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

Abstract

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.

Infections by the human parvovirus B19 virus (B19V) in pregnant women are often overlooked because these patients are frequently asymptomatic or exhibit symptoms commonly associated with pregnancy, such as fatigue and joint pain. However, B19V infections of pregnant women have been associated with poor fetal outcomes, including fetal anemia, nonimmune hydrops, spontaneous abortion, and stillbirth (1, 7, 10, 12, 18). The transplacental transmission rate of B19V has been estimated to be about 33% (17). Although the literature has reported studies with widely divergent rates of B19V-associated fetal death, the majority of the studies have shown an overall risk of from <5 to 9% (9, 17, 19). Therefore, it is important for the physician to have accurate clinical data regarding the immune status of a pregnant woman when an exposure to or infection by B19V is suspected.

In an immunocompetent individual, B19V infection has an incubation period of approximately 10 days (2), after which the virus can be detected within respiratory secretions and the blood of an infected individual. The peak of viremia occurs 5 to 7 days prior to the appearance of specific clinical symptoms, such as rash or arthalgia, and before the levels of antibodies in serum are measurable. B19V-specific immunoglobulin M (IgM) antibodies appear first, followed by IgG a few days later. On average, IgM antibodies will remain detectable for 1 to 2 months, but their levels may be elevated for up to 3 to 4 months. The presence of IgM antibodies in serum is an indicator of an acute or recent infection. In contrast, IgG antibodies remain elevated for years, and their presence without detectable IgM is usually interpreted as an indicator of a previous infection. The absence of both IgM and IgG antibodies would indicate that the patient has not been previously infected. Between 50 and 70% of women of childbearing age have IgG antibodies to B19V, inferring previous infection (5, 15).

During an acute B19V infection, B19V-specific antibodies to both linear and conformational epitopes are produced against the viral capsid proteins, VP1 and VP2 (20). Söderlund et al. (20) estimated that IgG antibodies to linear epitopes of VP2 disappear at approximately 6 months postinfection, leaving only those antibodies that recognize undenatured VP2. This loss of epitope recognition can be problematic when using Escherichia coli-based enzyme immunoassays (EIAs) for the detection of B19V-specific IgG antibodies because they employ only denatured antigen. Studies by Franssila et al. (8) later supported the loss of epitope recognition finding as well. More recently it was observed that approximately 16% of serum samples (12 of 75) that were confirmed to be positive for B19V IgG did not react with denatured VP1 or denatured VP2 (14). This lack of B19V IgG reactivity with denatured epitopes presents a limitation to serological assays incorporating denatured B19V antigens.

Based on these findings, both the source and nature of the viral antigen(s) used in a serological assay are important variables to consider in evaluating its performance. Capture EIAs employing native or recombinant antigens are the best choices for measuring these antibodies. Systems employing either an E. coli or baculovirus expression vector have been described and used to express B19V capsid protein. E. coli-based expression vectors produce linear proteins. In contrast, baculovirus-based expression vectors produce conformational proteins. In fact, baculovirus-expressed VP2 (BVP2) has been shown to self-assemble into empty capsids whose appearance, as evidenced by electron microscopy, is very similar to that of the native B19 virion capsids (13).

A split-sample study was conducted to compare the results of the BVP2 EIAs to those of the currently available E. coli-expressed VP1 (EVP1) EIAs for detecting B19V IgM and IgG. Both methods utilized an EIA format, but they differed in the type and nature of the viral antigen being used. Confirmatory testing for discrepant IgM or IgG data was accomplished by using B19V-specific immunofluorescent assays (IFAs) which incorporate conformational VP1 antigen expressed from a baculovirus vector (BVP1).

Study population.

Serum samples from 269 pregnant women were tested by both the BVP2 EIA and the EVP1 EIA for the detection of IgM and IgG antibodies to B19V. In some cases, more than one sample was collected from the same patient due to the physician's request for additional (follow-up) serological testing. Consequently, a total of 307 samples were tested for B19V IgM and 311 were tested for B19V IgG. The serological testing for B19V IgM and IgG antibodies was ordered for these women either because of a known or suspected exposure to B19V (65%) or the appearance of symptoms consistent with B19V infection (i.e., fever, rash, and/or arthalgia) (22.5%) or as a general request for B19V serological testing (12.5%).

Five-milliliter volumes of whole blood were drawn from patients and collected into red-top tubes. The serum fractions were allowed to clot at room temperature prior to centrifugation. One-milliliter volumes of sera were transferred aseptically to sterile containers before being shipped at 4 to 8°C to a reference laboratory for EVP1 EIA testing. The remaining (discarded) aliquots of sera were stored at −20°C until being batch tested by BVP2 EIA and BVP1 IFA at Magee-Womens Research Institute. Approval for use of discarded sera in this study was granted by the Magee-Womens Hospital Institutional Review Board.

MATERIALS AND METHODS

The MRL Diagnostics (Cypress, Calif.) enzyme-linked immunosorbent assays for parvovirus B19V IgM and IgG detection utilize a linear VP1 antigen generated from an E. coli expression vector. The EVP1 EIAs were performed at a national reference laboratory, and the data were reviewed by J.A.J. In the B19V IgM and IgG EVP1 EIAs, the microwells are coated with a recombinant B19V VP1 antigen. The B19V-specific IgM and IgG serological tests incorporate a peroxidase-conjugated anti-human IgM or IgG, respectively, along with tetramethylbenzidine (TMB) substrate. In the IgM EVP1 EIA, the patient sera and controls are diluted in a solution containing hyperimmune anti-human IgG-precipitating immunoglobulin to remove both free and complexed IgG from the sample.

The Biotrin International (Dublin, Ireland) enzyme-linked immunosorbent assays for B19V IgM and IgG both use an undenatured VP2 antigen generated from a baculovirus expression vector. These B19V IgM and IgG assays are sandwich EIAs. The B19V IgM BVP2 EIA is a μ capture assay. IgM antibodies present in the serum are captured by rabbit anti-human IgM (μ-chain specific) coated onto the surfaces of the wells of a microtiter plate. The assay incorporates a biotinylated B19V recombinant VP2 antigen, streptavidin-peroxidase, and TMB substrate. The B19V IgG BVP2 EIA utilizes recombinant B19V VP2 antigen coated onto the wells of a microtiter plate to capture B19V-specific antibodies from serum. The captured IgG antibodies are detected by a rabbit anti-human IgG–horseradish peroxidase conjugate and the TMB substrate.

The Biotrin B19V BVP1 IFAs utilize an indirect-immunofluorescence antibody technique. Patient serum is incubated with B19V recombinant VP1 antigen expressed in Spodoptera frugiperda cells stabilized on a glass slide. The B19V antibodies, if present, bind to the nondenatured VP1 antigen. Bound antibody reacts with a fluorescein-labeled anti-human IgM or IgG antibody, and the complex is visualized with the aid of a fluorescence microscope. To prevent interference from rheumatoid factor and to reduce IgG competition in the IgM assay, samples are pretreated with an adsorbent reagent prior to testing.

The BVP2 EIAs and BVP1 IFAs were performed at Magee-Womens Research Institute. The methods outlined in the package insert were followed precisely for all of the testing procedures. The BVP1 IFAs for detecting B19V-specific IgM and IgG antibodies were used as confirmatory tests to resolve discrepancies between the EVP1 EIA and BVP2 EIA results.

RESULTS

Agreement between the BVP2 EIAs and EVP1 EIAs for detection of B19V-specific IgM and IgG antibodies.

Over 300 serum samples, obtained from 269 pregnant women, were evaluated in a split-sample study for the detection of B19V-specific IgM and IgG antibodies, using BVP2 EIAs and EVP1 EIAs, respectively. Tables 1 and 2 respectively illustrate the high degree of agreement between these two different EIAs for detecting B19V-specific IgM (92.2%) and IgG (89.7%) in sera of pregnant women. The discordant results revealed 24 of 307 (7.8%) and 32 of 311 (10.3%) discrepancies for B19V IgM and IgG, respectively. A significant number of the IgM and IgG discrepancies, 17 of 24 (71%) and 16 of 32 (50%), respectively, resulted from equivocal data generated by the EVP1 EIAs. The percentages of EVP1 EIA IgM and IgG equivocal data seen in this study were similar to those seen historically with these assays.

TABLE 1.

Comparison of E. coli- and baculovirus-based EIAs for detecting B19V IgM antibodies

Baculovirus-based B19V IgM EIA result	E. coli-based B19V IgM EIA result			No. of concordant results/total	No. of discordant results/total
	Positive	Negative	Equivocal
Positive	21	2	0	21/23	2/23
Negative	5	262	17	262/284	22/284
Equivocal	0	0	0	0/0	0/0
Total				283/307a	24/307b

^a92.2% agreement. 

^b7.8% disagreement. 

TABLE 2.

Comparison of E. coli- and baculovirus-based EIAs for detecting B19V IgG antibodies

Baculovirus-based B19V IgG EIA result	E. coli-based B19V IgG EIA result			No. of concordant results/total	No. of discordant results/total
	Positive	Negative	Equivocal
Positive	171	15	15	171/201	30/201
Negative	1	108	1	108/110	2/110
Equivocal	0	0	0	0/0	0/0
Total				279/311a	32/311a

^a89.7% agreement. 

^b10.3% disagreement. 

Resolution of discrepant results by using the confirmatory BVP1 IFAs.

The specimens with discordant BVP2 EIA and EVP1 EIA results were retested by confirmatory BVP1 IFAs for B19V-specific IgM and IgG antibodies. The results of the comparison testing revealed good agreement between the BVP2 EIAs and BVP1 IFAs for both B19V IgM and IgG results (20 of 23 [87%] and 28 of 32 [87.5%], respectively). In stark contrast, the results of the EVP1 EIAs for B19V IgM and IgG agreed with the BVP1 IFA data for only 3 of 23 (13%) and 4 of 32 (12.5%) discrepant specimens, respectively. One of the 24 IgM discordant samples was not retested due to insufficient sample volume.

Of the 17 women with IgM discrepancies that were negative by the BVP2 EIA and equivocal by EVP1 EIA, 8 had clinical histories consistent with a possible exposure to an individual with fifth disease. Only one woman presented with a skin rash. Pregnancy outcomes were available for 11 of the 17 women, including the individual with the skin rash. All 11 women had term deliveries with good outcomes.

Similarly, of the 12 women with the 15 IgG discrepancies that were positive by the BVP2 EIA and equivocal by EVP1 EIA, 6 had clinical histories consistent with possible exposure to an individual with fifth disease. None of these 12 women demonstrated any symptoms consistent with B19V infection, i.e., fever, rash, or arthralgia. Pregnancy outcome data were available for 6 of the 12 women; all 6 had term deliveries with good outcomes.

DISCUSSION

B19V is a member of the family Parvoviridae, genus Erythrovirus (16). It was first identified as a human pathogen in 1975 (6). The major cellular receptor for B19V is the blood group P antigen, globoside (3). It is now accepted that P antigen-positive, B19V-seronegative women are susceptible to infection and, as such, are at risk of adverse fetal outcome if they become infected while pregnant (4). Although the majority of pregnancies complicated by B19V infection result in the delivery of healthy term infants (11), approximately 5 to 9% of them end in fetal death (9, 15, 17). Consequently, it is important to determine the B19V antibody status of pregnant women who may be at risk of infection by B19V or who may have been infected with the virus following exposure.

The data presented here support the effectiveness of the BVP2 EIAs in determining accurately the IgM and IgG statuses of pregnant women following known or suspected exposure to B19V. The analyte-to-analyte comparison revealed a high degree of agreement between the BVP2 EIAs and the EVP1 EIAs for detecting B19V-specific IgM and IgG antibodies in the sera of pregnant women. Despite this fact, the BVP2 EIAs had significantly fewer equivocal results than did the EVP1 EIAs. Equivocal data at best are not useful and at worst are misleading. It is not an understatement to say that unequivocal, or precise, data provide much more useful clinical information to the physician than do equivocal results.

Further confirmation of the accuracy of the BVP2 EIAs for B19V IgM and IgG determinations was obtained upon evaluation of the discordant specimens by the BVP1 IFAs. For the vast majority of discordant samples, the confirmatory BVP1 IFA results agreed with the BVP2 EIA results and not with the EVP1 EIA results. When considering the B19V IgM data alone, there was a trend toward BVP2 EIA-negative, BVP1 IFA-negative, and EVP1 EIA-equivocal data. The opposite was true for the B19V IgG data, with the trend being BVP2 EIA-positive, BVP1 IFA-positive, and EVP1 EIA-equivocal data.

From the clinician's viewpoint, an equivocal test result translates into the need for further patient visits, additional blood sampling, and repeat testing and thus a delay in confirming the patient's immune status. If the individual's serological status remains unresolved, the physician may have to initiate the use of expensive fetal ultrasound screening measures to monitor his/her patient.

In summary, this study illustrates the accuracy of assays utilizing conformational viral capsid proteins to detect B19V IgM and IgG in the sera of pregnant women. Providing fewer inconclusive test results will be helpful to the clinicians faced with making decisions on the extent to which follow-up care is necessary in these pregnant women. Ultimately, a serological assay which produces far fewer equivocal results will be more cost-effective. The savings will be realized by minimization of the need to do repeat serological testing and/or fetal ultrasonography.