Performance of an automated system for quantitation of hepatitis C virus core antigen

The performance of an automated system for the HCV core antigen assay using the Lumispot LS-2000 automated analyzer was evaluated against that of the COBAS AMPLICOR HCV MONITOR Test, version 2.0 (COBAS HCM-2) for the testing of sera from 155 chronic hepatitis C patients. The within-run coefficient of variations (CVs) and the between-day CVs were <9.6 and <8.4%, respectively. The analytical detection limit of the HCV core antigen assay was 5.0 fmol/l and it was linear up to at least 45000 fmol/l. No blood elements interfered with the assay. HCV core antigen levels were significantly correlated with HCV RNA levels in both serogroup 1 and serogroup 2 (r=0.829, P<0.001). It is estimated that 100 fmol/l of HCV core antigen level was equivalent to approximately 30000 IU/ml of HCV RNA level. Three sera had HCV core antigen levels below the detection limit of the assay and their HCV RNA levels as determined by COBAS HCM-2 assay were very low at 1000, 1100 and 1700 IU/ml, respectively. In six IFN responders among seven patients, HCV core antigen levels were in parallel with HCV RNA levels. In conclusion, since this assay demonstrated good reproducibility, a favorable dynamic range and adequate sensitivity, it may be useful as an alternative direct marker of viral level monitoring in patients with hepatitis C.     

Comparison of enzyme immunoassays and an immunofluorescence test for detection of antibody to human immunodeficiency virus in African sera

A total of 152 sera from African subjects were tested for presence of antibody to human immunodeficiency virus using four enzyme immunoassays (EIA) marketed by Abbott Diagnostics, Organon Teknika, Wellcome and Diagnostics Pasteur respectively, an indirect immunofluorescence assay (IFA) and an immunoblot assay (IBA) as reference test. The sensitivity (95 % confidence limits, CL) of the EIAs and the IFA ranged between 80.9 % and 99.1 %. The specificity of the Abbott EIA was lower (95 % CL: 38.1–72 %) than that of the other assays (95 % CL: 83.5–100 %). The use of an IFA or the Wellcome competitive EIA as confirmatory test on initially EIA positive sera yielded a specificity of 85.5–100% (95 % CL) compared with the IBA. The costs of screening by an EIA, followed by confirmatory testing of reactive sera with IFA or the Wellcome EIA and IBA on discrepant test results was similar for all combinations with the exception of initial screening with the Abbott EIA which was more expensive. Using a limited number of sera from African subjects no one test system yielded a significantly superior degree of specificity or sensitivity.     

Diagnosis of granulocytic ehrlichiosis in humans by immunofluorescence assay

Serodiagnostic tests are widely available for tick-borne diseases. We evaluated a cell-free antigen of the human granulocytic ehrlichiosis agent. Immunofluorescence assay (IFA) with this antigen is as efficient as with the MRL kit and allows a one-step IFA with other cell-free antigens that is useful when testing sera from patients bitten by ticks.     

How should a district general hospital immunology service screen for anti-nuclear antibodies? An ‘in-the-field’ audit

Anti‐nuclear antibody (ANA) testing assists in the diagnosis of several immune‐mediated disorders. The gold standard method for detection of these antibodies is by indirect immunofluorescence testing on human epidermoid laryngeal carcinoma (HEp‐2) cells. However, many laboratories test for these antibodies using solid‐phase assays such as enzyme‐linked immunosorbent assay (ELISA), which allows for higher throughput testing at reduced cost. In this study, we have audited the performance of a previously established ELISA assay to screen for ANA, making comparison with the gold standard HEp‐2 immunofluorescence test. A prospective and unselected sample of 89 consecutive ANA test requests by consultant rheumatologists were evaluated in parallel over a period of 10 months using both tests. ELISA and HEp‐2 screening assays yielded 40 (45%) and 72 (81%) positive test results, respectively, demonstrating lack of concordance between test methods. Using standard and clinical samples, it was demonstrated that the ELISA method did not detect several ANA with nucleolar, homogeneous and speckled immunofluorescence patterns. None of these ELISA^NEG HEp‐2^POS ANA were reactive with a panel of six extractable nuclear antigens or with double‐stranded DNA. Nonetheless, 13 of these samples (15%) originated from patients with recognized ANA‐associated disease (n = 7) or Raynaud's phenomenon (n = 6). We conclude that ELISA screening may fail to detect clinically relevant ANA that lack defined specificity for antigen.     

