Vol. 28, No. 4


0095-1137/90/040719-05$02.00/0 Copyright © 1990, American Society for Microbiology

Development of a Highly Specific and Sensitive Rubella Immunoglobulin M Antibody Capture Enzyme Immunoassay That Uses Enzyme-Labeled Antigen HELENA SEPPANEN Labsystems Research Laboratories, Pulttitie 8, SF-00810 Helsinki, Finland Received 30 June 1989/Accepted 14 December 1989

An enzyme immunoassay (EIA) for serum immunoglobulin M (IgM) antibodies to rubella virus based on enzyme labeling of viral antigen was developed. The sensitivity of the EIA for the detection of recent rubella virus infection.was evaluated by using 115 rubella-IgM-antibody-positive serum specimens, which were confirmed as positive by Rubazyme M (Abbott Diagnostics). In addition, 12 individuals, 2 of whom were exposed to rubella through vaccination and 10 of whom were exposed through natural infection, were studied, and the results were compared with those obtained by indirect EIA (Rubelisa M; Electro-Nucleonics, Inc.) and immunoblotting. The sensitivity of the newly developed EIA with sera from these individuals was 100%. Serum specimens from two patients indicated that the IgM antibodies were detected by the newly developed EIA at the same time as IgM antibodies were detected by immunoblotting and before positive reactions were detected by an indirect EIA. The reference population consisted of 564 healthy blood donors and hospitalized patients (150 serum specimens). In addition, 145 serum specimens commonly giving false-positive reactions in conventional rubella IgM EIAs were studied. With these specimens, no false-positive reactions were observed. Positive IgM responses, which could not be confirmed by immunoblotting, were observed in two samples from the reference population. However, these two samples were rubella IgG positive. The overall specificity of the EIA was 99.8%.

Rubella virus, a major human pathogen, is an enveloped, positive-strand RNA virus that belongs to the family Togaviridae (25). Viral infection normally causes a mild, selflimited disease (German measles), but if acquired during the first trimester of pregnancy, it may cause fetal damage, including congenital heart disease, deafness, and mental retardation. Thus, the precise and reliable diagnosis of recent rubella virus infections by demonstration of virusspecific immunoglobulin M (IgM) antibodies is important, particularly in cases of primary postnatal and congenital infections. Detection of rubella-specific IgM by sucrose density gradient fractionation (2, 29) with a hemagglutination inhibition test has been a standard method, but more sensitive immunoassays are used today. Two types of enzyme immunoassays (EIAs) are available for detecting rubella IgM antibodies: indirect and antibody capture. In an indirect EIA, the antigen is adsorbed to a solid phase and the specific IgM is detected by enzyme-labeled anti-human IgM antibodies. However, indirect rubella IgM tests have a tendency to give false-positive results due to rheumatoid factor (RF) associated with high levels of specific IgG (1, 4, 6). The level of specific IgG has more influence than the level of RF on the resulting false positivity, and in the presence of a low titer of specific IgG the influence of RF is not perceptible (6). Also, a reaction of a

antigen (4). The main advantage of all antibody capture assays is, in general, the elimination of false-positive results due to RF. In addition, capture tests are more sensitive than other tests since there is no competition between IgG and IgM for antigenic sites. However, in antibody capture ETAs using labeled antibody, the interactions between solid-phase adsorbed anti-human IgM and the Fc portions of labeled anti-rubella IgG antibodies may cause false-positive reactions (16). This can be overcome by removing altered IgG from the conjugate or by conjugating only the F(ab)2 portion (16). Another method that can be used to ensure specificity is to use a control well for each serum specimen and omit the addition of antigen. When virus is used as an enzyme conjugate, this kind of interaction is eliminated and less antigen is needed than in indirect assays (4). Here, I describe a simple and rapid three-step rubella IgM antibody capture EIA with purified labeled viral antigen as an enzyme conjugate, and I evaluate the sensitivity and specificity of the assay. MATERIALS AND METHODS Sera. The sera examined for rubella IgM antibodies included seroconversion serum panels (from Boston Biomedica Inc., Mansfield, Mass.) from two vaccinated people (patients 1 and 2) and 10 paired serum specimens from patients exposed to rubella through natural infection. Samples were collected between 1 and 35 days after the onset of rash. The seroconversion of these patients was confirmed by a single-radial hemolysis test (Orion Diagnostica, Helsinki, Finland) and by complement fixation. In addition, a panel of 115 serum specimens determined as rubella IgM positive by another commercial test kit (Rubazyme M; Abbott Diagnostics, North Chicago, Ill.) was tested. The reference specimens consisted of 564 serum specimens from healthy blood

