Recombinant parvovirus B19 capsids as a new substrate for detection of B19-specific IgG and IgM antibodies by an enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay for the detection of B19-specific IgG and IgM antibodies was established using B19 capsids synthesized in a baculovirus expression system. These B19 capsids, consisting of either coat protein VP2 alone or of both VP1 and VP2, have been shown to be similar to native virus in size and appearance. The results obtained for the detection of B19-specific antibodies showed good correlations with a radioimmunoassay which uses native B19 virus and an immunofluorescence assay based on insect cells expressing coat protein VP1. The course of the antibody response could be followed by determining the titers of sequential serum samples taken after a recent B19 infection. Both types of recombinant capsids form an excellent source of antigen for the detection of both B19 IgG and IgM antibodies and are a very promising substitute for native virus.     

New oligopeptide immunoglobulin G test for human parvovirus B19 antibodies.

A new, highly sensitive and specific enzyme immunoassay using oligopeptides as antigen (enzyme-linked immunosorbent assay [ELISA] B19-OP) for detecting parvovirus B19-specific immunoglobulin G (IgG) was established. As antigens, B19-specific oligopeptides of 24 and 30 kDa derived from a 196-kDa fusion protein of beta-galactosidase and viral capsid protein (VPI) of B19 after CNBr cleavage and separation by high-pressure liquid chromatography were used. Of 139 serum specimens tested in parallel for anti-B19 IgG by standard ELISA using B19 particles as antigen and by ELISA B19-OP, 73 (52.5%) were positive and 63 (45.3%) were negative in both tests, and 3 (2.2%) were negative by standard ELISA but positive by ELISA B19-OP and by immunoblot. By using ELISA B19-OP, it was possible to detect anti-B19 IgG in an asymptomatic blood donor 4 weeks after acute infection, and anti-B19 IgG titers of 10(-5) could be measured in convalescent-phase sera.     

Comparative evaluation of tests for detection of parvovirus B19 IgG and IgM

The aim of this study was to evaluate enzyme immunoassays (EIA) (Euroimmun, Lübeck, Germany) and chemiluminiscent immunoassays (CLIA) (Diasorin, Saluggia, Italy) in their application to detect B19V‐IgM and ‐IgG. For this purpose, one hundred and ninety samples were studied. Of them, 101 came from recent infection cases (B19V‐specific IgM (86) and/or PCR (87), 42 from past infections, 18 from non‐infected, and 29 from other viral recent infections (Epstein‐Barr virus, measles, and rubella). Samples were characterized by capture (for IgM), or indirect (for IgG) EIA (Biotrin, Dublin, Ireland); indeterminate samples were classified by indirect immunofluorescence (IIF) (Biotrin). All the samples were used for testing IgM assays, and all but the cases from other viral infections were used for IgG tests. For IgM, CLIA, and EIA identified 76 and 62 of 86 IgM positives, respectively (sensitivity 88.4% and 72.1%). Considering B19V IgM negative samples, negative result was obtained in 95 and 92 of 104, being the specificity values of CLIA and EIA 91.3% and 88.5%, respectively. For IgG, CLIA and EIA identified correctly 114 and 115 of the 122 positive samples (sensitivity 93.4% and 94.3%, respectively), and 39 and 36 of 39 negative samples (specificity 100% and 92.3%). As conclusion, CLIA methods can be used in clinical laboratories as adequate alternatives to the well‐established Biotrin EIAs.     

An immunofluorescence assay for the detection of parvovirus B19 IgG and IgM antibodies based on recombinant viral antigen

An indirect immunofluorescence assay for serum IgG and IgM antibodies to human parvovirus B19 was established using recombinant B19 viral antigen, the capsid protein VP1, which had been produced in a baculovirus expression system. This protein gives a strong and characteristic signal in the immunofluorescence assay, making it a suitable candidate for this test system. The test results showed a good correlation with results obtained with a solid-phase capture radioimmunoassay (Cohen et al., 1983). 76% of sera from a random selection of blood donors were positive for B19 IgG which agrees with previous findings. The course of the IgM and IgG antibody response to B19 infection could be followed with the immunofluorescence assay by determining the titers of series of sera taken after a recent B19 infection. Investigation of 24 sera containing rubella-specific IgM showed no cross-reactivity with the recombinant B19 VP1 used in this test system. The test described here has the advantage of being based on a renewable source of antigen and will be further evaluated for routine diagnostic use in comparison with radioimmunoassay.     

Evaluation of commercial enzyme linked immunosorbent assay for detection of B19 parvovirus IgM and IgG.

An indirect enzyme linked immunosorbent assay (ELISA) (Parvoscan-B19; Sweden) was compared with an in-house MACRIA for the detection of B19 specific IgM. A Parvoscan-B19 IgG test was also evaluated for its ability to detect a recent B19 infection in paired sera. Two hundred and twenty sera submitted to the laboratory for B19 serology and four MACRIA positive control sera were assayed for B19 IgM. Confirmation of the response of sera giving discordant results in the two assays was sought by the use of a "nested" polymerase chain reaction (PCR) for the detection of B19 DNA. The Parvoscan-B19 IgM test was 79% sensitive and 96% specific. Parvoscan-B19 was poor at detecting parvovirus infection in sera collected two to three months after the onset of symptoms. When sera collected more than seven weeks after the onset of symptoms were excluded from the analysis, Parvoscan-B19 IgM was 84% sensitive and 96% specific. Rubella specific IgM positive sera, rheumatoid factor positive sera, and heterophil antibody positive sera were also assayed for B19 IgM. No false positive results were encountered with these problematic sera. By using the cut off criteria for the Parvoscan-IgM test previously advocated by the manufacturers, 90% sensitivity and 87% specificity could be achieved. False positive results, however, occurred with six of the 17 rubella IgM positive sera, four of the 10 rheumatoid factor positive sera, and two of the 11 heterophil antibody positive sera tested. It is concluded that the Parvoscan-B19 was specific but insensitive when compared with in-house assays.     

Enzyme-linked immunosorbent assay for IgG and IgM antibodies against human parvovirus B19: use of monoclonal antibodies and viral antigen propagated in vitro

Enzyme-linked immunosorbent assay (ELISA) systems for serum IgG and IgM antibodies to human parvovirus B19 were established by utilizing anti-B19 monoclonal antibodies (mAbs) and human plasma B19 antigen. The specificities of IgG and IgM ELISA were confirmed by indirect immunofluorescence staining and Western blot immunoassay with panel sera. The series of serum specimens obtained from two B19-infected patients were examined with ELISA. The IgM antibody titers increased quickly after the onset of the symptoms and returned to a negative range after five months. The IgG antibody titers also increased just after the increasing of IgM titers and the elevated levels continued for more than a year. We also established the same ELISA systems by utilizing in vitro propagated B19 antigen and similar results were obtained.     

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.     

An amplified ELISA for the detection of parvovirus B19 IgM using monoclonal antibody to FITC

A new M-antibody capture ELISA for the detection of specific IgM against parvovirus B19 is described. The test uses a monoclonal anti-fluorescein isothiocyanate (FITC) antibody conjugated to horseradish peroxidase to amplify the positive reactions. Serum samples (N = 823) submitted for B19 IgM assay were tested in parallel in the new ELISA and the standard B19 M-antibody capture radioimmunoassay (MACRIA) test. By both tests B19 IgM was detected in 38 (4.6%) samples, and not detected in 771 (94%) samples. One sample was positive in the ELISA test but negative in the RIA and of the 13 sera giving 'equivocal' results in the MACRIA, 6 were positive and 7 negative. If the RIA equivocal results were excluded, the ELISA showed 100% (38/38) sensitivity and 99.9% (771/772) specificity compared to the MACRIA test. The B19 IgM ELISA is a sensitive and specific test with better discrimination between anti B19 IgM positive and negative specimens than MACRIA.     

Evaluation of five commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19.

The following commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19 were evaluated: Ideia Parvovirus B19-IgM, MRL Diagnostics Human Parvovirus B19 IgM ELISA, Parvoscan-B19, and Biotrin Parvo B19 IgM EIA and IF. A total of 203 serum specimens from patients who probably have current B19 infections or have other viral infections and sera with rheumatoid factor were investigated. Between 75 and 79 of 102 serum samples from patients thought to have current B19 infections yielded positive results with the different tests. Ideia had the highest specificity (94.8%), while Parvoscan showed a specificity of only 70.1%. Our evaluation results show that Ideia, MRL, and Biotrin EIA and IF can be recommended for diagnostic purposes.     

The detection of rubella-specific IgM antibodies by radioimmunoassay.

