Comparative evaluation of two commercially available antigen enzyme immunoassays (EIA) for the detection of Legionella pneumophila urinary antigen in frozen non-concentrated urine samples

We evaluated two commercial enzyme immunoassay kits, Binax EIA (for detection of soluble antigen of Legionella pneumophila serogroup 1) and Biotest EIA (for detection of antigens of Legionella pneumophila serogroups and other Legionella spp.) in order to introduce this test routinely for the diagnosis of Legionnaires' disease (LD) in our Laboratory. Frozen non-concentrated urine samples belonging to 45 patients with and without LD were tested. The sensitivity of Binax EIA and Biotest EIA was 47.4% and 42.1% respectively, the specificity was 95% by both tests. Biotest did not detect antigen from a patient with culture-proven infection of L. pneumophila serogroup 6. The detection of urinary antigen by both EIA tests is a useful tool for rapid diagnosis of LD, especially when samples are unavailable for culture; the sensitivity may be increased if the assay is performed on unfrozen and concentrated samples.     

Cross-reactivity and sensitivity of two Legionella urinary antigen kits, Biotest EIA and Binax NOW, to extracted antigens from various serogroups of L. pneumophila and other Legionella species

Legionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non-serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in-vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L pneumophila SG 1 antigens, but reacted to different sets of non-SG I L. pneumophila with different sensitivity. No cross-reactivity was observed to Legionella species other than L. pneumophila.     

鼠疫菌F1抗体/抗原酶联免疫诊断试剂盒的应用评价

目的对兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒进行临床应用评价。方法采用双抗原/抗体夹心酶联免疫吸附试验(ELISA)、间接血球凝集试验(IHA)、胶体金免疫层析试验(GICA)3种方法的诊断试剂对比检测云南省地方病防治所中心实验室保藏的和现场采集的血清样品和脏器样品,对血清样品做鼠疫菌F1抗体检测,对脏器样品做鼠疫菌F1抗原检测。结果在358份血清样品中,ELISA试剂检出F1抗体阳性52份(14.52%),IHA试剂检出阳性37份(10.34%),GICA试剂检出阳性45份(12.57%)。ELISA与IHA试剂的符合率为95.23%,与GICA试剂的符合率为96.92%。经统计学χ2检验,ELISA试剂检出F1抗体阳性率高于IHA试剂(χ2=11.53,P=0.000 7),与GICA试剂检出的差异无统计学意义(χ2=3.27,P=0.070 4)。进一步分析滴度差值频数,ELISA试剂检测人血清的敏感性高于IHA试剂的样品占87.5%。在117份脏器样品中,3种试剂均检出F1抗原阳性15份(12.82%),符合率100%。滴度差值频数比较,ELISA试剂检测敏感性高于反向间接血球凝集试验(RIHA)试剂的样品为78.57%。结论兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒性质特异,其敏感性优于IHA试剂盒和GICA试剂条,值得在鼠疫的监测和快速诊断中推广应用。     

基于环介导恒温扩增技术的大肠杆菌O157:H7快速检测试剂盒的评价

【目的】评价基于环介导恒温扩增技术(LAMP)的大肠杆菌O157:H7(Escherichia coli O157:H7)快速检测试剂盒的实效性。【方法】测定快速检测试剂盒的特异性、灵敏度、重复性、保质期以及运输稳定性,并与传统方法对比检测实际样品。【结果】大肠杆菌O157:H7标准菌株样品均检测为阳性,非大肠杆菌O157:H7标准菌株样品均检测为阴性,未发现有交叉反应;试剂盒最低检验限为29 CFU;该试剂盒的特异性、灵敏度及准确度与传统方法相比具有较高的一致性;试剂盒对高菌量目标菌和阴性菌样品的检测重复率均为100%,对低菌量目标菌样品的批间检测重复率为94%。试剂盒可在4°C保存9个月以上,并且可进行变温储存72 h以上。【结论】该试剂盒特异性好,灵敏度高,重复性好,储存方便,检测结果稳定、可靠,适用于对食品中大肠杆菌O157:H7的检测需求。     