Diagnosis of Granulocytic Ehrlichiosis in Humans by Immunofluorescence Assay

Serodiagnostic tests are widely available for tick-borne diseases. We evaluated a cell-free antigen of the human granulocytic ehrlichiosis agent. Immunofluorescence assay (IFA) with this antigen is as efficient as with the MRL kit and allows a one-step IFA with other cell-free antigens that is useful when testing sera from patients bitten by ticks.     

Evaluation of immunoassays for detection of antibodies to human herpesvirus 7.

An enzyme immunoassay (EIA), an immunoblot assay (IB), and an indirect immunofluorescence assay were developed for detection of human herpesvirus 7 (HHV-7) antibodies in human serum. Cross-absorption studies with EIA or IFA using HHV-7 and human herpesvirus 6 (HHV-6) antigens indicated that most human sera contain cross-reactive HHV-6 and HHV-7 antibodies and that the degree of cross-reactivity varies between individual serum specimens. Inhibition of homologous antibody activity by absorption with heterologous virus ranged from 0 to 57% by EIA. However, for every sample tested, absorption with homologous virus removed more activity than did heterologous virus. An 89-kDa protein was identified as an HHV-7-specific serologic marker by IB. Activity to this protein was not removed by absorption with HHV-6 antigen. Of the three assays, the EIA was the most sensitive (94%), while the IB was the most specific (94%). Approximately 80% of specimens collected from German adults and children older than 2 years were positive for HHV-7 antibodies by these assays.     

Immunodiagnosis of Human Granulocytic Ehrlichiosis by Using Culture-Derived Human Isolates

Human granulocytic ehrlichiosis (HGE) is an emerging infection caused by an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused by Ehrlichia chaffeensis, Rickettsia rickettsii, and Coxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples from R. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests for Borrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n = 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure to Ehrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.     

Comparison of a multiplexed bead-based assay with an immunofluorescence and an enzyme-immuno assay for the assessment of Epstein–Barr virus serologic status

We have compared a multiplexed bead-based assay (BBA) with an enzyme immunoassay (EIA) and immunofluorescence assay (IFA) for the assessment of the Epstein–Barr virus (EBV) serostatus. Three hundred and ninety-three sera, classified according to IFA results as seronegative (n = 100), acute infection (n = 100), past infection (n = 100) and indeterminate (n = 93), were tested by BBA and EIA. Overall, the three methods gave similar results with a relatively high (75.2%) concordance with the consensus interpretation of the serostatus. The most significant discordances were: (i) 58 samples had uninterpretable results for BBA, in majority due to the detection of non-antigen specific antibody binding by control beads. (ii) almost half the samples positive for anti-Epstein—Barr nuclear antigen (EBNA) IgG by BBA or EIA were negative by IFA. Among the latter, only a minority had a history of immunocompromise or treatment, or detectable anti-early antigen antibody. This discrepancy probably reflects a poor sensitivity of IFA for anti-EBNA IgG detection. EIA and BBA had a similar performance and had substantial practical advantages over IFA with respect to testing for EBV serostatus.     

Parvovirus B19 viral loads in relation to VP1 and VP2 antibody responses in diagnostic blood samples.

BACKGROUND: Human parvovirus B19 infection is characterised by high peak viral load levels followed by episodes of prolonged viremia. The risk of transmission of parvovirus B19 by blood or blood products has been increasingly recognised and parameters that can predict the risk of transmission are subject of interest. OBJECTIVES: This study aimed to study correlations between B19 viral DNA loads and antibody responses to the viral antigens VP1 and VP2 in clinical serum samples. STUDY DESIGN: A panel of 1610 serum samples from patients clinically suspected from acute B19 infection were analysed. Antibodies were measured by the parvovirus anti-VP1 immuno-fluorescence assay (IFA) and the anti-VP2 enzyme immunoassay (EIA) from Biotrin. B19 viral loads were measured by a real-time PCR using the external WHO standard for DNA quantification. RESULTS: Positive IgM responses were found in 154 (9.6%) of the 1610 sera tested. Based on the PCR results in a subset of 312 sera, the anti-VP2 EIA IgM showed a better combination of sensitivity/specificity (91%/94%) compared to the anti-VP1 IFA (66%/97%). B19 DNA levels in the sera strongly correlated with the levels of IgM antibodies, all sera with high viral loads (>10(6)IU/ml) having VP2 EIA IgM ratios above 3.0. CONCLUSIONS: The B19 VP2 IgM ELISA is superior to the B19 VP1 IgM IFA if verified by PCR. Anti-VP2 IgM antibodies in sera are indicative for the presence B19 DNA and can be used to predict high levels of B19 DNA in diagnostic sera.     

