广东地区严重急性呼吸综合征(SARS)患者血清的IFA检测

目的：证实新分离出的冠状病毒与SARS患者的关糸。方法：应用IFA诊断试剂做免疫荧光染色，检测2003-02-04-04-13期间，广东地区3所医院临床诊断为SARS的72例患者血清特异性抗体，以及直接参加救治SARS患者但末发生感染的109名医护人员的血清标本，并以疾病流行期间70例健康体检者作为正常对照组。结果：72例SARS患者中，62例血清特异性：IgG呈阳性；而109例密切接触但末发生感染的医护人员及70例SARS流行期间健康体检者，血清特异性IgG和IgM均呈阴性。结论：SARS冠状病毒是引发广东地区SARS的病原体。     

结核分枝杆菌三种特异性抗体ELISA检测方法的建立

目的 建立以结核分枝杆菌特异性蛋白ESAT6、CFP10-ESAT6、ESAT6-PPE68为包被抗原检测结核病人血清中特异性抗体的间接ELISA检测方法。方法 利用本室构建并纯化的结核分枝杆菌特异性抗原蛋白ESAT6、CFP10-ESAT6、ESAT6-PPE68,建立结核分枝杆菌特异性抗体检测的间接ELISA检测方法：ESAT6-ELISA、CFP10-ESAT6-ELISA、ESAT6-PPE68-ELISA。并与PPD-ELISA同时检测220例结核病人血清、30例非结核呼吸疾病病人血清和50例健康人群血清,分别对三种方法的敏感性与特异性进行统计学分析。结果 ESAT6-ELISA的敏感性为25%,特异性为95%;CFP10-ESAT6-ELISA的敏感性为45%,特异性为93%;ESAT6-PPE68-ELISA的敏感性为48%,特异性为85%。结论 三种结核分枝杆菌特异性抗体检测方法的敏感性均低于PPD-ELISA,但三种结核分枝杆菌特异性抗体检测方法的特异性均高于PPD-ELISA,其中CFP10-ESAT6-ELISA检测的效果最佳,PPE68可作为结核特异性诊断的候选抗原。     

GSTP1在前列腺癌组织中的表达及其甲基化检测

目的寻找前列腺癌患者血清中的生物标志物,为临床前列腺癌的早期无创性诊断提供依据。方法采用免疫组化SP染色法检测前列腺癌组织中GSTP1表达;运用甲基化特异性聚合酶链反应(MSP)方法检测前列腺癌患者癌组织和相应血清GSTP1基因启动子区5’端CpG岛甲基化程度。结果免疫组化结果:GSTP1在88例前列腺癌组仅有2例呈阳性反应且均为石蜡包埋标本;49例前列腺增生组均为阳性反应,其中38例呈现为强阳性反应。MSP检测结果:38例新鲜前列腺癌组织中,GSTP1基因高甲基化29例,此29例相应血清GSTP1基因高甲基化28例。对照组19例前列腺增生标本和对应的血清均没有检测到GSTP1基因甲基化。结论前列腺癌组织中GSTP1基因的表达与其启动子区甲基化程度呈负相关。前列腺癌患者的组织和血清中GSTP1甲基化检测结果相一致,检测血清中GSTP1的甲基化程度可反应癌组织中GSTP1基因的表达的情况。     

SARS病毒抗体的检测及其应用

为了解SARS患者血清抗体产生的规律 ,我们用间接免疫荧光法 (IFA)和酶联免疫吸附试验 (ELISA)对广州市第八人民医院的 4 3例住院治疗的SARS患者和 10例正常人的双份血清进行SARS抗体检测 ,同时比较了IFA和ELISA法的特点。10例正常人双份血清的SARS病毒IgM和IgG抗体均阴性。 4 3例SARS患者 ,其中 34例检出IgM占 79.1%。 37例检出IgG占 86 .10 %。SARS病毒IgG抗体的阳性率高于IgM。结果显示 ,发病的同时机体便可检测到SARS抗体IgM最迟离起病时间最长 (33d)的血清也能检测到 ,但也有患者发病第 9、2 3及 30天的第二份…     