virus-specific IgG with autologous IgM-RF may cause false

positivity (6). Consequently, confirmatory tests or sample pretreatments are essential in this type of assay. In addition, a high level of specific IgG may lead to false-negative tests when the IgM titer in a sample is low, because of the competition between IgG and IgM antibodies (6). In an antibody capture assay, antibodies to human IgM are adsorbed to a solid phase (11) and the specific antibodies are detected by using rubella antigen and labeled antirubella antibodies (1, 10, 16, 21, 28) or by using enzyme-labeled 719




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donors and 150 serum specimens from hospitalized patients. Other IgM assays were not used to test the reference population. To assess further the specificity and performance of the test, 30 serum specimens with heterophile antibodies (Paul-Bunnell positive), 64 serum specimens with RF, 10 serum specimens with hepatitis A virus IgM antibodies, 7 serum specimens with cytomegalovirus IgM antibodies, and 34 serum specimens with toxoplasma IgM antibodies were tested. Antigen. The Therien strain of rubella virus was grown in B-Vero cell cultures and purified as described previously (23). Purified virus was used as an antigen in immunoblotting and as an enzyme conjugate in the development of the EIA. Antigen labeling. Purified rubella virus was conjugated with horseradish peroxidase (Boehringer GmbH, Mannheim, Federal Republic of Germany) by using the periodate method (22).

REFERENCE POPULATION in the rubella IgM antibody capture EIA described in the text.

IgG EIA. Rubella IgG antibodies were measured with a Labsystems rubella IgG EIA kit (Rubella IgG EIA; Labsystems Oy, Helsinki, Finland). IgM EIA. Microdilution plates (Labsystems Oy) were coated with anti-human IgM antibodies. Sheep antibodies to human IgM were purified by using Sepharose 4B (Pharmacia, Stockholm, Sweden) affinity chromatography prepared as described previously (18). Test sera were diluted 1:100 with phosphate-buffered saline (10 mM sodium phosphate, 0.9% NaCl [pH 7.4]) containing 10% protein carrier and Tween 20 and were incubated for 1 h at 37°C. Unbound proteins were removed by rinsing three times with phosphate-buffered saline-Tween 20 (washing solution). Rubellaspecific IgM antibodies were detected by incubation for 2 h at 37°C with horseradish peroxidase-conjugated rubella virus. Unbound conjugate was removed by washing three times with washing solution, and the quantity of bound


VOL. 28, 1990

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FIG. 2. Characterization of the purified rubella virus used for labeling and IgM immunoblot analysis of seroconversion serum panels P2 (patient 2; lanes 1 to 6) and Pi (patient 1; lanes 1 to 6). Lanes 1 to 4 at left show the following. Lane 1, Molecular weight markers in thousands (K). Lane 2, Purified rubella virus under reducing conditions; major structural polypeptides El (molecular weight, 58,000) and E2 (molecular weight, about 42,000 to 47,000) and capsid protein C (molecular weight, 33,000). Lane 3, Immunoblotting of reduced rubella virus with rubella-specific IgG antibodies. Lane 4, Reactivity of rubella-IgM-positive serum with structural proteins of rubella virus under nonreducing conditions; dimeric forms of envelope proteins El-El and El-E2 and monomeric El are shown.

