Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.     

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。     

Enzyme-linked immunosorbent assay for the detection of Fusobacterium necrophorum antibody in bovine sera.

An enzyme-linked immunosorbent assay (ELISA) with HCl heat extracted antigen of Fusobacterium necrophorum was developed for the detection of antibody in bovine sera. Optimal conditions for antigen concentration and dilution of bovine serum were established. Pretreatment of positive reference serum with the antigens of different bacteria demonstrated no decrease, whereas the serum pretreated with F. necrophorum antigens revealed a decrease in the ELISA values. The apparent difference in ELISA values was observed between the sera derived from cattle infected and not infected with Fusobacterium necrophorum. These findings indicate that the ELISA detects the antibody to F. necrophorum in bovine sera.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

Detection of antibodies to Neospora caninum in cattle by enzyme-linked immunosorbent assay with truncated NcSRS2 expressed in Escherichia coli

The surface antigen 1-related sequence 2 of Neospora caninum (NcSRS2) is considered as an immunodominant antigen. In this study, the gene encoding truncated NcSRS2 (NcSRS2t) lacking an N-terminal signal peptide and C-terminal hydrophobic regions was expressed in Escherichia coli, and its diagnostic potential in an enzyme-linked immunosorbent assay (ELISA) was evaluated. ELISA could discriminate clearly between known N. caninum-positive and -negative sera from cattle. Field serum samples collected from cattle in Brazil were examined for the diagnosis of N. caninum infection using ELISA. Of the 197 samples analyzed, 64 (32.5%) samples were positive for antibodies to N. caninum. Of the 64 ELISA-positive samples, 58 (90.6%) were confirmed as positive by Western blot analysis with whole-parasite antigens. These results suggest that ELISA with recombinant NcSRS2t is an effective method for diagnosis of N. caninum infection in cattle.     

鸭瘟病毒gB蛋白N端主要抗原域的表达及间接ELISA检测

在分析鸭瘟病毒gB蛋白抗原性的基础上,设计一对引物克隆gB蛋白N端抗原性较好的抗原域编码基因.将克隆的基因定向插入pET-32a的EcoR Ⅰ和HindⅢ之间,构建了gB蛋白主要抗原域原核表达载体pET-gB1.将pET-gB1质粒转化BL(21)宿主菌后,对培养和表达条件进行了优化,实现了DPV gB蛋白主要抗原域的高效表达.免疫印迹试验表明获得的表达产物具有良好的反应原性.应用His·Bind亲和层析柱纯化重组DPV gB蛋白,以纯化的重组gB1蛋白作为检测抗原,初步建立了检测鸭瘟病毒抗体的igB1-ELISA.结果表明,抗原的最佳包被浓度为6.5μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:待检血清OD490＞0.4,且待检血清OD490/阴性血清OD490＞2.应用igB1-ELISA对鸭血清样品进行检测,结果表明igB1-ELISA与全病毒包被的iDPV-ELISA符合率达到95.6%.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

猪和牛轮状病毒抗原和抗体的检测

应用ELISA直接双抗体夹心法检查轮状病毒抗原,24份仔猪和29份犊牛的腹泻粪样,分别有12和16份阳性。用病毒RNA电泳分析检查阳性粪样,各出现两种病毒RNA电泳型,用中和试验检查17份成年牛和16份成年猪血清,分别有16和15份病毒抗体阳性。将其与ELISA间接法和结合法进行了比较。     

鼠疫F1抗原和V抗原双组分候选疫苗的免疫学评价

目的通过由提取的鼠疫F1抗原和重组鼠疫V(rV)抗原组成的鼠疫候选疫苗免疫豚鼠,对其免疫效果进行评价。方法将豚鼠随机分成5个试验组,在免疫后不同时间点采血进行抗体检测、MTT法淋巴细胞增殖试验以及皮肤迟发型超敏反应(DTH)检测。结果抗体检测结果显示,双组分鼠疫候选疫苗能诱导较强的体液免疫应答;MTT细胞增殖结果显示,脾脏淋巴细胞特异性增殖不明显;中剂量组、高剂量组针对F1和rV抗原DTH阳转率均为100%。结论双组分鼠疫候选疫苗能诱导豚鼠体液免疫和细胞免疫应答,该疫苗有希望成为我国新一代鼠疫疫苗。     

Technical note: a rapid diagnostic test detects plague in ancient human remains: an example of the interaction between archeological and biological approaches (southeastern France, 16th-18th centuries)

A rapid diagnostic test (RDT) that detects Yersinia pestis F1 antigen was applied to 28 putative plague victims exhumed from seven burial sites in southeastern France dating to the 16th-18th centuries. Yersinia pestis F1 antigen was detected in 19 of the 28 (67.9%) samples. The 27 samples used as negative controls yielded negative results. Soil samples taken from archeological sites related to both positive and negative samples tested negative for F1 antigen. The detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a preliminary retrospective diagnosis of Y. pestis infection in human remains. The high specificity and sensitivity of the assay were confirmed. For two sites positive to F1 antigen (Lambesc and Marseille), Y. pestis-specific DNA (pla gene) had been identified previously by PCR-sequence based analyses. Specifically, the positive results for two samples, from the Lambesc cemetery and the Marseille pit burial, matched those previously reported using PCR. Independent analyses in Italy and France of different samples taken from the same burial sites (Draguignan and Martigues) led to the identification of both Y. pestis F1 antigen and Y. pestis pla and gplD genes. These data are clear evidence of the presence of Y. pestis in the ancient human remains examined in this study.     

