利用华支睾吸虫的排卵规律和消炎利胆片提高虫卵检出率的研究

目的利用华支睾吸虫的排卵规律和服用消炎利胆片来提高华支睾吸虫虫卵的检出率。方法将300例血清肝吸虫免疫球蛋白G(IgG)抗体阳性的疑似华支睾吸虫感染者随机分成3组,第1组为常规方法留取标本的对照组,第2组为服用消炎利胆片组,第3组则根据华支睾吸虫的排卵周期,分别于第1天、10天、20天留取粪便标本;各组送检的粪便标本均采用改良加藤厚涂片法、水洗沉淀法和醚醛沉淀法三种方法检测华支睾吸虫卵,并对检测结果进行χ^2检验分析。结果第2组和第3组检测华支睾吸虫卵的检出率明显高于第1组,差异有统计学意义(P<0.05);第2组和第3组的检出率比较,差异无统计学意义(P>0.05)。结论利用华支睾吸虫的排卵规律留取标本和服用消炎利胆片均能明显提高华支睾吸虫虫卵检出率,有助于明确华支睾吸虫病的诊断,在临床上合理用药。     

实时定量PCR检测胆囊结石患者胆汁中华支睾吸虫DNA

摘要：目的：探讨用实时定量PCR技术检测胆囊结石患者胆汁标本中的华支睾吸虫DNA。 方法：随机选取40例实施内镜取石保胆手术胆囊结石患者的胆汁标本,对胆汁沉渣分别进行显微镜检及实时定量PCR检测。同时收集患者的血清、粪便标本,分别进行免疫学检测及粪便镜检。 结果：建立的实时定量PCR法特异性良好,对华支睾吸虫DNA的最低检测限为0.1 pg/μL;40例胆囊胆汁标本中有21例镜检发现虫卵（阳性率为52.5%）,实时定量PCR检测结果与镜检结果一致;免疫学检测抗体阳性20例（阳性率为50%）,与前二者的符合率为82.5%(33/40);粪便镜检虫卵阳性9例（阳性率为22.5%）,与其他3种方法比较,差异有统计学意义（χ²=9.12,P＜0.01）。 结论: 建立的实时定量PCR技术可用于胆囊结石患者华支睾吸虫感染的检测。     

灵芝孢子误判为华支睾吸虫感染病例分析及处理

目的分析将灵芝孢子误认为华支睾吸虫感染的原因,并提出防范误诊的对策。方法回顾性分析广西医科大学第一附属医院2007～2010年将灵芝孢子误认为华支睾吸虫感染的患者临床资料。镜检患者服用的灵芝保健品,对比观察华支睾吸虫卵示教标本,患者粪便集卵毛蚴孵化试验检测以及患者停服灵芝保健品后复查粪便。结果确认粪便中检出的是灵芝孢子,患者并未感染华支睾吸虫。结论检验人员应加强业务学习,掌握异常结果鉴别诊断的方法,参考其他检查项目,并及时与临床沟通,才能防止错误报告。     

嗜酸性粒细胞计数与免疫球蛋白E测定筛查华支睾吸虫病的研究

目的评价嗜酸性粒细胞计数(Eos)与免疫球蛋白E(IgE)在华支睾吸虫病中的临床诊断价值,为筛查华支睾吸虫感染提供依据。方法共有66例华支睾吸虫感染者与53例健康对照者入选,检测血清IgE水平、白细胞计数(WBC)、血红蛋白量(Hb)、Eos,通过Logistic逐步回归分析,建立华支睾吸虫病诊断的统计模型,并应用受试者工作特性(ROC)曲线评价统计模型的诊断效能和临界值。结果 Eos与IgE华支睾吸虫感染组明显高于对照组,差异有统计学意义(P<0.05),而WBC与Hb两组间差异无统计学意义(P>0.05);Eos和IgE联合诊断华支睾吸虫病的ROC曲线下面积为0.884,明显高于IgE的0.814和Eos的0.783,差异有统计学意义(P<0.05),其回归方程P=1/[1+e-(-2.374+6.062Eos+0.006IgE)],当临界值为0.16,其诊断华支睾吸虫病的敏感度为100.0%,特异性为30.2%,阳性预测值为64.1%,阴性预测值为100.0%。结论联合Eos和IgE检测有助于提高华支睾吸虫病的诊断效能,明显优于单一指标的检测,而且可以排除部分真阴性标本,对降低劳动力、节约成本具有重要意义。     