人心脏型脂肪酸结合蛋白检测试剂盒的临床评价

目的评价人心脏型脂肪酸结合蛋白(H-FABP)检测试剂盒(胶体金法)在急性心肌梗死(AMI)诊断中的价值。方法采用平行、盲法、对照的对比试验设计,比较其试验产品和对比产品对诊断AMI的敏感性、特异性、准确性。结果共测定240份临床血液标本。试验产品和对比产品的阳性符合率为100%,阴性符合率为96.15%,总符合率为97.92%。对比产品和试验产品结果不一致的5例标本以临床诊断结果为标准进行验证后,试验产品与临床诊断结果的阳性符合率为100%,总符合率为100%。采用Kappa检验考核两种产品测定结果的一致性,Kappa指数为0.958。经McNamara's test分析,两产品之间无差异,χ2=3.20,P>0.05。结论试验产品显示出较好的诊断价值,可以作为AMI早期诊断标志物,试验产品与对比产品等效。     

五种国产与进口小鼠病毒ELISA抗体检测试剂盒的比较研究

目的评价国产小鼠病毒抗体ELISA检测试剂盒。方法选择国产与进口小鼠淋巴细胞脉络丛脑膜炎病毒（LCMV）、肝炎病毒（MHV）、仙台病毒（SV）、腺病毒（MAV）、细小病毒（MPV）ELISA抗体检测试剂盒,进行敏感性、特异性、精密性、稳定性、可信度试验比较。结果国产与进口试剂盒：同种试剂盒之间灵敏度相差最低为2倍,差异显著（P〈0.05）,最高为16倍,差异极显著（P〈0.01）;特异性试验显示每种试剂盒,与其他4种病毒均无交叉反应;精密性试验显示5种试剂盒批内平均变异系数均小于10%;稳定性试验显示5种试剂盒相对偏差均小于25%;分别选择已知36份小鼠血清进行检测,国产和进口LCMV、MHV、SV、MPV符合率均为100%;国产MAV符合率为86.1%,进口MAV符合率均为100%,二者之间差异极显著（P〈0.01）。结论除国产MAV试剂盒敏感性、可信度低于进口外,国产LCMV、MHV、SV、MPV试剂盒与进口同种试剂盒相比,在敏感性、特异性、精密性、稳定性和可信度方面均良好。     

新型HCV EIA诊断试剂盒的研制

丙型肝炎病毒（ＨＣＶ）基因组结构区核壳蛋白（Ｃ）区抗原、膜蛋白Ｅ１和Ｅ２区抗原,以及非结构区ＮＳ３－ＮＳ５区抗原的区段,已经在原核细胞中获得有效的表达。同时,相应区段中的优势抗原表位肽也经化学合成法大规模地制备。ＨＣＶ基因组上各区段抗原性的分析发现,由Ｃ区和ＮＳ３区分别编码的Ｃ抗原和Ｃ３３ｃ抗原是ＨＣＶ基因组上两个优势抗原区段。其相应的抗体出现早（感染后６周可检出抗Ｃ３３ｃ抗体）,阳转率高（约９９％阳性检出率）,特异性和重复性均优于其它区段抗原。以中国人ＨＣＶ的Ｃ３３ｃ重组蛋白和分支状合成肽ＭＡＰ－Ｃ－１９为复合抗原,研制了适合我国抗ＨＣＶ抗体检测的新型丙型肝炎病毒酶免疫测定（ＨＣＶＥＬＡ）诊断试剂盒。它同当代美国Ａｂｂｏｔｔ／ＵＢＩＨＣＶＥＬＡ诊断试剂的符合率约９８％,同加拿大ＹＥＳ公司ＨＣＶＥＩＡ诊断试剂的符合率约９７．８％,阳性检出率提高了约２％,３次重复性达１００％,表明其特异性、敏感性和重复性均达到了当代第二代ＪＣＶＥＬＡ诊断试剂的水平。我国人群中抗ＨＣＶ抗体的分布情况为：正常人群的检出率１％－２％;外科类住院病人检出率约２８．８％;肝炎患者抗ＨＣＶ阳性率为３４．４％,慢活肝、肝硬化和重症肝炎患者     

BDV核蛋白FQ RT-PCR试剂盒的评价与应用

为评价博尔纳病病毒(Borna disease virus,BDV)核蛋白荧光定量PCR(FQRT-PCR)试剂盒的各项指标,比较分子信标探针相对普通探针的优势,并了解其实际检测效果,本课题组使用BDVOL持续感染细胞株、非BDV病毒序列转染的OL细胞、正常的OL细胞,对BDVRT-PCR试剂盒的敏感性、特异性、重复性和稳定性进行评估,同时检测部分临床病人和动物外周血液RNA。实验结果显示:试剂盒可以检测出的病毒RNA最低浓度为2.5×101,相当于1个病毒拷贝数,无非特异检出;不同批次的试剂盒的检测结果变异系数小于0.7;加速破坏的试剂盒和正常试剂盒检测结果之间变异系数在2以内;对临床病人检测阳性率为3.6%,对动物检测阳性率为4.2%(猪)和1.5%(马)。可见该试剂盒重复性和稳定性均好;敏感性、特异性优于普通探针试剂盒,是BDV基础研究、流行病学调查和临床检测的良好工具。     

Usefulness of immunoenzyme diagnostic kits and latex tests for isolation of rotaviruses

The aim of this study was to compare characteristic parameters of 4 diagnostic kits available in Poland-immunoenzymatic Rotazyme II and Enzygnost kits and latex kits Rotalex and Slidex. Studies were performed on 67 samples of feces of children treated because of diarrhea. The sensitivity, specificity, frequency of positive tests, false positives and false negatives and the accuracy of the tests under evaluation were determined. The results obtained were further verified using a reference test electrophoresis of RNA of rotavirus. The highest sensitivity was found for Enzygnost, Rotazyme II and Rotalex, 97%, 92% and 90%, respectively, and the lowest for Slidex- 79%, while the specificity was higher for latex kits than for immunoenzymatic kits. The accuracy of the results was highest for Rotalex kit (92%), next for Enzygnost kit (88%), Rotazyme (87%), and Slidex (84%). The significant correlation between OD value readings in spectrophotometer in Rotazyme II kit and the results of visual reading in latex test was found. All tested kits were found to be useful for diagnostic purposes. Rotalex kit due to the high accuracy of the results obtained, methodological simplicity, short time of testing and relatively low price could be a based test in hospital laboratories.     

丙型肝炎病毒抗体化学发光免疫检测方法的建立及其临床应用简

目的：建立丙型肝炎病毒（rmv）抗体化学发光免疫检测方法,并分析其临床应用价值。方法：应用基因工程重组的HCV抗原包被微孔板,以辣根过氧化物酶标记的羊抗人IgG为二抗,并结合鲁米诺化学发光底物系统,建立HCV抗体化学发光免疫检测方法;应用HCV抗体诊断试剂国家参考品分析所建立方法的特异性、灵敏度、稳定性和精密性,并-9北京万泰公司的ELISA试剂盒同时检测临床血清样本350份,比较检测结果。结果：检测结果符合国家参考品质量标准。批内变异系数5．1％。6．6％,批间变异系数9-5％;试剂盒置37℃考核3d,其稳定性良好;与万泰公司的ELISA检测结果对照,阳性符合率分别为99．0％,阴性符合率分别为100％,总符合率为99．4％;Kappa值为0．986,一致性强度最强。结论：建立了特异、敏感和稳定的HCV抗体化学发光免疫检测方法,适用于HCV感染的批量筛查,具有较大的临床应用价值。     

Improved sensitivity and specificity of enzyme immunoassays with P1-adhesin enriched antigen to detect acute Mycoplasma pneumoniae infection

An in-house P1-enriched (168-kDA protein) Mycoplasma pneumoniae antigen preparation was compared in IgG, IgA and IgM enzyme immunoassays (EIAs) to the respective EIAs employing crude antigen lysate, antigen prepared by Triton X-114 partition and two commercial antigens, one of which was an ether-extracted antigen and the other a P1-enriched antigen. In addition, three commercial kits from Sanofi Pasteur, Novum Diagnostica and Savyon Diagnostics were also assessed for comparison. Diagnostic sensitivity was studied with paired samples from adults (n=37) with acute respiratory illness interpreted as acute, recent or past infection to M. pneumoniae on the basis of the results of complement fixation test (CFT). If the consensus of at least two methods is taken as the true positive for acute infection, the diagnostic sensitivities of combined IgG and IgM EIAs were 100% for the Platelia(R), Sero MP and in-house EIAs whereas for the Novum EIAs and CFT- 97% and 74%, respectively. Moreover, the sensitivity of the P1-enriched antigen was proven superior on the basis of systematically highest OD(405 nm) ratios between convalescent and acute serum samples.Analytical specificity was studied by screening serum samples from 92 Finnish blood donors and 111 serum samples from cord blood. Diagnostic specificity was studied in a blind testing of 30 paired serum samples from infants with pneumonia of variable etiology. No single misinterpretation of acute infection from the group of samples with other respiratory diseases did occur.The present study confirmed and extended the earlier observations of the usefulness of P1-enriched antigen for reliable serologic diagnosis of acute M. pneumoniae infection.     

Binax NOW® Streptococcus pneumoniae test of middle ear fluid for detecting causative pathogens in children with acute otitis media

We investigated rapid diagnosis of acute otitis media, (AOM) with the Binax NOW® Streptococcus pneumoniae test kit. Middle ear fluid specimens were obtained from 38 children with AOM (mean age: 1.1 years). Binax NOW® demonstrated 100% sensitivity and 72% specificity, suggesting it is a useful auxiliary test for AOM.     

Evaluation of an indigenous ELISA for diagnosis of Johne’s disease and its comparison with commercial kits

Country lacks sensitive and indigenous diagnostic kits for the screening of goats and sheep against Johne’s disease. Therefore an indigenous ELISA kit was developed using protoplasmic antigen from native Mycobacterium avium subspecies paratuberculosis ‘Bison Type’ strain of goat origin (Kit 1). In the present study, kit 1 and two commercial kits (kit 2 and 3) were evaluated with respect to ‘Gold Standard’ fecal culture in 71 animals (55 goats and 16 sheep). Kit 1 using indigenous antigen (protoplasmic antigen) was sensitive at very low concentration (0.1 μgm / well) as compared to purified commercial protoplasmic antigen (4 μgm / well) used in kit 2, in the Type 1 reactors (strong positive as positive). Screening of 71 animals by fecal culture detected 38.0% animals (goats-40.0%, sheep-31.2%) as positive (clinical shedders of bacilli) from these farm animals. Of the farm animals located at Central Institute for Research on Goats, herds of goat were endemic whereas, sheep flocks were comparatively resistant to Johne’s disease. The 29.5 and 61.9, 15.4 and 57.7 and 4.2 and 14.0% animals (goats and sheep) were in the category of sero-reactors type 1 and 2 of the ELISA kits 1, 2 and 3, respectively. In the type 1 sero-reactors, sensitivity and specificity of kit 1, 2 and 3 was 53.7 and 86.0, 17.8 and 86.0 and 3.5 and 94.7%, respectively. Indigenous ELISA test (kit 1) was significantly superior for the screening of goatherds and sheep flocks against JD as compared to commercial ELISA kits (Kit 2 and 3). In comparison to kit 2 and 3, kit 1 had highest sensitivity, comparable specificity and substantial to nearly perfect proportional agreement (Kappa Scores) with respect to ‘Gold standard’ fecal culture in goats and sheep. Disease being endemic in herds and flocks screened using ELISA kits, Type I sero-rectors had better correlation with fecal culture in comparison to Type II sero-reactors therefore, used for estimation of sero-prevalence. Newly developed Indigenous ELISA kit was simple, inexpensive, sensitive and reliable for screening of goats and sheep population against Johne’s disease. The study reports high prevalence of Johne’s disease in farm goatherds and sheep flocks, using sensitive tests (fecal culture and ELISA kit). Results of Type 1 reaction in kit 1 were optimally correlated with culture and were good for estimating the sero-prevalence. For controlling Johne’s disease in endemic herds initial removal of the animals in strong positive category (Tyep 1 reactors), may help to remove heavy shedders.     

重组人巨细胞病毒(HCMV)优势表位嵌合抗原的构建表达以及捕获法检测IgM抗体

勘误：重组人巨细胞病毒(HCMV)优势表位嵌合抗原的构建表达以及捕获法检测 IgM 抗体     

Comparison of three ELISA kits for the differentiation of foot-and-mouth disease virus-infected from vaccinated animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals. Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits, Ceditest® FMDV-NS (Ceditest® kit), UBI® FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI® kit) and a FMDV 3ABC-I-ELISA kit developed at the Lanzhou Veterinary Research Institute. The test parameters (sensitivity and specificity) of the three kits were determined, and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits. The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest® kits was 98.05%, and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI® kits was 94.4%; the sensitivity of both Ceditest® and FMDV 3ABC-I-ELISA kit was 100%. However, the sensitivity of the UBI® kit was only 81.8%. With sera from naive or vaccinated non-infected animals, the specificity of all tests exceeded 90%.     

Comparison of Three ELISA Kits for the Differentiation of Foot-and-mouth Disease Virus-infected from Vaccinated Animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals.Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits,Ceditest(R)FMDV-NS (Ceditest(R) kit),UBI(R) FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI(R) kit) and a FMDV 3ABC-I-ELISA kitdeveloped at the Lanzhou Veterinary Research Institute.The test parameters (sensitivity and specificity) of the three kits were determined,and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits.The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest(R) kits was 98.05%,and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI(R) kits was 94.4%; the sensitivity of both Ceditest(R) and FMDV 3ABC-I-ELISA kit was 100%.However,the sensitivity of the UBI(R) kit was only 81.8%.With sera from naive or vaccinated non-infected animals,the specificity of all tests exceeded 90%.     

Toxoplasma gondii: expression pattern and detection of infection using full-length recombinant P35 antigen

A complete P35 surface antigen of Toxoplasma gondii was sequenced (GenBank ). Immunoblot found that it reacted specially with T. gondii acute infected sera, and the recombinant P35 signal was specific for P35 antigen. The P35-GST protein was used as antigen to detect 125 sera samples by double-sandwich ELISA. P35-IgM positive rate in a chronic infected group, a persistent IgM positive chronic group, a recently seroconvered group and an acute infected group were 4% (1 out of 25), 16% (4 out of 25), 88% (22 out of 25), and 100% (25 out of 25), respectively. The sensitivity and specificity of the recombinant full-length P35 antigen were 100 and 96%, respectively. The detailed expression patterns of P35 antigen were studied in 36 IgM and IgG positive sequential samples from 10 recently seroconvered patients. Results showed that the P35-IgM positive rate decreased as the time after the first seroconversion increased. P35-IgM positive samples in the first, second, third, fourth, and fifth month after the first seroconversion test were 90, 78, 57, 50, and 33%, respectively. P35-IgG positive samples in the first, second, third, fourth, fifth, sixth, and seventh month after the first seroconversion test were 70, 100, 100, 100, 67, 100, and 100%, respectively. All samples were P35-IgM negative after the fifth month, and P35-IgG negative after the seventh month from seroconversion. P35-IgM existed the shortest time and was a more specific marker for T. gondii acute infection than P35-IgG, IgM, and IgG to whole tachyzoites antigens.     

广西人类免疫缺陷病毒感染者/艾滋病患者合并马尔尼菲篮状菌感染的特征及Mp1p抗原快速筛检的研究

为调查广西人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者/艾滋病(acquired immunodeficiency syndrome,AIDS)患者合并马尔尼菲篮状菌(Talaromyces marneffei,TM)感染的特征并评价TM Mp1p(一种甘露糖蛋白)抗原试剂...     

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria. To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit’s performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.