer by using AIBN as initiator and PAA as stabilizator. A series of characterization experiments were done. In the second step of the study the characterized particles were used as microcarriers for the immobilization of the natural antigen for the purpose of diagnosing syphilis. The covalent binding method was used and GA was utilized as a spacer arm. The optimum immobilization conditions for the binding of natural antigen onto the particles were determined. In the third and the last step of the study the agglutination tests were realized with in vitro experiments by using both serum samples taken from syphilis patients and positive controls. After these experiments the kits chosen among the ones showing high performance were compared with the commercial RPR kit being used widely by reacting with serially diluted positive controls and patient serums. After this comparison the performances of the synthesized kits were found as satisfactory as the commercial kits.     

Synthesis of diagnostic test kits for syphilis with polymeric particles

This investigation aimed to derive alternative guidelines for syphilis. In the first step of this study monodisperse PGMA particles were synthesised by dispersion polymerization of the GMA monomer by using AIBN as initiator and PAA as stabilizator. A series of characterization experiments were done. In the second step of the study the characterized particles were used as microcarriers for the immobilization of the natural antigen for the purpose of diagnosing syphilis. The covalent binding method was used and GA was utilized as a spacer arm. The optimum immobilization conditions for the binding of natural antigen onto the particles were determined. In the third and the last step of the study the agglutination tests were realized with in vitro experiments by using both serum samples taken from syphilis patients and positive controls. After these experiments the kits chosen among the ones showing high performance were compared with the commercial RPR kit being used widely by reacting with serially diluted positive controls and patient serums. After this comparison the performances of the synthesized kits were found as satisfactory as the commercial kits.     

HBsAg ELISA试剂盒筛检结果的评估

目的 评估部分国产与进口HBsAg ELISA试剂盒筛查血源的价值.方法 选用部分国产和进口HBsAg ELISA试剂,对中国生物制品检定所120份HBsAg临床科研组合血清样本及随机抽取400份东莞市无偿献血者血清标本为验证样本进行同步平行检测,以中国生物制品检定所组合血清及献血者HBsAg阳性并经中和试验证实者为金标准.结果采用四格表法计算两者对等指标并进行评价.结果 国产HBsAg ELISA试剂和进口HBsAg ELISA试剂的灵敏度分别为85.71%（72/84）和100%（84/84）;特异性分别为100%（436/436）和96.55%（421/436）;尤登指数分别为0.86、0.97;两种试剂重复合格率均为100%.结论 试剂的灵敏度以进口HBsAg ELISA试剂较好,但特异性比国产试剂差,两种试剂的重复性均好,两种试剂联合筛检献血者血样标本可提高临床输血的安全性.     

Evaluation of rapid kits for detection of HIV, HBsAg and HCV infections

Diagnostic kits (J. Mitra Co. Ltd) for rapid detection of HIV, HBsAg & HCV were evaluated against respective enzyme immunoassay (Ortho Diagnostics). All rapid kits were 100% specific for HIV, HBsAg & HCV. Sensitivity for HIV, HCV & HBsAg was 80%, 87.5% & 93.4% respectively.     

丙型肝炎病毒抗体酶标试剂盒的评价

目的了解国内丙型肝炎病毒抗体酶标诊断试剂盒的质量。方法应用中国国家丙型肝炎病毒抗体参考品及美国ＢＢＩＡｎｔｉ－ＨＣＶｐａｎｅｌ对Ａｂｂｏｔ等四家国外及六家国内丙型肝炎病毒抗体试剂盒进行了评价。结果１９８份中国国家丙型肝炎病毒抗体参考品中的１００份阳性参考品的检出率，Ａｂｂｏｔ试剂盒为９８％，国产试剂盒为９５％～９７％。对２５份ＢＢＩｐａｎｅｌ中的２３份阳性血清，ｐａｎｅｌ检出率Ａｂｂｏｔ等国外试剂盒为８７％～１００％，国产试剂盒为７０％～９１％。结论国产试剂盒所用丙型肝炎病毒抗原结构区抗原与国外试剂一致，但非结构区抗原较弱。提高国产丙型肝炎病毒抗体试剂盒的质量，应增加高质量的非结构区抗原的应用     

两种巨细胞病毒IgG抗体ELISA诊断试剂性能评价

目的对国内两种巨细胞病毒IgG抗体ELISA诊断试剂进行评价。方法用两种试剂对BBI公司的质控盘QTC711,血清转化盘PTC901,BIOMEX公司血清转化盘SCP—CMV-001（RP-003）、SCP—CMV-002（RP-019）,儿童、孕妇和门诊患者2163份标本进行检测,对不一致的样本用DiasorinELISA试剂盒和Mikrogen重组免疫印迹试剂盒进行复测,比较两种试剂的灵敏度、特异性和对不同人群的检测差异。结果A试剂对3个血清转化盘的检出时间均早于B试剂,平均提前25d,与AbbottImxCMVIgG检测灵敏度一致。两种试剂检测孕妇样本607份,8份不一致,总符合率98．68％;门诊样本512份,7份不一致,总符合率98．63％;儿童样本1044份,74份不一致,总符合率92．91％,两试剂对儿童人群的符合率低于孕妇和门诊人群。两试剂对三个人群检测均为阴性的161份样本,和A试剂检测为阳性B试剂检测为阴性的89份样本,用意大利Diasorin公司CMV—IgGELISA试剂复测,A试剂与Diasorin的阳性符合率为100％,阴性符合率为93．06％,总符合率为95．22％;B试剂与Diasorin的阴性符合率为100％,78份阳性未检出,总符合率为68．92％。选择A试剂与Diasorin结果不一致的样本12份,一致的样本6份,用重组免疫印迹复测,其中14份阳性,4份阴性。结论国内A试剂对血清转化盘的检测窗口期较B试剂显著提前。两试剂对孕妇和门诊人群的符合率高于儿童人群。两试剂不符合的样本用进口试剂盒复测,A试剂与进口试剂盒的符合率高于B试剂,显示了较高的灵敏度。     

Mutation in the 3'untranslated region of APP as a genetic determinant of cerebral amyloid angiopathy

Aβ-related cerebral amyloid angiopathy (CAA) is a major cause of primary non-traumatic brain hemorrhage. In families with an early onset of the disease, CAA can be due to amyloid precursor protein (APP) pathogenic variants or duplications. APP duplications lead to a ~1.5-fold increased APP expression, resulting in Aβ overproduction and deposition in the walls of leptomeningeal vessels. We hypothesized that rare variants in the 3''untranslated region (UTR) of APP might lead to APP overexpression in patients with CAA and no APP pathogenic variant or duplication. We performed direct sequencing of the whole APP 3''UTR in 90 patients with CAA and explored the functional consequences of one previously unreported variant. We identified three sequence variants in four patients, of which a two-base pair deletion (c.*331_*332del) was previously unannotated and absent from 175 controls of same ethnicity. This latter variant was associated with increased APP expression in vivo and in vitro. Bioinformatics and functional assays showed that the APP c.*331_*332del variant affected APP messenger RNA (mRNA) structure and binding of two microRNAs (miR-582-3p and miR-892b), providing a mechanism for the observed effects on APP expression. These results identify APP 3''UTR sequence variants as genetic determinants of Aβ-CAA.     

Intraductal papilloma with "comedo-like" necrosis, a diagnostic pitfall

Necrosis in the breast is more frequently associated with malignancies than benign conditions. In the latter, it may be diffuse as seen in infarcts, postpartum changes, and after anticoagulant therapy, or focal as in florid adenosis and juvenile papillomatosis. The goal of this study was to find other benign entities associated with necrosis. Using our pathology database from January 1994 to March 2003, we identified 10 cases of florid duct hyperplasia associated with luminal necrosis. Clinical information was collected and all slides were reviewed. The age of the patients ranged from 36 to 94 years (mean, 53.2 years). All patients underwent an initial excisional biopsy except for one who had a core biopsy. Patients presented with a palpable mass (6 cases) a radiographic density (3 cases), or nipple discharge (1 case). Findings correlated with the presence of an intraductal papilloma with adjacent or associated florid duct hyperplasia. Within the central luminal spaces of the florid duct hyperplasia, cellular debris associated with necrosis was present and was morphologically identical to that seen in comedo intraductal carcinoma. In fact, in two of the four consultation cases, these lesions were initially diagnosed as comedo carcinoma. Re-excision performed in three cases showed no atypia or malignancy. It is important to recognize the presence of comedo-like necrosis in florid duct hyperplasia associated with or adjacent to intraductal papillomas of the breast and not to misinterpret these findings as intraductal carcinoma. The necrosis may be secondary to reactive changes within the intraductal papilloma.     

A glycopeptide containing the group-specific antigenic determinant of hepatitis b surface antigen (HBsAg)

The treatment of purified 22 nm structures of HBsAg with pronase in the presence of SDS resulted in the appearance of light fragments which contain predominantly group-specific ‘a’ antigenic determinant. By size and mobility in SDS-polyacrylamide gel electrophoresis the isolated material corresponds to globular proteins with molecular weight of 12,000 (or less) daltons. The analysed structures and HBsAg had different isoelectric points and spectrum in UV light. The released material in contrast with HBsAg was inactivated during the treatment with neuramindase. The aggregates of the obtained glycopeptide turned out to be immunogenic for guinea pigs. The immune sera contained antibodies against ‘a’ and ‘y’ specificities of HBsAg but not against normal human plasma proteins. The possibility of using the minimal structures carrying protective antigenic determinants of HBsAg as a vaccine is discussed.