conjugate was measured by incubation with 3,5,3',5'-tetramethylbenzidine (Sigma Chemical Co., St. Louis, Mo.) and H202 as substrates. The enzyme reaction was stopped after 30 min with H2SO4, and the amount of bound rubella-specific IgM antibody was determined by measuring the A450 (Muttiscan MC; Labsystems Oy, Helsinki, Finland). A reagent blank, negative serum, and calibrator serum were included in each test run. The cutoff value was defined as 0.1 + the absorbance value of the reagent blank. It was calculated to be twice the mean value of negative samples plus three standard deviations. Immunoblotting. Purified rubella virus proteins (2 ,ug per lane) were separated under reducing or nonreducing conditions by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% [wt/vol]) as described by Laemmli (17) and were transferred to a nitrocellulose sheet (27) essentially as described previously (24). Serum dilutions were 1:100 for IgG and 1:50 for IgM. Other commercial tests. Rubenz M II (Northumbria Biologicals, Ltd., Northumberland, England), Rubelisa M (Electro-Nucleonics, Inc., Columbia, Md.), and Rubazyme M (Abbott Diagnostics) were also used to determine the amount of rubella IgM antibody. The tests were carried out according to the instructions of the manufacturers. RESULTS AND DISCUSSION The distribution of absorbance values for 115 serum specimens known to contain anti-rubella IgM antibodies and for 714 serum specimens from blood donors and hospitalized patients is summarized in Fig. 1. Low-IgM positive samples were separated well from negative samples. Over 99% of the IgM-negative serum samples gave values less than half of the cutoff level. The overall specificity of the test was 99.8%, and the sensitivity was 100% with the material used. One serum sample from a blood donor and one from a hospitalized patient were determined as low positives repeatedly. In immunoblot analysis, these two serum specimens were found to contain rubella IgG antibodies but not IgM antibodies. One explanation for this reactivity may be IgG-IgG interactions in which specific IgG antibodies from sera physically or immunologically react with IgG antibodies

against human IgM (5). It is also known that low levels of IgM antibodies may be detected after rubella virus reinfection (19) and that rubella virus may persist in lymphocytes (7) or it may form immunocomplexes that persist for at least 2 years after rubella vaccination (8). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and IgG immunoblotting results used for controlling the quality of purified rubella antigen are shown in Fig. 2. Under reducing conditions, three major protein bands with apparent molecular weights of approximately 58,000 (El), 42,000 to 47,000 (E2), and 33,000 (C) were resolved with the purified rubella antigen. These proteins are the major structural proteins of rubella virus (23). In addition, dimeric forms of El-El and El-E2 can be observed in the figure. These major bands were immunoreactive with rubella-specific IgG antibodies in immunoblotting (Fig. 2). Under nonreducing conditions, the C polypeptide is known to migrate as a disulfide-linked dimer with a molecular weight of 68,000 (12). Also, the El and E2 proteins migrate as dimeric forms linked by disulfide bonds (15), which can be separated only under strong reducing conditions (14). Under nonreducing conditions, the following structural viral polypeptides were immunoreactive with IgM antibodies: the El-El dimer, the El-E2 dimer, and the El polypeptide (Fig. 2). Serial serum samples from two vaccinated individuals (patients 1 and 2) indicated that the rubella IgM antibodies could be detected with the test described here at the time IgM seroconversion was observed in immunoblotting (Fig. 2). The IgG antibody level in these two cases remained relatively low in the EIA (Table 1). It should be noted that in one of the two samples (that from patient 1), IgM antibodies were detected earlier with the developed IgM capture test than with the indirect rubella IgM test (Table 1). The results of the test described here with 10 paired serum specimens from patients exposed to rubella through natural infection are shown in Table 2. IgM antibodies could be detected in one sample as early as the first day after the onset of rash as well as in one sample taken 35 days after the onset of rash. It is probable that IgM antibodies could have been detected for a longer period of time, but suitable serum samples were not available. Serial serum specimens from these 10 patients were not taken.