酶联免疫吸附试验检测抗-HBe与抗-HBc假阳性分析

目的 对酶联免疫吸附试验(ELISA)检测乙肝病毒血清抗-HBe与抗-HBc出现假阳性进行分析,并提出一些解决办法.方法 对521例血清标本采用酶联免疫吸附试验进行检测,先用一种试剂初检,再用另外两种试剂复检阳性标本,初检呈阳性反应而两种试剂复检均为阴性者判为假阳性.结果 在521份标本中用第一种试剂检测到抗-HBc阳性434份,抗-HBe阳性451份.用两种试剂复查阳性标本后,其中65份标本抗-HBc均为阴性,假阳性占15.98%,60份抗-HBe标本均为阴性,假阳性占13.30%.结论 酶联免疫吸附试验检测抗-HBc、抗-HBe,有许多影响因素可导致结果假阳性结果,对怀疑假阳性的标本除了消除其影响因素外,还应使用两种试剂进行复检.     

探讨乙肝两对半模式与乙肝病毒DNA定量的相关性

目的：探讨乙肝两对半模式与乙肝病毒DNA定量的相关性,为临床治疗乙肝提供参考和借鉴。方法对我院2012年1月~2014年6月收治的320例乙肝患者的血清标本进行乙肝两对半及乙肝病毒核酸扩增酶联定量检测,对乙肝两对半不同模式及乙肝病毒DNA之间的关系进行分析和探讨。结果265例患者血清HBV DNA呈阳性,阳性率为82.81%;小二阳72例,其中54例HBV DNA呈阳性,阳性率为75%;小三阳138例,其中120例HBV DNA呈阳性,占86.96%;大三阳110例,其中91例HBV DNA呈阳性,占82.72%。大三阳样本含量超过107例数超过小二阳及小三阳病例。结论乙肝两对半是基层医疗检验项目,其不足之处在于无法充分反映乙肝患者的病情状况,HBeAg与HBV-DNA拷贝数呈正相关性,HBV-DNA检测可判断乙肝患者是否存在传染性,为制定合理治疗方案提供了依据。     

检测抗人疱疹病毒6型IgG的间接免疫荧光试验的建立及其应用

目的 :建立检测血清中人疱疹病毒 6型 (HHV 6 )IgG的间接免疫荧光试验 (IFA)。方法 :用HHV 6国内分离株感染人脐带血单个核细胞制备抗原片 ,建立检测HHV 6IgG的IFA方法 ,并用于育龄期妇女血清流行病学调查。结果 :建立的IFA具有特异性。对 116份育龄期妇女血清标本检测表明 ,HHV 6IgG的阳性率为 72 .4 % ,几何平均滴度 (GMT)为 1∶6 1;在孕妇和正常未孕妇女之间 ,以及不同孕期的孕妇之间 ,HHV 6IgG的阳性率和GMT均无差异 (P >0 .0 5 )。结论 :建立了具有特异性的IFA法 ,可用于对育龄妇女HHV 6感染率的流行病学调查     

微斑免疫酶技术在检测肾综合征出血热病毒及其特异抗体中的应用

本文报道一种简便且快速的方法—微斑免疫酶技术(MPIPA),检测Vero-E6细胞培养的肾综合征出血热(HFRS)病毒及其特异抗体,并与免疫荧光技术(IFA),酶联免疫吸附技术(ELISA)进行比较。结果,本法用于检测HFRS患者血清IgG抗体和抗HFRS病毒单克隆抗体(McAb),其敏感性介于ELISA和IFA之间;用于检测Vero—E6细胞培养的病毒,阳性结果比IFA和细胞病变(CPE)出现早。MPIPA具有简单,快速、敏感性高、特异性好,结果易判定诸优点,便于基层医疗单位推广应用,适合于临床HFRS血清标本的检测和血清流行病学调查。     