Undenatured parvovirus B19 antigens are essential for the accurate detection of parvovirus B19 IgG

Recombinant versions of parvovirus B19 capsid proteins VP1 and VP2 are used for immunodiagnostic assays for detection of antiviral antibodies. The immune response to B19 is characterized by a gradual loss of antibodies directed against linear epitopes of VP2. A similar occurrence for antibodies raised against VP1 protein would represent a limitation to serological assays incorporating denatured versions of either viral antigen. Four detection systems for B19 Ig detection have been developed, including an IgG enzyme immunoassay (EIA) based on undenatured VP2, an immunofluorescence assay (IFA) based on undenatured VP1, a Western blot assay incorporating denatured VP1 and VP2, and an alternative blot system using denatured VP1 but undenatured VP2. Specimens (n = 108) were tested by all four systems and identical results were obtained by EIA, IFA, and alternative blot systems, whereby 75/108 (69%) were B19 IgG-positive. Twelve B19 IgG-positive specimens, representing 16% (12/75) of the confirmed positives, did not react to either viral antigens when tested by Western blot. It is concluded that these sera do not react with linear epitopes of VP1 and VP2 antigens. Eighty-five different specimens, which had previously been shown to be both B19 IgM- and IgG-positive by EIA and IFA, were positive by B19 IgM and IgG Western blot. In the IgG Western blot assay, 69 reacted with both VP1 and VP2 and 16 with VP1 only. It is concluded that there is a requirement for at least one undenatured antigen for the immunological detection of B19 IgG. J. Med. Virol. 57:179–185, 1999. © 1999 Wiley-Liss, Inc.     

Development and clinical evaluation of two immunoassays for prostatic acid phosphatase in serum

Polystyrene tubes were coated with IgG, isolated from antiserum against human prostatic acid phosphatase, and incubated with a known amount of purified antigen as a standard, and sera from patients. The amount of bound prostatic acid phosphatase was established by using its enzyme activity. This was performed either spectrophotometrically with p-nitrophenylphosphate (enzyme immunoassay) or fluorometrically with alpha-naphthylphosphate as a substrate (immunofluorescence assay). Results of both methods were compared with those of the conventional method and were evaluated in four groups of patients with: non-prostatic disease, hypertrophy, treated and untreated prostatic carcinoma. The first group was used to establish the upper limit of normal. In the hypertrophy group, the specificity of the immunological methods when compared with the conventional method, improved from 79% to 91%. Sensitivity, calculated from the untreated prostatic carcinoma group, was 74% for the enzyme immunoassay (EIA) and 71% for the immunofluorescence assay (IFA). The probability of having either carcinoma or hypertrophy, given observed EIA or IFA values, was calculated by the statistical method of the logistic regression.     

Comparison of a baculovirus-based VP2 enzyme immunoassay (EIA) to an Escherichia coli-based VP1 EIA for detection of human parvovirus B19 immunoglobulin M and immunoglobulin G in sera of pregnant women

A split-sample study was conducted to evaluate the clinical performance of an enzyme immunoassay that detects the human parvovirus B19 virus (B19V) immunoglobulin M (IgM) or IgG in the sera of pregnant women. The initial study compared a baculovirus-expressed VP2 enzyme immunoassay (BVP2 EIA) (Biotrin International Inc., Dublin, Ireland) with the currently available and commonly used Escherichia coli-expressed VP1 enzyme immunoassay (EVP1 EIA) (MRL Diagnostics, Cypress, Calif.). There was a high degree of agreement between the two assays in the detection of IgM antibodies (283 of 307 [92.2%]) or IgG antibodies (279 of 311 [89. 7%]), with the majority of discrepancies (IgM, 17 of 24 [71%]; IgG, 16 of 31 [50%]) being due to equivocal data obtained with the EVP1 EIA. Specimens with discordant BVP2 EIA and EVP1 EIA results (23 of 24 IgM and 32 of 32 IgG results) were analyzed further by baculovirus-based VP1 immunofluorescence assays (BVP1 IFAs) (Biotrin International). The BVP2 EIA and BVP1 IFA results for 20 of 23 and 28 of 32 specimens for IgM and IgG, respectively, were concordant. In contrast, the EVP1 EIA and BVP1 IFA data for only 3 of 23 and 4 of 32 specimens for IgM and IgG, respectively, were in agreement, despite the fact that the same capsid antigen was used. Both the BVP2 EIAs and BVP1 IFAs utilize a conformational viral capsid antigen, while the EVP1 EIA uses a denatured viral capsid antigen. In conclusion, the BVP2 EIAs produced far fewer equivocal results for IgM and IgG, correlating more closely to the confirmatory BVP IFAs, than did the EVP1 EIAs and proved to be more accurate for detecting B19V antibodies in the sera of pregnant women.     

Evaluation of Pooled and individual components ofBordetella pertussis as antigens in an enzyme immunoassay for diagnosis of pertussis

Six different antigen preparations for use in an enzyme immunoassay (EIA) to detect IgM, IgA and IgG antibodies toBordetella pertussis were evaluated using sera from 13 randomly selected culture-positive patients and from 87 patients with suspected pertussis during a pertussis outbreak. Based on results in 80 healthy control sera a specificity limit of 99.9 % was selected. Sera from all culture-positive patients reacted with at least one of the antigens. The sensitivity of the EIA using the individual antigen preparations was 85 % for filamentous hemagglutinin, 92 % for pertussis toxin, 62 % for 69 kDa outer membrane protein, 85 % for a pool of these three antigens, 54 % for sonicated whole bacteria and 69 % for 21 kDa pertussis toxin subunit S1. In the outbreak patient group 49 (56 %) of the initial sera reacted with at least one of five antigen preparations. The EIA using sonicated bacteria detected only 41 % of all seropositive cases compared with 51 % using filamentous hemagglutinin, 61 % using pertussis toxin, 65 % using 69 kDa OMP and 65 % using pooled antigen. It is concluded that either the pooled antigen or pertussis toxin antigen are suitable antigen preparations for use in the EIA for diagnosis of pertussis.     

Diagnostic usefulness of antineutrophil cytoplasmic autoantibody serology. Comparative evaluation of commercial indirect fluorescent antibody kits and enzyme immunoassay kits

Antineutrophil cytoplasmic autoantibodies (ANCAs) are increasingly used as serologic markers for pauci-immune crescentic glomerulonephritis and small vessel vasculitis. Many hospital laboratories and referral laboratories use commercial assay kits to detect ANCAs, despite inadequate documentation in the medical literature of kit performance. We evaluated the diagnostic sensitivity, specificity, and predictive value of 3 commercial indirect immunofluorescence assay (IFA) kits and 7 commercial enzyme immunoassay (EIA) kits for several ANCA subtypes. Serum samples from 396 patients with a variety of renal diseases were analyzed, including 146 patients with pauci-immune crescentic glomerulo-nephritis with or without systemic vasculitis. With 1 exception, the kits had more than 90% agreement with the reference standard and gave results similar to those of research laboratories. IFA diagnostic sensitivity ranged from 81% to 91% and EIA sensitivity from 75% to 84%. Maximum specificity was obtained with combined IFA and EIA. Diagnostic specificity was more than 70% for 2 of 3 IFA kits and at least 90% for 5 of 7 EIA kits. Predictive values varied with clinical manifestations. Most commercial IFA and EIA kits that were evaluated provide acceptably accurate analytic results.     

Use of a receiver operating characteristic in the evaluation of two commercial enzyme immunoassays for detection ofHelicobacter pylori infection

Two novel commercial IgG enzyme immunoassay (EIA) systems based on acid-glycine-extracted (Pyloriset IgG EIA, Orion Diagnostica) or fast protein liquid chromatography-purified (Cobas Core Anti-H. pylori EIA, Roche Diagnostic Systems)Helicobacter antigens were evaluated in a prospective study involving 127 patients. All patients underwent upper endoscopy with biopsy, and biopsies were examined for the presence ofHelicobacter pylori by a rapid urease test, microscopy and culture. Of the 71 patients found to be infected withHelicobacter pylori, 69 (97.2 %) and 65 (91.5 %) tested positive with the Cobas Core and Pyloriset test, respectively. A detailed receiver operating characteristic analysis of the two tests showed that the Cobas Core assay was more sensitive and specific at every possible cut-off level; gave a better resolution of individual results, indicating a greater fine-sensitivity; and had no grey zone compared to a large grey zone encompassing 13.4 % of the serum samples tested with the Pyloriset EIA. The Cobas Core assay appears to be a valuable tool for epidemiological purposes as well as for pre-endoscopic screening of dyspeptic patients.     

Enzyme immunoassay for detection of antibodies against isolated flagella ofLegionella pneumophila

Flagella ofLegionella pneumophila serogroup 1, strain Philadelphia (ATCC 33152), were isolated and used as antigen to detect antibodies by enzyme immunoassay (EIA) in sera of patients with significant titers againstLegionella as determined by the indirect immunofluorescence assay (IFA). Healthy blood donors served as a control group. Whereas the sera of blood donors were negative in both assays, sera of patients showed two patterns of reactivity. Sera with elevated titers againstLegionella pneumophilasero-group 1 orLegionella gormanii in IFA usually demonstrated high titers in EIA. However, high IFA titers againstLegionella bozemanii were associated with low titers in EIA. The data show that flagella protein ofLegionella pneumophila serogroup 1 strain Philadelphia is of limited value for the detection of antibodies againstLegionella other thanLegionella pneumophila serogroup 1, although in vitro studies revealed genus-specific epitopes. This might be due to unpredictable variabilities in the expression of flagella in vivo.     

Evaluation of Cytomegelisa immunoglobulin M assay and comparison with indirect fluorescent antibody testing of QAE-Sephadex A50-treated sera

The Cytomegelisa IgM assay (M. A. Bioproducts, Walkersville, MD) was evaluated as an alternative to indirect fluorescent antibody (IFA) testing of QAE-Sephadex A50-treated sera for detection of cytomegalovirus (CMV)-specific IgM. The Cytomegelisa assay was consistently more sensitive than IFA testing for detection of CMV-specific IgM. Of 101 rheumatoid factor-negative sera that were IFA positive before column separation, only 47 (46%) remained positive after column separation, while 72 (71%) were positive using Cytomegelisa. Nine transplant patients who had primary CMV infection develop were also evaluated. Two of the nine did not form sufficient IgM to remain IFA positive after column separation, however, all had IgM detected by Cytomegelisa. In all cases, Cytomegelisa provided laboratory evidence of CMV infection earlier or at the same time as IgG seroconversion or IgM testing by column/IFA. Comparison of both methods with 100 consecutive sera received for CMV testing yielded five sera positive for CMV-IgM with the column/IFA method and 20 using Cytomegelisa. The specificity of Cytomegelisa was evaluated with 56 RF-positive/CMV IgG-positive sera. Four of 56 (7%) appeared positive after column separation; however, of concern, 22 of 56 (39%) remained positive in the Cytomegelisa.     

Evaluation of a multiplex flow immunoassay for detection of epstein-barr virus-specific antibodies

Conventional methods for the detection of Epstein-Barr virus (EBV)-specific antibodies include the immunofluorescence assay (IFA) and enzyme immunoassay (EIA). While sensitive and specific, these methods are labor-intensive and require separate assays for each analyte. This study evaluated the performance of a multiplex bead assay (BioPlex 2200; Bio-Rad Laboratories, Hercules, CA) for the simultaneous detection of immunoglobulin G (IgG) and IgM class antibodies to the EBV viral capsid antigen (VCA) and IgG class antibodies to Epstein-Barr virus nuclear antigen-1 (EBNA-1). Serum specimens (n = 1,315) submitted for routine EBV-specific antibody testing by EIA (Grifols-Quest, Inc., Miami, FL) were also tested by the multiplex bead assay using the BioPlex 2200 automated analyzer. Specimens showing discordant results were tested by IFA. Following IFA resolution, the BioPlex VCA IgM, VCA IgG, and EBNA-1 IgG assays demonstrated 97.9%, 91.4%, and 96.9% agreement, respectively, with the results obtained by EIA. Furthermore, the BioPlex assays showed an overall agreement of 94.1% with the EIA when the specimens were categorized by disease state (susceptible, acute, or past infection) based on the EBV-specific antibody profiles. These findings indicate that the BioPlex EBV assays demonstrate a performance comparable to that of the conventional EIA, while allowing for a more rapid (2.3 h for 100 samples versus 4.5 h by the EIA) and higher-throughput ( approximately 400 samples per 9 h versus 200 samples by the EIA) analysis of the EBV-specific antibody response.     

Binding of sperm-reactive antibodies in human sera to surface-associated antigens on human sperm compared by indirect immunobead, immunofluorescence, and immunogold assays

Human sera were identified as positive or negative for sperm-reactive antibodies in a solid-phase enzyme-linked immunosorbent assay (ELISA). Of these, 28 positive and 22 negative sera were blind-coded and used as first antibody to compare three immunoassays, a modified liquid-phase indirect immunobead assay (IBA); a liquid-phase indirect immunofluorescence assay (IFA); and a solid-phase indirect immunogold assay (IGA). These three immunoassays perform both as sperm-reactive antibody detection assays and as sperm-associated antigen localization assays. As antibody detection assays, the IBA, IFA, and IGA gave 37, 27, and 28 positives and 13, 23, and 22 negatives, respectively. The usefulness of the IBA as an antigen localization assay was limited by the size of the marker, while the smaller IFA and IGA markers enabled increased resolution of binding patterns of sperm-reactive antibodies to surface-associated sperm antigens. Although the antigen-antibody binding patterns were almost identical for IFA and IGA, suggesting the same sperm-associated antigens were detected by both assays, the IGA reaction product was stable, higher in resolution, and visible by light microscopy.