TABLE 1. Rubella-specific IgG and IgM antibodies in two seroconversion serum panels

Optical density Opiadest Serum panel Sample Rubella at 450 nm with patc405density source and date IgG value rubella IgM with Rubelisa sample no. (day-mo-yr) (EIU)l antibody capture Mc EIAb Patient 2 1 2 3 4 5 6

Patient 1 1 2 3 4 5 6

05-11-87 10-11-87 16-11-87 23-11-87 30-11-87 02-12-87

1 2 15 52 57 67

0.000 0.045 1.238 1.445 1.133 1.114

0.018 0.034 0.505 0.803 0.655 0.642

05-11-87 11-11-87 13-11-87 16-11-87 24-11-87 01-12-87

0 6 17 56 62 77

0.000 0.319 1.301 1.242 0.885 0.656

0.025 0.096 0.665 >1.000 0.461 0.321

a EIU, Enzyme immunoassay units. Cutoff value, 15 EIU. b Cutoff value, 0.200. C Cutoff value, 0.280.

The high specificity of the capture assay described in this paper is clearly demonstrated with sera known to have a tendency to give false-positive reactions in rubella IgM assays. None of the 30 heterophile-antibody-positive serum specimens were positive in this assay, although it has been found that sera from patients with Epstein-Barr virus infectious mononucleosis may give false-positive results in some IgM immunoassays (3, 20). It has been assumed that rubellavirus-specific IgM antibodies derived from sera of patients with Epstein-Barr virus may result from Epstein-Barr virus stimulation of B lymphocytes already committed to prior stimulation of rubella virus. Alternatively, this IgM reactiv-

ity may be due to the presence of cellular impurities in the semipurified antigen preparations (9). All 64 rubella-IgGpositive serum samples with RF tested negative in the antibody capture assay described here, whereas one of these samples gave a low positive result in Rubenz M II. This serum gave a high positive value (149 enzyme immunoassay units) in the rubella IgG test, and the RF-latex titer was 4,096. It may be assumed that there is no question of recent rubella virus infection in this case. In indirect enzyme-linked immunosorbent assays, false-positive IgM results are obtained mainly with cytomegalovirus-IgM-positive sera derived from patients with primary cytomegalovirus infections (26). In the rubella IgM antibody capture assay, all seven of these serum specimens remained negative. All 10 antihepatitis-A-virus-IgM-positive serum specimens and all 34 anti-toxoplasma-IgM-positive serum specimens were negative in the antibody capture assay. An antibody capture assay seems to be a very sensitive and specific method for the detection of IgM antibodies, as shown by others (4, 13). The capture test developed by Bonfanti et al. (4) is also based on enzyme-labeled antigen. However, they used semipurified rubella virus for labeling. In my experiments, the specificity was clearly affected when semipurified antigen was used. In their assay, the incubation time for the sample was 2 h at 37°C and the conjugate was incubated overnight at 4°C, but the incubation time of the conjugate could be reduced to 3 h at a temperature of 45°C. In my assay, good results were obtained when the sample and the conjugate were incubated for 1 and 2 h, respectively, at 37°C. Rubenz M IL, which is also an antibody capture assay, uses a 1-h incubation time for the sample, overnight incubation for the antigen, and a 3-h incubation time for the conjugate at room temperature. When Rubazyme M is used, samples are pretreated for 1 h at 45°C and then incubated for 1.5 h with coated beads and enzyme conjugate, respectively, at the same temperature.


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ACKNOWLEDGMENTS I thank Klaus Hedman (Department of Virology, University of Helsinki, Helsinki, Finland) and Jukka Suni (Aurora Hospital, Helsinki, Finland) for providing serum samples. I also thank MarjaLiisa Huhtala and Christian Oker-Blom for valuable comments on the manuscript.


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Development of a highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses enzyme-labeled antigen.

An enzyme immunoassay (EIA) for serum immunoglobulin M (IgM) antibodies to rubella virus based on enzyme labeling of viral antigen was developed. The ...
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