RPHI法检测患者及不同种属动物血清中HFRSV总抗体

本研究用亲和力高,反应谱广的HFRSV-McAb致敏绵羊红细胞,以Tween-80和乙醚处理HFRSV陈株感染鼠脑制备特异性抗原,建立了检测HFRSV总抗体的RPHI试验。检测拟诊HFRS血清82例156份,阴性对照人血清256份,黑线姬鼠、褐家鼠、大白鼠、小白鼠血清127份,豚鼠血清8份,兔血清12份,并与IFA法进行对比,结果如下:     

新型抗蝰蛇毒血清对不同蝰蛇毒作用的实验研究

目的探讨新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的作用. 方法采用免疫电泳、SDS-聚丙烯酰胺电泳及动物实验等方法. 结果国产圆斑蝰蛇毒的LD50(腹腔注射)为0.306±0.003mg/kg,缅甸蝰蛇毒的LD50(腹腔注射)为0.290±0.003mg/kg.免疫电泳结果表明国产圆斑蝰蛇毒和缅甸蝰蛇毒均和新型抗蝰蛇毒血清产生明显的免疫沉淀线.体外中和实验表明抗蝰蛇毒血清对国产圆斑蝰蛇毒的中和抗体效价为2750μg/ml(约450LD50), 对缅甸蝰蛇毒的中和抗体效价为2490μg/ml(约430LD50).体内保护实验表明给予0.05ml抗蝰蛇毒血清可抵御254μg(约41.5LD50)国产圆斑蝰蛇毒或116μg(约20LD50)缅甸蝰蛇毒的攻击,使小鼠全部存活. 结论新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的中和抗体效价均较高,有较大的应用价值.     

Detection of Salmonella contamination in food samples by dot-ELISA, DNA amplification and bacterial culture.

A dot-blot enzyme-linked immunosorbent assay (dot-ELISA) employing a genus Salmonella specific monoclonal antibody (MAb) was used for detection of the bacteria in food samples in comparison with the conventional culture method and the DNA amplification. Among the 200 chicken and pork samples (100 each) tested, 9% and 33%, 7% and 20% and 7 and 23% were positive for salmonellae by the dot-ELISA, the culture method and the DNA amplification, respectively. Statistical analyses revealed that the sensitivity, specificity, efficacy, and positive and negative predictive values of the detection of Salmonella in the food samples by dot-ELISA compared with the culture method were 93.33%, 91.76%, 92%, 66.66% and 98.73%, respectively. Comparison of the DNA amplification and the culture method revealed the sensitivity, specificity, efficacy, and positive and negative predictive values of 100%, 91.58%, 92%, 65.21% and 100%, respectively. The dot-ELISA and the DNA amplification results were in a better agreement when the two assays were compared. The sensitivity, specificity, efficacy, positive and negative predictive values of the dot-ELISA compared to the DNA amplification were 91.3%, 100%, 98%, 100% and 97.5%, respectively. From this study, the dot-ELISA is rapid, simple, sensitive, specific at low cost with limited amount of infectious waste to be disposed and offers another advantage in that it detects only the smooth LPS of Salmonella which implies the possible presence of the virulent organisms.     

组合单克隆抗体夹心斑点-ELISA检测日本血吸虫循环抗原的研究

本研究将抗不同表位单抗组合，首次用于夹心斑点-ＥＬＩＳＡ检测血吸由感染。对慢性日本血吸虫病患者的阳性检出率为８５％，１２例急性患者均阳性，１０例晚血病人４例阳性，非疫区正常人的假阳性率为２．２％。用单盲法检测的-组血清其符合率亦较满意。在基本消灭血吸虫病地区检测了１１００人，用夹心斑点-ＥＬＩＳＡ测抗原，阳性率为６．５％，以ＩＨＡ测抗体阳性率为２５．２％。在低年龄组人群，斑点ＥＬＩＳＡ与ＣＯＰＴ符合率为８８．９％，明显高于ＩＨＡ与ＣＯＰＴ的符合率（３５．７％），Ｐ＜０．０５。动物实验中，组合单抗夹心班点ＥＬＩＳＡ对低感染兔的阳性检出率高于单个单抗直接斑点-ＥＬＩＳＡ。     