Evaluation of immunodiagnostic antigens in the excretory-secretory products of Fasciola hepatica

The metabolic antigens of F. hepatica have been shown to be a source of potential immunodiagnostic antigens. We have fractionated F. hepatica excretory-secretory (ES) antigens by conventional gel filtration and HPLC, analyzed these fractions in PAGE, and evaluated their immunogenicity by ELISA with sera from experimentally infected rabbits to identify potential serodiagnostic antigens for fascioliasis. A fraction enriched in high molecular weight components of ca. 150-160 kDa was found to be very reactive with sera from early fascioliasis. This fraction was successfully adapted to the DOT-ELISA, where titers up to 1:16,000 still appeared visually as positive. Both acute and chronic fascioliasis sera also recognized, in the enzyme-linked immunoelectrotransfer blot technique (EITB), prominent 25-30-kDa polypeptides that have previously been shown to be recognized by infected rabbits, cows, and sheep. We have therefore employed conventional gel filtration and HPLC gel exclusion chromatography as a 1-step procedure to obtain fractions enriched in antigens recognized in early fascioliasis. In addition, these antigens have been successfully applied to a sensitive, visual immunodiagnostic technique that can be easily employed in field studies.     

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS).

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.     

Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods

An extract of Candida albicans was used as an antigen on microtitre plates in the enzyme-linked immunosorbent assay (ELISA) to measure IgM, IgG and IgA class antibodies in the sera of hospitalized patients. It was found that of these patient sera that reacted positively in Ouchterlony immunodiffusion (ID) when undiluted, 58% were also positive in the ELISA against the same antigen preparation. However, all the sera with an ID titre of 1:2 or higher were ELISA-positive, demonstrating especially IgG and IgA. Of the sera positive by counterimmunoelectrophoresis against somatic and metabolic antigens of C. albicans, 86% were positive by ELISA. Reactions in precipitin-negative sera, if they occurred, usually demonstrated IgM or IgA. The sera with high passive haemagglutination or indirect immunofluorescence titres against surface antigens of C. albicans were positive in the IgG and IgA assays, while approximately one third were positive in the IgM assay.     

Comparison of heterologous adult Brugia malayi and homologous Onchocerca volvulus antigen in the enzyme-linked immunosorbent assay (ELISA) for Guatemalan onchocerciasis

To determine the susceptibility of a heterologous filarial antigen for measuring Onchocerca volvulus antibodies, worms were compared using an enzyme-linked immunosorbent assay (ELISA) test. Control serum samples from helminth-free U.S. residents and from helminth-infected but filariae-free Salvadoran residents were tested and compared with serum obtained from microfilariae-positive and -negative Guatemalan residents living in an area of endemic onchocerciasis. The results showed that none of the sera from U.S. residents had positive O. volvulus ELISA titers (greater than or equal to 1:160); however, 8.51% (4/47) had positive B. malayi ELISA titers (greater than or equal to 1:640). The geometric mean titers with the B. malayi ELISA test were higher than with the O. volvulus ELISA test--in sera from 47 U.S. residents (1:219 vs. 1:49), from 108 Salvadoran residents (1:92 vs. 1:71), and from 145 microfilariae-negative (1:539 vs. 1:167) and 303 microfilariae-positive (1:1,270 vs. 1:561) Guatemalan residents. The B. malayi ELISA test exhibited slightly less sensitivity than the homologous O. volvulus ELISA test; nevertheless, a good correlation (r = 0.74) was found between the 2 test antigens, indicating that the B. malayi antigen could be used to measure O. volvulus antibodies.     

Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

Anti-thyroglobulin anti-idiotypic antibodies in sera of patients with Hashimoto's thyroiditis and Graves' disease

The objective of this study was to find naturally occurring anti-idiotypic (anti-Id) antibodies to anti-human thyroglobulin (anti-hTg) idiotype in sera of patients with autoimmune thyroid disease. Sera from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) and sera from normal subjects were tested for the presence of anti-Id antibodies against mouse anti-hTg monoclonal antibodies (McAb) in indirect ELISA and in indirect solid-phase RIA. Microtitration plates were coated with six McAb, five of them directed against different epitopes on hTg molecule, and then incubated with patients' sera. The bound antibody was detected with either peroxidase or 125I-labeled anti-human IgG. The specific positive reaction was observed in four of 40 patients with HT, in two of 26 patients with GD, in seven of 58 patients with RA, and in none of 20 normal subjects. The detected binding was due to the presence of anti-hTg anti-Id antibodies and not to Tg-anti-Tg circulating immune complexes, as the positive sera did not contain hTg when resolved on SDS-PAGE, nor did they bind to all anti-hTg McAb tested. The binding was dose dependent, and titers of anti-Id antibodies varied from 1:243 to 1:2187. The binding could be inhibited up to 50% by hTg, but not by the thyroid microsomal antigen, indicating that some of those anti-Id might represent the internal image of the antigen. Serum from the patient 3403, showing the strongest reactivity against McAb A-3, was chosen for IgG purification and F(ab')2 fragment isolation. The 3403 F(ab')2 fragment, but not the Fc fragment, was found to react specifically with four mouse anti-hTg McAb but not with the control mouse IgG. Thus, the obtained results permit the conclusion that anti-hTg anti-Id antibodies could occur naturally during the course of thyroid autoimmune disorders.     

Detection of Antibody against Fungal Glucosylceramide in Immunocompromised Patients: A Potential New Diagnostic Approach for Cryptococcosis

We have developed an ELISA to determine the value of anti-glucosylceramide antibody for the prediction of disseminated cryptococcosis in immunocompromised subjects and performed a clinical prospective study at the Medical University of South Carolina. The study enrolled a total of 53 patients who were free of active fungal diseases at the time of enrollment but at risk of developing one because they were all immunocompromised, e.g., (1) patients positive for HIV and (2) patients post- or awaiting solid organ transplantation. Among 53 patients enrolled, two patients developed invasive cryptococcosis, and in both patients, IgM anti-GlcCer was detected in sera using the ELISA at least 6 weeks prior to the clinical presentation of the brain disease. These results were corroborated by a cryptococcal antigen lateral flow assay, which was also positive in serum prior to the development of meningoencephalitis. However, a high number of positive results were also detected in patients with no evidence of cryptococcosis. This study highlights the potential utility of this new assay in early diagnostic testing algorithms for patients at risk for cryptococcosis, but further investigations are needed to validate the sensitivity and specificity of the glucosylceramide ELISA as a predictor of cryptococcosis.     

Development of an up-converting phosphor technology-based 10-channel lateral flow assay for profiling antibodies against Yersinia pestis

In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was developed to profile antibodies against Yersinia pestis. Ten expressed Y. pestis proteins were covalently conjugated with an up-converting phosphor particle to develop double-antigen sandwich immunochromatographic strips to detect corresponding antibodies. After optimization one by one, each strip was integrated into a TC-UPT-LF disc for simultaneously detection of different antibodies. A scanning biosensor was also developed to acquire the results. The performance of the TC-UPT-LF assay was evaluated by using standard samples and plague monkey serum samples. Fifty-one patient serum samples were detected by the TC-UPT-LF assay. The TC-UPT-LF disc could be stable for 10 days at 37 °C with an average CV of 10.3%. Its sensitivity and qualitative results are comparable to those of ELISA. Its linearity fitting coefficient of determination (R²) for different antibody detection is between 0.93 and 0.99. Besides F1 antibody, the LcrV and YopD antibodies also showed higher positive ratio than the other seven antibodies, as 100% (13/13) and 92% (12/13) in monkey sera and 86.3% (44/51) and 66.7% (34/51) in patient sera, respectively. It is suggested that the TC-UPT-LF assay has been successfully developed for multi-detection and LcrV and YopD can be the potential diagnostic markers of the plague.     

Evaluation of antibody responses in American visceral leishmaniasis by ELISA and immunoblot

American visceral leishmaniasis (AVL) is an important disease among children of northeast Brazil. In order to characterize antibody responses during AVL, sera of hospitalized patients were analyzed by ELISA and Western blot using a Leishmania chagasi antigen preparation. The ELISA was positive (absorbance greater than or equal to 0.196) at a serum dilution of 1:1024 in all patients at presentation, and fell to ward control levels over the following year. Only one of 72 control subjects tested positive, and that donor had a sibling with AVL. Immunoblots of the patients' sera recognized multiple bands, the most frequent of which were at approximately 116 kDa, 70 kDa, and 26 kDa. Less frequently observed were bands at approximately 93 kDa, 74 kDa, 62 kDa, 46 kDa and 32 kDa. The ELISA responses and patterns of banding were distinctive for AVL, and could be used to differentiate patients with AVL from those with Chagas' disease or cutaneous leishmaniasis. Sera from six AVL patients followed for up to six weeks after treatment identified no new bands. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of surface iodinated parasite proteins showed one major band and four minor bands, whereas SDS-PAGE of biotinylated parasite proteins revealed a banding pattern similar to those of patient sera. AVL appears to produce characteristic immunoblot patterns which can be used along with a sensitive screening ELISA to diagnose AVL.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.