Laboratory Diagnosis of Toscana Virus Infection by Enzyme Immunoassay with Recombinant Viral Nucleoprotein

A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and/or previous infections. The rEIA, based on the recombinant nucleoprotein of TOSV expressed in Escherichia coli, was evaluated with 97 serum samples collected in an area where TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.     

Diagnosis of Oropouche Virus Infection Using a Recombinant Nucleocapsid Protein-Based Enzyme Immunoassay

Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America.     

A Novel Antigen Detection Immunoassay for Field Diagnosis of Hepatitis C Virus Infection

Abstract

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (?5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.     

Diagnosis of Penicillium marneffei Infection by Quantitation of Urinary Antigen by Using an Enzyme Immunoassay

Penicillium marneffei is a major cause of opportunistic infection in patients with AIDS in north and northeastern Thailand. A method for the quantitation of P. marneffei antigen in urine was developed by using fluorescein isothiocyanate-labelled purified rabbit hyperimmune immunoglobulin G in an enzyme-linked immunosorbent assay. This method was evaluated with 33 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects, 248 hospitalized patients without penicilliosis) from the same area in which penicilliosis is endemic. Urinary antigen was found in all 33 (100%) patients with penicilliosis, with a median titer of 1:20,480. With undiluted samples, 67 (27%) of 248 hospital patients and 3 (6%) of 52 healthy controls were reactive. At a cutoff titer of 1:40, the urine antigen detection assay had a diagnostic sensitivity of 97% and specificity of 98% (positive predictive value, 84%; negative predictive value, 99.7%). This test offers a valuable and rapid method for the diagnosis of penicilliosis in patients with AIDS and could be a useful addition to conventional diagnostic methods in areas in which penicilliosis is endemic.     

Blastomyces dermatitidis Antigen Detection by Quantitative Enzyme Immunoassay

The second-generation MVista Blastomyces antigen enzyme immunoassay was not quantitative; therefore, specimens obtained previously were tested in the same assay as new specimens to assess the change in antigen levels. Furthermore, the sensitivity in serum had not been fully evaluated. The purpose of this study was to evaluate a quantitative Blastomyces antigen assay and detection of antigen in serum. Calibrators containing known concentrations of Blastomyces galactomannan were used to quantify antigen in urine and serum from patients with proven blastomycosis and from controls. Paired current and previously obtained urine specimens were tested to determine if quantification eliminated the need for concurrent testing to assess change in antigen. Pretreatment of serum with EDTA at 104°C was evaluated to determine if dissociation of immune complexes improved detection of antigenemia. Antigenuria was detected in 89.9% of patients with culture- or histopathology-proven blastomycosis. Specificity was 99.0% in patients with nonfungal infections and healthy subjects, but cross-reactions occurred in 95.6% of patients with histoplasmosis. Change in antigen level categorized as increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in ng/ml determined from different assays. Pretreatment increased the sensitivity of detection of antigenemia from 35.7% to 57.1%. Quantification eliminated the need for concurrent testing of current and previously obtained specimens for assessment of changes in antigen concentration. Pretreatment increased the sensitivity for detection of antigenemia. Differentiation of histoplasmosis and blastomycosis is not possible by antigen detection.     

Identification of Parainfluenza Virus Serotypes by Indirect Solid-Phase Enzyme Immunoassay

An indirect enzyme immunoassay was used to detect and identify parainfluenza virus serotypes 1 and 3 in cell culture residuals from which infectious virus could no longer be recovered.     

用重组SAG1抗原检测弓形虫抗体

目的探讨重组SAG1抗原对弓形虫IgG和IgM抗体的检测效果。方法用rSAG1作抗原建立免疫印迹方法（rSAG1-WB）,与玻片虫体过氧化物酶免疫染色试验（TSHE）平行检测不同来源血清。结果15例病原学检查阳性小鼠血清和5例免疫兔血清的IgG抗体均为阳性,30例正常小鼠血清和10例正常兔血清均未出现阳性反应。rSAG1-WB检测可疑弓形虫病患者血清阳性率为60．3％（38／63）,献血员血清阳性率为6％（3／50）,与TSHE检测结果（65．1％和4％）均无统计学差异（P〉0．05）。1例IgM强阳性血清和13例IgM弱阳性血清在Western—blot检测中分别出现相应的强阳性与弱阳性反应,50例献血员血清均未出现IgM阳性反应,结果与TSHE一致。结论rSAG1-WB检测弓形虫IgG和IgM抗体均具有高度的敏感性和特异性．与TSHE的符合率高。     

Rapid Detection of Campylobacter Antigen by Enzyme Immunoassay Leads to Increased Positivity Rates

Campylobacter antigen detection by enzyme immunoassay (EIA) provides rapid results compared to traditional culture. However, concern exists regarding specificity. Verification studies of an EIA compared to culture revealed a positive predictive value (PPV) of 91%, whereas PPV fell to 42% during routine diagnostic testing. We suggest all positive EIA results be confirmed via culture.     

Use of a Novel Enzyme Immunoassay Based on Detection of Circulating Antigen in Serum for Diagnosis of Helicobacter pylori Infection

Recently, noninvasive diagnostic tests for Helicobacter pylori infection have gained in significance. We have developed a sensitive and specific noninvasive immunoassay based on the detection of an H. pylori circulating antigen (HpCA) in sera from H. pylori-infected individuals. Monospecific antibody and Western blot analyses were used to demonstrate the presence of the target antigen in H. pylori cell lysate and serum samples. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HpCA in serum. Endoscopic biopsy specimens from the gastric antra of 221 individuals (143 males and 78 females) with dyspeptic symptoms were evaluated for H. pylori infection, with culture used as a “gold standard” for diagnosis. The target H. pylori antigen was identified at 58 kDa. HpCA has been detected by ELISA with high degrees of sensitivity, specificity, and efficiency (>90%), and ELISA results show no significant difference (P > 0.05) from results of H. pylori culture of gastric biopsy specimens. The test's positive and negative predictive values were also high (95 and 86%, respectively). In conclusion, a sensitive and specific immunoassay was developed for the detection of HpCA in human serum. This test can be applied for noninvasive laboratory and field diagnoses of H. pylori infection.     

An Enzyme Immunoassay for the Detection of Antisperm Antibodies

ABSTRACT: A simple and reliable enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies has been developed in our laboratory. The antigen for the solid phase was produced by sperm sonication while antihuman globulin conjugated to alkaline phosphatase was used as the developing reagent. The conditions and reagents of the assay were chosen to give a mild treatment of the antigen, simple manipulation during washing steps, and nontoxic and readily available reagents. The results were compared to a conventional microscopical method routinely used in our laboratory that detects agglutinating antibodies to human spermatozoa. In 96% of all cases antibodies detected by the microscopical method were also detected by ELISA. Moreover there were some cases where no antisperm antibodies could be demonstrated by microscopy, but gave a positive reaction with ELISA. These were usually cases of unexplained oligospermia, agglutinates in the ejaculate, and bad motility or low viability of the sperms. These results, and also titration experiments of positive samples demonstrate the higher sensitivity of the ELISA by comparison with microscopical methods.     

Detection of Norwalk Virus and Other Genogroup 1 Human Caliciviruses by a Monoclonal Antibody, RecombinantAntigen-Based Immunoglobulin M Capture Enzyme Immunoassay

Sera obtained from two groups of adult volunteers infected with Norwalk virus (NV) and two groups of patients involved in two natural outbreaks were tested for NV-reactive immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant-antigen-based IgM capture enzyme immunoassay (EIA). No NV-reactive IgM was detected in the preinoculation sera of 15 volunteers, and 14 of 15 showed NV-reactive antibodies postinfection with NV. All of the volunteers showed IgG seroconversion to NV. In the outbreak studies, all 9 persons in one outbreak and 19 of 24 in another outbreak had NV-reactive IgM. In the first outbreak, only three of nine seroconverted to NV, which was likely due to late collection of acute-phase sera. In the second outbreak, 21 of 24 showed IgG seroconversion to NV. Sequencing of viruses isolated from five stool samples selected from those in the second outbreak showed that they were human calicivirus (HuCV) genogroup 1 viruses related, but not identical, to NV. In the volunteer studies, NV-reactive IgM was first detected 8 days postinoculation. The time of development of NV-reactive IgM antibodies in natural outbreaks was estimated to be similar to that found in the volunteer studies. Sera from three Hawaii virus-infected volunteers, four Snow Mountain virus patients, and 80 healthy individuals were negative for NV-reactive IgM, indicating test specificity for HuCV genogroup I infections. This capture IgM EIA is suitable for diagnosis of NV and other HuCV genogroup I infections and is especially useful when sera and fecal samples have not been collected early in the course of an outbreak.     

Detection of Helicobacter pylori in Stool Specimens by PCR and Antigen Enzyme Immunoassay

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom H. pylori had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.     

Highly Sensitive Detection of Hepatitis B Virus Surface Antigen by Use of a Semiautomated Immune Complex Transfer Chemiluminescence Enzyme Immunoassay

The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-day-shorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients.     

Quantitation of Immunoglobulin to Hepatitis E Virus by Enzyme Immunoassay

We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and intertest coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.     

Comparison of a Chemiluminescent Immunoassay with Two Microparticle Enzyme Immunoassays for Detection of Hepatitis B Virus Surface Antigen

A comparative evaluation of the following commercial immunoassays for the detection of hepatitis B virus surface antigen (HBsAg) was performed: the Abbott AxSYM, Abbott IMx, and DPC IMMULITE assays. The specificity was 100% for all assays. Twelve samples were identified and were confirmed to be positive for HBsAg by all three methods. One additional sample was identified as reactive and was confirmed to be positive by the Abbott AxSYM assay only. Prior to confirmation testing the DPC IMMULITE assay produced significantly fewer false-positive results than the Abbott AxSYM assay (P < 0.05).     

Comparative Study of Antinuclear Antibody Detection by Indirect Immunofluorescence and Enzyme Immunoassay in Lupus Patients

To evaluate the value of immunofluorescent and ELISA techniques in early diagnosis of systemic lupus erythematosus (SLE) and to find out whether there is a correlation between Antinuclear antibodies (ANA) pattern and prognosis by observing clinical score changes using British Isles Lupus Assessment Group score. The study included 75 SLE patients, 11 disease control group, and 18 healthy control group. ANA and ds-DNA antibodies detection were done by ELISA and Immunofluorescence for all groups. Immunofluorescence technique is more sensitive for ANA and ds-DNA detection than ELISA technique (100% versus 90.7%,and 93% versus 89.3% respectively); ELISA showed 89.7 % specificity for ANA detection compared to 86.2% for Immunofluorescence, and both have 100% specificity for ds-DNA detection ;homogenous ANA pattern, showed statistically significant higher BILAG score compared to speckled pattern either at the start of the study or after the follow up period (p = 0.000); and ds-DNA titer showed statistically significant decrease in titer after therapy (p = 0.01). ANA detection by Immunofluorescence is more sensitive and effective screening assay in patients with clinical features of SLE and both ds-DNA titer by ELISA and BILAG score for severity index are considered the best markers for follow-up.     

Development of Enzyme Immunoassay of Glial Fibrillary Acidic Protein on the Basis of Recombinant Antigen

Quantitative enzyme immunoassay of glial fibrillary acidic protein of astrocyte intermediate filaments opened new prospects for highly selective diagnosis and monitoring of pathological processes in the CNS. Immunochemical screening of glial fibrillary acidic protein in biological fluids helps to adequately evaluate the permeability of the blood-brain barrier in CNS diseases associated with violation of its functions, such as hypoxic and ischemic disorders, neuroinfections, glial tumors, brain injuries, etc. Wide-scale introduction of enzyme immunoassays into clinical laboratory practice implies the development of biotechnological approaches to the creation of methods for obtaining EIA components. This paper presents a method for creation of a test system for EIA of glial fibrillary acidic protein (GFAP) on the basis of recombinant GFAP and antibodies obtained by immunization with recombinant GFAP. Due to this approach, a highly standardized test system for the analysis of GFAP in human biological fluids was created. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 11, pp. 535–540, November, 2008