肾综合征出血热Ⅰ型灭活疫苗免疫后4年抗体水平监测

目的 比较肾综合征出血热（HFRS）Ⅰ型灭活疫苗接种组和对照组免后4年抗体水平,了解HFRSⅠ型灭活疫苗有无增强接种组隐性感染发生情况。方法 1994年7月-1998年7月在建德市肾综合征出血热Ⅰ型灭活疫苗随机对照试验现场,采集接种组和对照组双份血清各305和283人。分别按对照组免前间接荧光抗体阳性和阴性的第二份血清间接荧光抗体滴度分布情况确定阳性判断阈值（cut-off值）,由此判断免前间接荧光抗体阳性和阴性的接种组人群发生隐性感染情况。结果 以不同的cut-off值来比较接种组和对照组免前间接荧光抗体阳性人群的抗体阳性率,差异无显著性,在免前间接荧光抗体阴性接种组和对照组中,其差异也无显著性,未见接种组和对照组免后抗体几何平均滴度和抗体阴转率差异有显著性。结论 接种组同对照组免后4年荧光抗体水平差异无显著性,未发现HFRSⅠ型灭活疫苗增强接种人群隐性感染发生率。     

Comparison of rapid immunofluorescence assay to cell culture isolation for the detection of influenza A and B viruses in nasopharyngeal secretions from infants and children.

In the hospital setting it is often critical to isolate patients appropriately in order to prevent nosocomial infection. This is especially true with respiratory infection in infants and young children. At the present time a rapid immunofluorescence assay (IFA) for respiratory syncytial and parainfluenza viruses is routinely carried out in our laboratory. During January and February of 1990 we used monoclonal antibodies specific for influenza A and B viruses (Baxter-Bartels, Bellevue, WA) in this rapid IFA. 152 samples of NPS were tested by cell culture isolation (CCI) and IFA for the presence of influenza antigens. Twenty-seven samples were positive by both methods, and 114 were negative by both. Three samples were positive by IFA and negative by CCI, while eight samples were positive by CCI and negative by IFA. Five of these eight samples were not positive until 10 to 14 days after inoculation into cell culture, suggesting that the virus inoculum was small. Using CCI as the 'gold' standard, IFA was 90% sensitive and 93% specific. Because of its turn-around time (2-4 h) and acceptable sensitivity and specificity, IFA for influenza viruses will be a routine test in our diagnostic laboratory during the influenza season.     

Validation of Legionella pneumophila indirect immunofluorescence assay with epidemic sera.

Sera from six outbreaks of legionellosis and four outbreaks of pneumonia of other etiologies were tested with the indirect immunofluorescence assay (IFA) as currently performed. The current IFA is at least as sensitive as the original test in detecting cases of Legionnaires disease (78 to 91%). By using Center for Disease Control criteria for a positive (fourfold increase in titer during convalescence to greater than or equal to 128) or presumptive (single titer greater than or equal to 256) serological test, the specificity exceeded 99%. No cross-reactions against Legionella pneumophila antigens were observed among sera from epidemic cases of Q fever, tularemia, and psittacosis; the only positive L. pneumophila IFA titer among the epidemic Mycoplasma pneumonia sera was reduced to a negative titer with an immunosorbent extracted from Escherichia coli strain O13:K92:H4. The slight increase in specificity (to 100%), however, was offset by a slight decrease in sensitivity. The sensitivity of the IFA was maximal when a conjugate that detected immunoglobulins G, M, and A was used. IFA titers were not significantly altered by replacing the monovalent serogroup 1 antigen with a polyvalent antigen (serogroups 1 through 4) nor by the presence of rheumatoid factor or heat-labile serum factors.     