An indirect solid-phase radioimmunoassay (RIA) has been developed for the detection of immunoglobulin (Ig) class-specific rubella antibodies. A commercial rubella haemagglutinin is dried and fixed on to the wells of flexible microtitre plates and allowed to react with serial dilutions of whole or fractionated human sera. Class-specific rubella antibodies are then detected by determining the specific binding of 125I-labelled anti-human IgG or IgM. The RIA was first evaluated by comparison with the haemagglutination-inhibition (HI) test for the detection of rubella-specific IgM in gel-filtration fractions. RIA was found to be as specific as HI but 10-150 times more sensitive. Rubella-specific IgG antibodies did not interfere in specific IgM determinations by RIA and therefore the latter technique was applied to unfractionated sera. The results obtained indicate that RIA on unfractionated sera is a practical, sensitive and specific technique which could provide a reliable method for the diagnosis of rubella. The rubella-specific IgM titres obtained by RIA were not increased by the removal of IgG by pretreatment of sera with Staphylococcal Protein A.     

Immunofluorescence assay for detection of human metapneumovirus-specific antibodies by use of baculovirus-expressed fusion protein

Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (kappa=0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.     

Evaluation of four commercial enzyme immunoassays for detection of immunoglobulin M antibodies to human parvovirus B19

Four commercial enzyme immunoassays (ElAs) for the detection of parvovirus B19-specific immunoglobulin M (IgM) antibodies [Biotrin Parvovirus B19 IgM (Biotrin International, Ireland); Parvoscan B19 IgM (Euro-Diagnostica, Sweden); Parvovirus IgM (Immunobiological Laboratories [IBL], Germany); and human parvovirus B19 IgM (Hillcrest Biologicals, USA)] were compared to indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR). Using IFA as the reference test, high sensitivities (97%) were observed with all four ElAs, though the specificities of the Biotrin and IBL ElAs (99% and 96% respectively) were significantly higher than those of the Hillcrest and Euro-Diagnostica ElAs (81% and 79% respectively).     

Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscopy and DNA hybridization and had no detectable B19 antibody. Antigen was not detected in serum specimens that had low titers of B19 DNA and had B19 antibody. With the IgM ELISA, we detected B19 IgM in over 85% of clinical cases of aplastic crisis and fifth disease and less than 2% of controls. The prevalence of B19 IgG antibodies increased with age. Approximately 2% of children less than 5 years of age and 49% of adults greater than 20 years of age had B19 IgG antibodies. The B19 antibody ELISAs are sensitive and specific tests to detect B19 infections.     

A simple adherence test for detection of IgM antibodies in typhoid

A simple adherence test to detect IgM antibodies in patients with typhoid is described. The test utilises the IgM-"capture" approach, in which the test serum is applied to microtitration plate wells previously coated with anti-human IgM, followed by application of a stained Salmonella typhi antigen suspension which shows adherence in positive cases. By this test, 58 (95%) of 61 sera from confirmed cases of typhoid possessed IgM antibodies to the H or O or both antigens of S. typhi. In patients for whom a diagnosis of typhoid was based only on a significant Widal-test titre, 31 (41%) of 76 sera had IgM antibodies to the H or O or both antigens of S. typhi. Some cross-reactivity of the IgM antibodies was detected, especially with the O antigens of S. paratyphi A and B. A total of 82 sera from non-typhoidal fevers (leptospirosis, typhus, dengue fever) showed no reactivity in this test. In normal sera there was no detectable IgM to the O antigen of S. typhi and only a small number (3.9%) had low levels of IgM to the H antigen. The significance and potential importance of this simple, sensitive, specific and economical test is discussed.     

Human parvovirus B19 infected fetal liver as a source of antigen for a radioimmunoassay for B19 specific IgM in clinical samples.

A radioimmunoassay for human parvovirus B19 IgM was developed using virus antigen derived from infected fetal liver obtained post mortem. The specificity and sensitivity of this assay, compared with an established radioimmunoassay using serum antigen, was determined by testing 126 sera by both techniques. The results obtained demonstrated close concordance. False negative results were not obtained using fetal liver antigen in 58 tests on known B19 IgM negative sera. Sixty-four IgM positive sera gave positive results using fetal liver derived antigen and the results obtained were quantitatively similar. Four sera gave false positive results using liver antigen but at a very low level. In view of these results we were able to establish a routine diagnostic service for B19 IgM using fetal liver derived antigen, and the results obtained on the first 459 clinical specimens are presented.     

Persistence of parvovirus B19 IgM antibodies and DNA in pure red cell aplasia resulting in myelodysplasia--a case report

Human erythrovirus B19 (B19), previously known as parvovirus B19, is a small spherical, non-enveloped single stranded DNA virus. It has been shown to cause a wide spectrum of clinical conditons including various hematological disorders. We report here for the first time from Inida a case of pure red cell aplasia in a 45-year-old female for last 7 years due to chronic persistent B19 infection leading to myelodysplasia after 4 years. Her sera were positive for two times 4 months apart for B19 IgM and B19 DNA at the initial stage. Presently the patient is on repeated blood transfusion on every 15-20 days.     

High-sensitivity PCR detection of parvovirus B19 in plasma

Parvovirus B19 (B19) is a human pathogen transmitted to susceptible individuals via respiratory secretions and contaminated blood or blood products. B19 levels in pooled plasma of less than 10(4) genome equivalents/ml may not be infectious, while those greater than 10(7)/ml are capable of transmitting infection. A World Health Organization (WHO) B19 DNA international standard has been recently introduced. The purpose of the present work was to develop a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) calibrated against the WHO B19 DNA international standard which could easily and reliably detect B19 DNA levels in plasma above 10(4) IU/ml (6.5 x 10(3) genome equivalents/ml). A B19 PCR-ELISA system was developed which uses a dinitrophenylated oligonucleotide probe to detect immobilized biotinylated amplicons following single-round PCR amplification. The level of B19 DNA (in international units per milliliter) in individual and pooled plasma specimens was evaluated. Proteinase K treatment of plasma was found to be sufficient to quantitatively release B19 DNA. The B19 PCR-ELISA had a sensitivity of detection of 1.6 x 10(3) IU/ml B19 DNA and a dynamic range extending from 8 to 1,000 IU of B19 DNA (equivalent to 1.6 x 10(3) to 2 x 10(5) IU of B19 DNA/ml). Furthermore, the antibody profile of pooled plasma products was determined in terms of B19 immunoglobulin G (IgG) (in international units per milliliter). The B19 IgG level was found to be 64.7 +/- 17.5 IU/ml (mean +/- standard deviation). The B19 PCR-ELISA, which is calibrated against the B19 DNA international standard, may have an application for the rapid screening of plasma minipools for B19 DNA, thereby leading to an improvement in blood product safety.     

Dot immunoperoxidase assay for detection of parvovirus B19 antigens in serum samples.

We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus B19 capsid antigens in human sera. The assay was performed with serum specimens dotted onto nylon membranes. VP1 and VP2 B19 antigens, which represent 4 and 96% of the capsid, respectively, were detected with a pool of monoclonal antibodies directed against the two proteins, and the complex was visualized by immunoperoxidase staining. The assay could be performed in about 4 h, and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane at the site of positive specimens. A total of 541 serum samples from different subjects and with different laboratory evaluations with regard to B19 infection were analyzed. The results obtained by the dot immunoperoxidase assay were compared with the results obtained for the presence of B19 DNA by dot blot hybridization and nested PCR. With optimized working conditions, the dot immunoperoxidase assay was able to detect the presence of B19 with a sensitivity comparable or slightly higher than that achieved by dot blot hybridization but less than that achieved by nested PCR. Since the level of sensitivity of the dot immunoperoxidase assay proved to be appropriate for the detection of acute B19 infection, and since the cost, time to a result, and versatility of the assay are important issues, from our evaluation, the dot immunoperoxidase assay described may be particularly suitable for large-scale screening of samples and a good alternative to DNA detection methods in the routine laboratory evaluation of B19 infection.     

Diagnosis of human parvovirus B19 infections by detection of epitope-type-specific VP2 IgG.

In the B19 VP2 molecule an immunodominant heptapeptide epitope has been detected, recently for which IgG antibodies are synthesized exclusively in the acute phase of B19 infection. Using this acute-phase-specific epitope (KYVTGIN) a 2(nd)-generation epitope-type EIA was developed, which compares serum IgG activity for native VP2 capsids exhibiting conformational VP2 epitopes with IgG activity for the KYVTGIN epitope. In this study the diagnostic performance (clinical sensitivity and specificity) of the 1st and 2nd-generation epitope-type EIAs and of a peptide-based EIA utilising as antigen the KYVTGIN epitope alone was assessed in comparison with various high-quality IgM- and IgG- based B19 assays. Serum samples from 489 patients with B19-related symptoms and asymptomatic controls from three countries were studied. Among 323 patients with B19-IgG, 20% were diagnosed as acute infection, 73% had past immunity and 7% were not classified due to contradictory results among the different assays. The unclassified samples were explored for viral strain diversity by PCR and DNA sequencing but all sequences obtained were B19-like with variance of only a few per cent. The 2nd-generation epitope-type EIA had a diagnostic sensitivity of 98% and a diagnostic specificity of 94%. In combination with conventional approaches, the epitope-type assays increase greatly the accuracy of B19 serodiagnosis.