R-Dot-IGS法、IFAT法与ELISA法诊断华支睾吸虫病的比较研究

目的：探讨同一条件下快速斑点免疫金染色法（R-Dot-IGS）、间接荧光抗体法（IFAT）和酶联免疫吸附法（ELISA）诊断华支睾吸虫病的比较。方法：取华支睾吸虫成虫可溶性抗原点加于微孔滤膜．经预作用和封闭．依次加入待检血清和金标记二抗．后观察结果，并与IFAT和ELISA法进行比较。结果：用R-Dot-IGS法、IFAT法和ELISA法同步检测83份华支睾吸虫病患者血清，75例健康人血清和10例日本血吸虫病人血清。三种方法的阳性率分别为98．79％（82／83）．96．38％（80／83）和97．59％（81／83）：检测75例健康人血清均为阴性：检测10例日本血吸虫病人血清．有1份日本血吸虫病人血清在IFAT反应为阳性。结论：检测结果证明R-Dot-IGS法具有敏感特异、快速简便的特点．适用于华支睾吸虫病的现场流行病学调查和临床快速诊断。     

日本血吸虫6种粗抗原和组分抗原的比较

目的比较日本血吸虫6种抗原(粗抗原及其组分抗原各3种)的敏感性和特异性.方法①用阳性钉螺逸出的尾蚴感染新西兰家兔,制作日本血吸虫病动物模型.②于感染后42天分别收集雌、雄成虫和虫卵,制作粗抗原及其组分抗原.③采用ELISA法检测抗原的敏感性和特异性,并以X2检验作统计学处理和分析.结果除雌虫组分抗原的敏感性(51%)较差外,都显示出日本血吸虫成虫及其虫卵组分抗原的较好敏感性和特异性.结论日本血吸虫组分抗原用于免疫诊断,具有较好的敏感性和特异性,但抗原分离、纯化方法有待进一步探讨.     

5例华支睾吸虫卵空壳的发现与探讨

目的：在受检者大便标本中查找华支睾吸虫空壳,探讨其出现的意义,完善华支睾吸虫生活史.方法：通过大便常规镜检在受检者大便标本中查找华支睾吸虫卵空壳,另选取同期健康体检者30例作正常对照组,对查找到华支睾吸虫卵空壳的患者及健康体检者均检测ALT、AST、TBIL、DBIL、GGT、PA、ALB和E％等肝吸虫病病情诊断指标.结果：在656例找到华支睾吸虫卵的病例中,发现其中5例大便中有华支睾吸虫卵空壳,这5例患者ALT、AST、GGT、PA和ALB的检测结果的均数与正常对照组均数的差异有统计学意义（P＜0.05）.结论：华支睾吸虫虫卵可以在人体内孵出毛蚴,这在华支睾吸虫生活史上是一个新的发现.因此在遇到可疑物时,一定要多次留标本或用多种方法找虫卵,同时查看病人相关的检验指标,只有这样才不会漏检、误诊.     

浙江省西部丘陵地区华支睾吸虫感染现状研究

目的 了解浙江省西部丘陵区华支睾吸虫病的流行现状和流行因素。 方法 以多阶段整群随机抽样的方法,在浙江省西部4个县(区)20个镇对1530名常住居民进行面对面问卷调查,并对其中1505人用ELISA法检测华支睾吸虫抗体。同时调查第二中间宿主华支睾吸虫囊蚴感染情况。 结果 人群血清抗体阳性率为0.67%。87.0%的居民不知道生食淡水鱼虾会感染华支睾吸虫,14.1%的居民有生食淡水鱼虾史。检测第二中间宿主20种鱼虾2812尾,其中12种鱼虾感染华支睾吸虫,囊蚴感染率为6.7%。 结论 浙西丘陵区华支睾吸虫感染率不高,但依然存在感染风险,需进一步加强健康教育。     

双抗原夹心ELISA检测抗HCV总抗体

目的建立检测抗HCV抗体的双抗原夹心ELISA法,评价其可行性。方法将与His或GST融合表达的HCV基因工程抗原,分别用作ELISA的包被和酶标记抗原,建立用于抗HCV总抗体检测的双抗原夹心ELISA。用此方法检测1 968份临床血清标本,并以间接ELISA(北京万泰试剂)与之对照;此外,用套式RT-PCR检测部分血清的HCV RNA。结果有1 761份血清2种ELISA检测均为阴性,有190份血清均为阳性,两种方法符合率为99.1%;有17份血清的检测结果不相符,间接法阳性而本法阴性的14份,其中HCV RT-PCR阳性1份;本法阳性而间接ELISA阴性的3份,其中RT-PCR阳性2份。双抗原夹心ELISA与间接ELISA的敏感性分别为99.48%、98.96%,特异性分别为99.94%、99.27%。结论新研制的检测抗HCV总抗体的双抗原夹心ELISA具有较高的敏感性和特异性,值得作进一步的深入研究。     

酶联免疫吸附试验检测人疱疹病毒7型抗体的研究

目的建立一种简便、快速、可靠的检测人疱疹病毒7型(HHV 7)特异性IgG和IgM酶联免疫吸附试验(ELISA)。方法应用HHV 7标准株G lasgow感染SupT1细胞,制备并纯化细胞工程抗原和重组pp85抗原;对325份血清HHV 7抗体进行检测,建立HHV 7特异性IgM和IgG间接ELISA,并与Q iagen公司ELISA试剂盒检测结果进行比较。结果采用细胞工程抗原和重组抗原制备的HHV 7 IgG和IgM ELISA诊断试剂敏感性、特异性、稳定性及重复性[变异系数(CV)<10%]较好;以Q iagen公司ELISA试剂盒为参比,重组抗原IgG ELISA检测上述标本的敏感性、特异性和符合率分别为98.1%、94.1%和97.8%,IgM ELISA分别为84.6%、99.7%和99.1%;与细胞工程抗原相比,重组抗原诊断试剂的特异性高而敏感性略低。结论自制HHV 7 IgG和IgMELISA检测试剂敏感特异,可用于临床HHV 7感染的诊断及流行病学调查。     

Potent epitopes derived from Fasciola gigantica cathepsin L1 in peptide-based immunoassay for the serodiagnosis of human fascioliasis

A peptide-based enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for its diagnostic ability to detect human IgG antibodies against Fasciola gigantica cathepsin L1. Two previously identified B-cell epitopes of cathepsin L1 were synthesized as single synthetic peptides (acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide and acetyl-DKIDWRESGYVTELKDQGNC-carboxamide) and their diagnostic potential was evaluated. The peptide-based ELISA was compared with an indirect ELISA with crude excretory-secretory products or with partially purified specific 27-kDa (FG27) antigen from adult F. gigantica. In an analysis of the sera of 13 patients infected with F. gigantica, 212 patients with other parasitic infections, 32 patients with cholangiocarcinoma, and 57 healthy controls, the sensitivity, specificity, accuracy, and positive and negative predictive values of this peptide-based ELISA with both peptides had the same performance and were shown to be 100%, 97.3%, 97.5%, 61.9%, and 100%, respectively. When 4 different ELISAs were compared, the results revealed that the specificity, sensitivity, accuracy, and negative predictive values of all antigens were similar except for the positive predictive value that was highest in the ELISA with the FG27 antigen. These results demonstrated that peptide antigens can be used in the serodiagnosis of human fascioliasis with the additional advantage that they are relatively cheap and easy to produce. This rapid, highly sensitive and specific peptide-ELISA has the potential to be used in future large-scale prevalence surveys throughout Southeast Asia.     

Serodiagnostic applicability of recombinant antigens of Clonorchis sinensis expressed by wheat germ cell-free protein synthesis system

We evaluated the diagnostic applicability of recombinant proteins from Clonorchis sinensis, the human liver fluke. Four recombinant proteins, 7-kDa protein (Cs7P), 28-kDa cysteine protease (Cs28CP), and 26- and 28-kDa glutathione s-transferases (Cs26GST and Cs28GST), were expressed by wheat germ cell-free protein synthesis system. In ELISA, crude antigen showed the highest sensitivity (92.7%). However, sensitivities of r7P (47.3%), r28CP (30.9%), r26GST (21.8%), and r28GST (14.5%) were dramatically lower. The overall specificities of the crude antigen, r7P, r28CP, r26GST, and r28GST, were 100%, 94.5%, 96.7%, 94.5%, and 98.9%, respectively. Taken together, r7P and r28CP showed moderate sensitivities and high specificities, whereas r26GST and r28GST revealed low sensitivities and high specificities. We demonstrated that recombinant antigens, when used as a single antigen for ELISA, are not sensitive enough to diagnose clonorchiasis. Cocktail or chimeric antigens may be useful to increase the sensitivity of each antigen and may improve the serodiagnosis of clonorchiasis.     

Echinococcus granulosus in Jordan: assessment of various antigenic preparations for use in the serodiagnosis of surgically confirmed cases using enzyme immuno assays and the indirect haemagglutination test

The Enzyme linked immunosorbent Assay (ELISA), indirect haemagglutination (IHA), and immunoblot techniques (IB) were used for the serodiagnosis of surgically confirmed cystic echinococcosis (CE) caused by the tapeworm Echinococcus granulosus. Antigens used for the detection of IgG or total antibodies included crude sheep hydatid fluid (CSHF), autoclaved antigen B (AAB), boiled antigen B (BAB), and homogenate protoscoleces antigen (HPA). The overall sensitivity of the ELISA and IHA tests used for the serodiagnosis of 57 surgically confirmed human cases was 91.2% and 68.4%, respectively. The sensitivity of both tests was comparable in groups whose sera were collected one week before surgery and up to one year after surgery at 95.8% and 87.5%, respectively. In contrast, the sensitivity of the ELISA was significantly higher than that of IHA for sera of patients collected after one year of surgery. There was a positive correlation (r = 0.61) between the titers of antibodies detected by the ELISA and IHA. Using the IB technique, antigen B fractions (8/12, 16, and 24 KDa) were detectable by sera of 68.4% using either CSHF or AAB, 49.1% using BAB and 22.8% using HPA as detecting antigens. The overall sensitivity of the three AgB fractions was identical or similar to that of the 8/12 KDa fraction alone, indicating that the detection of the latter fraction is sufficient for the serodiagnosis of CE infection in humans. In conclusion, the ELISA is the test of choice for the serodiagnosis of CE and the follow up of cases following surgery using CSHF as an antigen. The IB test is a confirmatory test when antigen B fractions of CSHF or AAB are detected.     

Comparative study of three different mycobacterial antigens with a novel lipopolysaccharide antigen for the serodiagnosis of tuberculosis

Demonstration of Mycobacterium tuberculosis in a smear or culture is the most reliable method for diagnosing tuberculosis (TB). In the last 10 years, several enzyme-linked immunosorbent assays (ELISAs) based on mycobacterial antigens (such as antigen 60, 38 kDa antigen, and antigen Kp90) have been used for the rapid diagnosis of TB. In this study, we report the isolation of an immunodominant lipopolysaccharide (LPS) antigen from M. tuberculosis H(37)Rv, which can be used for the serodiagnosis of TB. The LPS antigen was compared with three commercially available mycobacterium-specific antigens for the detection of TB. The antigens were evaluated using serum samples obtained from 59 Indian patients (19 patients with active pulmonary TB, 20 with extrapulmonary TB, and 20 with nontuberculous pulmonary disease) and 20 healthy adults. Antigen 60 IgG (sensitivity 89%, specificity 97%) and LPS (sensitivity 84%, specificity 97%) were more sensitive and specific than 38 kDa antigen IgG (sensitivity 79%, specificity 97%) and Kp90 IgA (sensitivity 82%, specificity 40%). These results indicate that the LPS antigen can be used as a sensitive tool for the serodiagnosis of TB and could be utilized to develop an ELISA for the screening of patients for TB.     

Comparative sensitivity of six serological tests and diagnostic value of ELISA using purified antigen in hydatidosis

Most serodiagnostic techniques have been evaluated for diagnosis of cystic hydatid disease caused by Echinococcus granulosus. Each, to varying degrees, has been shown to give false results, with considerable variation between laboratories. The comparative study was made concerning the sensitivity of the immunodiagnostic methods based on 58 sera from hydatid disease with different cyst locations. Latex agglutination, immunoelectrophoresis (IEP), and specific IgE, IgG enzyme-linked immunosorbent assay (ELISA) tests were studied. Specific IgG ELISA AgB (antigen B-rich fraction) was the most sensitive test (96.5%) and the least sensitive tests were specific IgE ELISA (24.1%) and IEP (25.8%). The low sensitivity of these two tests was due partly to the low reactivity detected in the sera of patients with lung hydatidosis. Initial laboratory studies showed purified antigens to be preferable to crude cyst fluid, regardless of the type of test used. For this reason, we evaluated the sensitivity and specificity of ELISA by using the purified antigen-B-rich fraction. In all, 117 sera were examined: 78 sera from patients with hydatidosis surgically confirmed, 15 sera from healthy control subjects, and 24 sera from patients with diseases other than hydatidosis. The method gave good results: 93.5% sensitivity, 89.7% specificity, and 92.3% diagnostic efficacy.     

重组抗原联合用于弓形虫病IgG免疫诊断的研究

目的以纯化的重组弓形虫RH株主要表面抗原SAG1、次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶（HXG-PRT）和腺苷激酶（AK）作为诊断抗原，建立间接ELISA法检测抗弓形虫IgG抗体。方法诱导表达本实验室保种的SAG1／pET28a／BL21、HXGPRT／pET28a／BL21和AK／pET28a／BL-21，获得rSAG1、rHXGPRT和rAK三种重组抗原。分别用不同浓度的上述抗原包被聚苯乙烯酶标板，检测疑似弓形虫感染者血清。结果混合重组抗原ELISA法的最佳rSAG1包被浓度为500ng／ml、rHXGPRT和rAK均为200ng／ml；人血清稀释度为1：20；血清1：200稀释仍示阳性；特异性试验的抑制率为72．36％，变异系数11．2％。结论成功表达了弓形虫三种重组诊断抗原，并应用组合抗原（rSAG1＋rHXGPRT＋rAK）建立了ELISA检测方法，具有高度特异性和敏感性，本试剂具有弓形虫病辅助诊断价值。     

A beta-turn rich oats peptide as an antigen in an ELISA method for the screening of coeliac disease in a paediatric population

BACKGROUND: ELISA methods for the measurement of IgA antigliadin antibodies (AGA), both home-made and commercial systems, routinely employ wheat gliadin fractions as coating antigens. We investigate the sensitivity and specificity for CD diagnosis of a new ELISA method using a highly immunoreactive beta-turn rich gamma3-avenin peptide as an alternative coating antigen. METHODS: The assay was standardized with antihuman IgA peroxidase-conjugated as the second antibody. Alternatively, an ELISA based on the use of protein A-peroxidase was assayed to measure both IgG plus IgA antibodies. Sixty-three sera from healthy controls were analyzed to establish the system's cut-off point. Sera from 103 coeliac and from 65 noncoeliac children were tested; for diagnosis purposes, a small intestinal biopsy had been performed in all of them. RESULTS: For the IgA class antibodies assay a high sensitivity and specificity of 90.3% and 98.5%, respectively, was obtained, comparable to those achieved for IgA antiendomysium antibodies (EmA) with the same sera. CONCLUSIONS: In view of the high sensitivity and specificity obtained together with water solubility of the peptide and easiness for large-scale reproducible synthesis, the new AGA IgA avenin peptide ELISA represents a significant improvement in CD diagnosis in comparison with conventional established AGA IgA ELISA using crude gliadins as coating antigens.     

Particle agglutination assay for sm antibodies using purified sm antigen

Sm antibodies are specific serum markers for the diagnosis of systemic lupus erythematosus (SLE) and are identified by immunodiffusion (ID), counterimmunoelectrophoresis (CIE), and passive hemagglutination (PHA) in the clinical laboratories. These current methods have certain disadvantages of sensitivity, specificity, and complexity. In this report, Sm antigen free from other nuclear antigens was purified. Synthetic particles were coated with Sm antigen and used to test for Sm antibodies in patient's sera. The particle agglutination test was specific for only Sm antibodies among different antinuclear antisera from patients with systemic rheumatic diseases. The particle agglutination test had the same sensitivity and specificity as the enzyme-linked immunoassay (ELISA) test for Sm and RNP antibodies. The correlation coefficient between the ELISA quantitation and the titers obtained by particle agglutination was 0.82. The particle agglutination test can be used for rapid screening and titer determination of anti-Sm antibodies.     

重组泡球蚴主要表面抗原在棘球蚴病免疫诊断中的效果

目的 研究非融合表达重组抗原用于棘球蚴病免疫诊断的效果。方法 将泡球蚴主要表面抗原基因编码序列克隆于原核非融合表达载体pQE30（＋）并进行表达。以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）及免疫印迹（WB）试验对重组抗原鉴定。亲和层析纯化重组抗原，用于酶联免疫吸附法（ELISA）检测患者血清特异性抗体。结果 SDS—PAGE及WB分析显示泡球蚴主要表面抗原基因在大肠埃希菌内得到了高效非融合表达。以此重组抗原进行ELISA试验，检测棘球蚴病68例，阳性率达97．1％，检测囊虫等其他感染性疾病80例及健康人血清20份，阴性符合率达98％。结论 泡球蚴主要表面抗原基因在大肠埃希菌中获得了高效非融合表达，以该重组抗原建立的ELISA试验，对棘球蚴病诊断具有较高的敏感性和属特异性。     

Evaluation of vero cell lysate antigen for the ELISA of flaviviruses

The vero cell lysate antigen for the enzymelinked immunosorbent assay (ELISA) of flaviviruses was evaluated for sensitivity, specificity including cross-reactions, and background by comparing with the standard ELISA. Human sera, in serial dilutions, were taken from subjects 14, 35, and 210 days postvaccination with 17D antigen. Early after injection, high sensitivity (82.9%) was shown by the cell lysate antigen method. Late after infection, high sensitivity was achieved by the standard method (96.2% and 94%), with significant difference (P = 0.0001). However, sensitivity achieved by the cell lysate antigen method was also acceptable (91.7% & 88.9%). The cell lysate antigen method showed high specificity and low cross reactivity early after infection. At 35 days postvaccination, no difference in specificity was observed between the two methods, but higher cross-reactions were observed for the standard method. This pattern continued at 210 days postvaccination, with significantly higher cross-reactions with the standard ELISA. The optical density differences by the two methods did not show significant relationship with the serial dilutions of human sera. No difference was observed in early and late infections in the background values of the negative control (Western equine encephalitis) between the two methods. The ELISA by the cell lysate antigen, within the limits of the experiments done, was found to be a good replacement for the ELISA by the standard method. © 1993 Wiley-Liss, Inc.