Comparable evaluation of serological diagnostic tests (ELISA, IFA and PA methods) for the detection of anti-Borrelia burgdorferi antibody]

We have evaluated the usefulness of the enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody assay (IFA) and particle agglutination (PA) method as serological screening tests for Lyme-borreliosis. Serum samples obtained from two patients with Lyme-borreliosis showed marked high antibody titers for Borrelia burgdorferi when measured by these methods. Of the serum of 368 healthy members of the Self-Defense Force in north-eastern Japan screened for the antibody to B. burgdorferi, 8.4%, 3.7%, 4.6% were found positive by the ELISA, IFA, and PA method, respectively. However, Western blot analysis of these "positive" sera demonstrated no identical bands to those seen in the serum from the patients with Lyme-borreliosis. While 85% and 15% of Treponema pallidum hemagglutination test (TPHA)-positive sera (20 samples) showed a false-positive reaction by the ELISA and IFA method, respectively, no cross-reaction to the anti-B. burgdorferi antibody was observed in these sera by the PA method. The analysis of the serum of the patients with autoimmune diseases (rheumatoid arthritis; 11 cases, systemic lupus erythematosus; 46 cases) by the ELISA and PA methods resulted in a cross-reaction to some extent, which suggested that the antibodies produced by autoimmune mechanisms such as the anticardiolipin antibody can cause a cross-reaction to the anti-B. burgdorferi antibody. These findings indicate that the PA and ELISA rather than the IFA method should be recommended for rapid and conventional screening of Lyme-borreliosis and that serum "positive" for the anti-B. burgdorferi antibody determined by these tests should be confirmed by Western blot analysis to negate the cross-reactions.     

Diagnosis of herpes simplex virus infection by immunofluorescence.

The utility of the indirect immunofluorescent antibody (IFA) technique for diagnosis of herpes simplex virus (HSV) infection was examined by testing specimens for this agent from 31 patients with encephalitis or meningitis, 17 with conjunctivitis, 19 with genital disease, and 1 with genital disease and meningitis. Brain biopsy tissue from four patients with encephalitis was positive by IFA and virus culture for HSV. Leukocytes in cerebrospinal fluid from these four patients and one with HSV meningitis were also positive by IFA, but virus isolation attempts on the fluid were all negative. Conjunctival scrapings from two patients with conjunctivitis were positive for HSV by both IFA and virus culture. Eleven of 12 culture-positive lesions of herpes progenitalis were positive by IFA, and 1 dark field-positive syphilitic chancre was also positive for HSV by both IFA and culture. Evidence for specificity of the results was provided by internal controls in each test and negative results from patients with other diagnoses. Thus, the IFA technique constituted a rapid, sensitive, and specific diagnostic method for the diagnosis of HSV infections.     

New criteria for immunofluorescence assay for Q fever diagnosis in Japan

A study was made to evaluate the cutoff value of indirect immunofluorescent-antibody (IFA) test for Q fever diagnosis in Japan. We used 346 sera, including 16 from confirmed Q fever cases, 304 from Japanese pneumonia patients, and 26 from negative cases. Thirteen sera from the confirmed Q fever cases with an immunoglobulin M (IgM) titer of > or =1:128 and/or IgG titer of > or =1:256 by the IFA test were positive by both enzyme-linked immunosorbent assay (ELISA) and Western blotting assay (WBA), whereas 298 sera from pneumonia patients and 26 negative sera with an IgM titer of < or =1:16 and an IgG titer of < or =1:32 by the IFA test were negative by both ELISA and WBA. In the proposed "equivocal area," with an IgM titer of > or =1:32 and < or =1:64 and/or an IgG titer of > or =1:64 and < or =1:128, we found 9 sera, 3 from confirmed Q fever cases and 6 from Japanese pneumonia patients, by the IFA test. Three sera from the confirmed Q fever cases and one of the sera from pneumonia patients were IgM and/or IgG positive by both ELISA and WBA. These results suggest that a single cutoff value for the IFA test may cause false-positive and false-negative results. In conclusion, this study showed that an "equivocal area" should be used for the IFA test rather than a single cutoff value and that sera in the equivocal area should be tested by additional serological assays for confirmation.     

Comparison of two recombinant major outer membrane proteins of the human granulocytic ehrlichiosis agent for use in an enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.     

Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals.