Simultaneous detection of three arboviruses using a triplex RT-PCR enzyme hybridization assay

Arboviruses represent a serious problem to public health and agriculture worldwide.Fast,accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surv...     

A Novel Immunochromatographic Test Applied to a Serological Survey of Japanese Encephalitis Virus on Pig Farms in Korea

Among vertebrate species, pigs are a major amplifying host of Japanese encephalitis virus (JEV) and measuring their seroconversion is a reliable indicator of virus activity. Traditionally, the hemagglutination inhibition test has been used for serological testing in pigs; however, it has several limitations and, thus, a more efficient and reliable replacement test is required. In this study, we developed a new immunochromatographic test for detecting antibodies to JEV in pig serum within 15 min. Specifically, the domain III region of the JEV envelope protein was successfully expressed in soluble form and used for developing the immunochromatographic test. The test was then applied to the surveillance of Japanese encephalitis (JE) in Korea. We found that our immunochromatographic test had good sensitivity (84.8%) and specificity (97.7%) when compared with an immunofluorescence assay used as a reference test. During the surveillance of JE in Korea in 2012, the new immunochromatographic test was used to test the sera of 1,926 slaughtered pigs from eight provinces, and 228 pigs (11.8%) were found to be JEV-positive. Based on these results, we also produced an activity map of JEV, which marked the locations of pig farms in Korea that tested positive for the virus. Thus, the immunochromatographic test reported here provides a convenient and effective tool for real-time monitoring of JEV activity in pigs.     

Evaluation of Rapid IgG4 Test for Diagnosis of Gnathostomiasis

Human gnathostomiasis is a parasitic disease caused by Gnathostoma nematode infection. A rapid, reliable, and practical immunoassay, named dot immuno-gold filtration assay (DIGFA), was developed to supporting clinical diagnosis of gnathostomiasis. The practical tool detected anti-Gnathostoma-specific IgG4 in human serum using crude extract of third-stage larvae as antigen. The result of the test was shown by anti-human IgG4 monoclonal antibody conjugated colloidal gold. The sensitivity and specificity of the test were both 100% for detection in human sera from patients with gnathostomiasis (13/13) and from healthy negative controls (50/50), respectively. Cross-reactivity with heterogonous serum samples from patients with other helminthiases ranged from 0 (trichinosis, paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis) to 25.0% (sparganosis), with an average of 6.3% (7/112). Moreover, specific IgG4 antibodies diminished at 6 months after treatment. This study showed that DIGFA for the detection of specific IgG4 in human sera could be a promising tool for the diagnosis of gnathostomiasis and useful for evaluating therapeutic effects.     

Rapid dot sputum and serum assay in pulmonary tuberculosis

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.     

人抗乙脑病毒IgG抗体检测试剂盒的研制和应用

乙脑病毒SA14-14-2株疫苗原液经β丙内酯灭活Sepharose 4FF纯化后作为包被抗原,制备阳性替代品,应用间接ELISA法检测人血清中乙脑病毒抗体。建立内部质量控制血清标准,比较蚀斑减少中和试验(PRNT)与ELISA的相关性。检测46份乙脑相关血清的结果与国内同类试剂进行比较,阳性符合率为93.1%,阴性符合率为89.5%。在咸安地区3万多名2~14岁人群中进行乙脑病毒IgG抗体水平普查,阳性率22.5%,与国内同类试剂的符合率为95.7%,使用效果很好。     

Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.     

Development of a chemiluminescent imaging assay for the detection of anti‐erythropoietin antibody in human sera

Measuring low amounts of anti‐erythropoietin antibodies (anti‐EPO Abs) is important to evaluate the therapeutic safety of recombinant human erythropoietin (rhEPO). In this work, a simple, sensitive and high‐throughput chemiluminescent (CL) imaging assay was developed for the detection of anti‐EPO Abs in human sera. The influence of several physicochemical parameters, such as coating conditions, incubation time, detergent concentration and exposure time, were investigated. A calibration curve was established and the range of quantitative detection was 0.12–13.91 ng/mL. The limit of detection (LOD, 3σ) for the CL‐imaging assay was 0.033 ng/mL. Compared to conventional colorimetric enzyme‐linked immunosorbent assay (ELISA), the LOD of the CL‐imaging assay is 50‐fold lower. The recoveries of anti‐EPO Abs in the fortified serum were in the range 87.1–116.9% using the present method, which highlighted the validity of the CL‐imaging assay system to accurately determine the anti‐EPO Abs in serum samples. CL‐imaging assay was used to evaluate the presence of anti‐EPO Abs in serum samples obtained from chronic renal failure (CRF) patients treated with rhEPO. Contrary to what was expected, the sera from CRF patients did not contain anti‐EPO Abs. Copyright © 2008 John Wiley & Sons, Ltd.     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

Detection of porcine parvovirus using a taqman-based real-time pcr with primers and probe designed for the NS1 gene

A TaqMan-based real-time polymerase chain reaction (PCR) assay was devised for the detection of porcine parvovirus (PPV). Two primers and a TaqMan probe for the non-structural protein NS1 gene were designed. The detection limit was 1 x 102 DNA copies/μL, and the assay was linear in the range of 1 x 102 to 1 x 10? copies/μL. There was no cross-reaction with porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The assay was specific and reproducible. In 41 clinical samples, PPV was detected in 32 samples with the real-time PCR assay and in only 11 samples with a conventional PCR assay. The real-time assay using the TaqMan-system can therefore be practically used for studying the epidemiology and management of PPV.     

Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.     

乙型脑炎病毒（JEV）ELISA检测方法的建立

目的建立猪乙型脑炎病毒（JEV）抗体ELISA检测方法。方法培养BHK21细胞,接种JEV病毒,制备BHK21正常抗原和JEV特异抗原,滴定酶结合物和抗原最佳工作浓度,并进行精密性、敏感性、稳定性、特异性实验。结果正常、特异抗原和酶结合物最佳工作浓度分别为0.2μg/mL、10μg/mL和1∶20000;正常、特异抗原批内变异系数分别为8.3%和6.4%,批间平均变异系数分别为9.7%和11.5%;检测灵敏度为1∶1280;与猪瘟病毒（CSFV）、猪细小病毒（PPV）均无交叉反应。稳定性试验相对偏差小于25%。结论建立的ELISA方法重复性、稳定性好,特异性、敏感性强。可用于猪JEV抗体的检测。     

应用ELISA法检测风疹病毒IgG抗体

实验证明,将0.1％脱氧胆酸钠制备的风疹病毒粗制抗原,用于ELISA法检测风疹病毒IgG抗体,效果较满意,方法的特异性好,与常规血凝抑制试验(HI)的相关性也好,所测抗体的几何平均值为HI的4倍。用本法初步调查了北京市不同年龄人群的风疹感染率,证明随年龄增长风疹感染率迅速上升,18岁以上人群达94％。检测河北省沧州地区孕妇的风疹IgG阳性率为99％。用於风疹病人的血清学诊断,获得较好结果。     

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).     

Comparison of JEV neutralization assay using pseudotyped JEV with the conventional plaque-reduction neutralization test

We previously reported the development of a neutralization assay system for evaluating Japanese Encephalitis Virus (JEV) neutralizing antibody (NAb) using pseudotyped-JEV (JEV-PV). JEV-PV-based neutralization assay offers several advantages compared with the current standard plaque-reduction neutralization test (PRNT), including simplicity, safety, and speed. To evaluate the suitability of the JEV-PV assay as new replacement neutralization assay, we compared its repeatability, reproducibility, specificity, and correlated its results with those obtained using the PRNT. These analyses showed a close correlation between the results obtained with the JEV-PV assay and the PRNT, using the 50% plaque reduction method as a standard for measuring NAb titers to JEV. The validation results met all analytical acceptance criteria. These results suggest that the JEV-PV assay could serve as a safe and simple method for measuring NAb titer against JEV and could be used as an alternative approach for assaying the potency of JEV neutralization.     

Detection of antibodies to Staphylococcus aureus Toxic Shock Syndrome Toxin-1 using a competitive agglutination inhibition assay

AIMS: To develop a competitive agglutination inhibition assay (CAIA) for the detection of anti-Toxic Shock Syndrome Toxin-1 (TSST-1) antibody in serum samples using a commercially available reverse passive agglutination assay (RPLA) kit. METHODS AND RESULTS: TSST-1 toxin and sera were incubated together, so that anti-toxin IgG would complex with the toxin. Latex particles sensitized with rabbit IgG anti-TSST-1 were added to test for un-complexed toxin. The sensitivity and specificity of the CAIA assay was determined relative to positive and negative ELISA results. The sensitivity (proportion of positive ELISA sera which tested positive by CAIA) was 66% whilst the specificity (proportion of ELISA negative sera which tested negative by CAIA) was 75%. Seven sera (14%) were negative by ELISA but positive for CAIA and 12 (18.8%) were positive for ELISA but negative for CAIA, suggesting some interference with the assays. Statistical analysis showed no significant difference between the methods in terms of the numbers of individuals testing positive (chi2, P = 0.04). CONCLUSIONS: The CAIA assay allowed detection of anti-TSST-1 within 18 h and was simple to read visually. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is a useful test for individual serum samples and a preliminary investigation for medical screening of suspected toxic shock syndrome and is applicable in situations where antibody assays are not routinely used for anti-TSST-1 and also where sophisticated equipment (e.g. microtitre plate reader) is not available.     

一种联合检测乙型肝炎病毒前S1抗原与核心抗原方法的建立及其与病毒核酸检测结果的一致性

本研究以与血清中HBV DNA含量高度相关的两种HBV抗原(前S1抗原与核心抗原)为靶标,建立了联合检测这两种HBV核酸相关抗原(NRAg)的双抗体夹心法ELISA试剂.对系列稀释血清的检测表明,该试剂的平均分析灵敏度为103.2基因组拷贝/mL(95%可信限102.2-4.2基因组拷贝/mL),显著高于前S1抗原或核心抗原的单独检测.对994份HBsAg阴性血清的检测结果表明NRAg ELISA的特异性为99.7%(95%可信限:99.1%～99.9%).对271份临床慢性肝炎血清进行检测,结果NRAg ELISA与HBV DNA结果的总符合率达96.3%(95%可信限:93.3%～98.2%),NRAg ELISA的读值/临界值比(S/CO)与HBV基因组拷贝数呈正相关.利用NRAg试剂,发现了1例HBsAg"a"抗原表位突变的变异株.这些结果显示HBV NRAg ELISA与HBV DNA具有高度相关性,并能够检测出HBsAg抗原变异株,有望成为HBsAg变异株筛选的有力工具,并为广大基层医疗单位提供一种便捷的替代HBV DNA定性检测的手段.     

Anti-VP1 and anti-VP2 antibodies detected by immunofluorescence assays in patients with acute human parvovirus B19 infection

Acute human parvovirus B19 infection is followed by an antibody response to the structural proteins of the viral capsid (VP1 and VP2). We used 80 sera collected from 58 erythema infectiosum and 6 transient aplastic crisis patients to test IgM and IgG antibodies against these two proteins in an immunofluorescence assay (IFA) using Sf9 cells infected with recombinant baculovirus expressing either VP1 or VP2 antigen. Although less sensitive than IgM capture enzyme immunoassay using native antigen (MACEIA), we could detect anti-VP1 or anti-VP2 IgM antibodies by IFA in 49 patients with acute infection (76.6%). Detection of IgG anti-VP1 and anti-VP2 by IFA, however, was as sensitive as IgG detection by indirect enzyme immunoassay. By applying IgG avidity IFA to sera of the 15 IgM IFA negative patients we were able to confirm acute infection in further 12 cases by IFA. Overall, acute infection was confirmed by IFA in 61 (95.3%) of the 64 patients.