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.

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Human erythrovirus (parvovirus) B19 (B19) is a common human pathogen. The recent discovery of three genotypes, 1 to 3, raised issues related to the ability of genotype-specific antigens to cross-react with antibodies elicited by other genotypes. This study assessed antibody capture and immunoglobulin G (IgG) cross-reactivity between genotype 1 and genotype 3 recombinant antigens and analyzed the potential gain of adding VP1 protein to VP2 capsid antigen. Plasma samples from genotype 1-or genotype 3-infected populations were blindly tested with blindly prepared reagents. The IgG reactivity was assessed with baculovirus-expressed VP2 or VP1 and VP2 recombinant genotype 1 or genotype 3 proteins in a standardized enzyme immunoassay (EIA). A high degree of agreement (>95%) between EIA results was observed, with Spearman correlation coefficient and kappa reliability coefficient results of >0.95 for samples from the United Kingdom and >0.77 for samples from Ghanaian children, respectively. Most discrepant results were related to equivocal reactivity. The addition of VP1 to VP2 capsids did not significantly impact antibody detection. These data suggest that the currently available genotype-1-based IgG EIA is suitable to detect antibody to B19 genotype 3 in Ghanaian children. However, samples from the Ghanaian adult population exhibited atypical results in the assay, possibly due to the high levels of nonspecific IgG antibodies found in adults living in this region. Within these limitations, the study demonstrates that genotype 1 and genotype 3 antigens are equally effective in detecting either antibody species.Human erythrovirus (parvovirus) B19 (called B19 in this report) is a nonenveloped single-stranded DNA virus (9, 32). B19 causes a range of diseases and conditions in humans, including fifth disease of childhood, fetal death, various forms of anemia, and other conditions (reviewed in reference 38).Three relatively long polypeptides encoded by the virus are two structural proteins (VP1 and VP2) and one nonstructural protein (NS-1) (8, 27). VP1 (781 amino acids [aa]) and VP2 (554 aa) are encoded by the same open reading frame and translated from two differentially spliced messages (25-27). VP1 is identical to VP2 except for a 227-aa N-terminal extension called the VP1 unique region. These two proteins form capsids (diameters, 19 to 25 nm) containing approximately 96% of VP2 and 4% of VP1 (27). In the eukaryotic baculovirus expression system, VP2 protein spontaneously forms virus-like particles that, under electron microscopy, are indistinguishable from the capsid structure of the native virions (4, 17). VP1 protein alone forms these structures very inefficiently, but when cotransfected with VP2-and VP1-containing recombinant baculovirus, chimeric capsids with VP2 and VP1 are produced (4, 17, 37). Up to 41% of VP1 was reported to be possible to incorporate in the capsid (3).The nature of the immune response to the virus is characterized by the predominance of antibodies against structural proteins. Immunoglobulin G (I...

Parvovirus B19 infection can cause severe effects in high-risk groups including pregnant women and immunocompromised individuals. Although serological detection of B19 infection is commonplace, minimal information is available on the absolute performance characteristics of various tests for the detection of B19 IgM. The performance of the first parvovirus B19 IgM enzyme immunoassay to be cleared by the US Food and Drug Administration (FDA) is described. The immunoassay cut-off has been established using receiver operating characteristic (ROC) analysis giving a sensitivity and specificity of detection of 89.1 and 99.4%, respectively. No cross-reactivity is observed with rubella or other viral disease IgM which cause similar symptomologies to parvovirus B19. Multi-site reproducibility studies have shown high immunoassay reproducibility with detection rates (observed/expected result) of 100% for nonreactive specimens (N=324) and strongly reactive (N=403), respectively. Immunoassay reproducibility ranged from 11.76 to 17.46% coefficient of variation for all reactive specimens tested (N= 12) whereby each specimen was assayed a total of 81 times. Parvovirus B19 IgM seroprevalence of 1% was observed in a US blood donor population (N= 399). In the absence of international performance criteria, this study will be of major benefit to the clinical virologist in assessing immunoassay reliability for the detection of recent infection with parvovirus B19.

Background and ObjectivesAlthough parvovirus B19 is a significant blood product contaminant, few methods other than polymerase chain reaction (PCR) have been developed to detect the presence of the virus.Material and Methods A B19 antigen enzyme immunoassay (EIA) has been developed and the sensitivity of detection is ascertained using dilutions of the B19 capsid protein VP2 and 10-fold dilutions of B19 viraemic serum. Once the assay cut-off was established, a panel of viraemic donations ( n = 70) was screened by the antigen EIA. The B19 immunoglobulin M (IgM) and IgG status of these specimens was also determined. During screening of blood donor units by quantitative PCR, 70 individuals were identified with levels of B19 DNA greater than 10 6 IU/ml at the time of blood donation. ResultsThe sensitivity of the B19 antigen EIA was estimated to be equivalent to between 10 8 and 10 9 IU/ml B19 DNA or 1-10 pg/ml of recombinant capsid protein. B19 detection was significantly enhanced when viraemic specimens were pretreated with a low pH proprietary reagent. Unlike other virus-detection assays, detection of the B19 antigen was not affected by the presence of B19 IgM or IgG antibodies. In addition, the assay was capable of detecting all three genotypes of human erythrovirus. Combined specimen analysis by the B19 antigen assay and a B19 IgM assay facilitated the detection of 91% of acute B19 infections in the test population. ConclusionIn combination with B19 IgM detection, application of the B19 antigen EIA is a flexible and efficient method of detecting recent B19 infection and can be used as an alternative to PCR.