Vol. 29, No. 7

JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1991, P. 1376-1381 0095-1137/91/071376-06$02.00/0 Copyright ©D 1991, American Society for Microbiology

Evaluation of a Synthetic-Peptide Enzyme-Linked Immunosorbent Assay for Immunoglobulin M to Human Parvovirus B19 E. FRIDELL,l.2* B. J. COHEN,3 AND B. WAHREN"2 Department of Virology, National Bacteriological Laboratory,' and Department of Virology, Karolinska Institute,2 S-105 21 Stockholm, Sweden, and Virus Reference Laboratory, Central Public Health Laboratory, London NW9 SHT, United Kingdom3 Received 29 May 1990/Accepted 15 April 1991

A synthetic peptide corresponding to a part of the virus protein 1-virus protein 2 overlapping region of human parvovirus B19 was used in an indirect enzyme-linked immunosorbent assay. Antibodies of the immunoglobulin (Ig) M class were measured in serum samples from patients with erythema infectiosum and controls. In comparison with an IgM assay using native B19 viral antigen, the peptide antigen assay was 92% sensitive and 87% specific. B19 IgM reactivities were seen in a limited number of children with other viral diseases. Specific IgM reactivities to short synthetic viral peptides have previously been reported only with Epstein-Barr virus. Since other sources of viral antigen are limited, the peptide antigen assay may be a useful alternative for the diagnosis of B19-associated disease in human beings.

peptide as antigen (peptide antigen assay) has therefore been developed and is described in this article.

The human parvovirus B19 was found by chance in sera from blood donors in 1975 (7). About 5 years later, two men with mild fever were shown to have B19 virus in their blood (27). In 1982, the virus appeared to be the main cause of aplastic crisis in patients with sickle cell anemia (24) and later in patients with other chronic hemolytic diseases (36). When cared for in the hospital, these patients have transmitted the B19 virus to other patients (3, 11). In the spring of 1983, an outbreak of erythema infectiosum occurred in a London school and B19 virus was identified as the etiological agent (1). The virus causes arthritis or arthralgia in about 50% of infected adult women (26). Some of these women experience the arthralgia for years, and rheumatoid arthritis has been a common differential diagnosis. During widespread outbreaks of B19 infection, nonimmune women of childbearing age are at some risk of acquiring an intrauterine infection. Hydrops fetalis and fetal loss may ensue during any part of the pregnancy (23). However, intrauterine B19 infection rarely if ever causes fetal anomalies (31, 33). During 1988 and 1989, persistent (i.e., lasting months to years) infections with B19 virus were described as causing intermittent anemia in patients with damaged or weakened immunity, e.g., leukemic children, bone marrow recipients, or patients with congenital immunodeficiency or AIDS (12, 17, 18, 32). These patients have intermittent or chronic virus shedding for long periods and may constitute a risk for other immunodeficient patients. Since B19 is a stable virus and is difficult to inactivate, it has also been spread through transfusion with factor VIII concentrates (2, 21). Investigations of erythema infectiosum outbreaks will confirm the diagnosis of B19 virus infection and identify people at risk of becoming infected through contact. The B19 virus can only be cultivated in limited amounts, and therefore antibody assays have been based on whole virions prepared from the plasma of viremic blood donors (viral antigen assay). These donors are rarely found, and the assays have been limited to a few laboratories. An assay for B19 immunoglobulin (Ig) M and IgG using a synthetic

*

MATERIALS AND METHODS

Peptide ELISAs for B19 virus IgM and IgG. The peptide enzyme-linked immunosorbent assays (ELISAs) for IgM and IgG were performed with a synthetic peptide. The peptide was identified by systematically synthesizing peptides, 10 amino acids long with a 2-amino-acid overlap, corresponding to the whole sequence of B19 virus protein 2 (VP2; 8, 14, 22, 25). VP2 was chosen since this is the major capsid protein of B19 virus. The amino acid sequences of neighboring reactive peptides included two cysteines. A 24-amino-acid-long peptide (FSPAASSCHNASGKEAKV CTISPI), containing the initially identified reactive peptide (underlined) and stabilized through a sulfur bridge between the cysteines, was prepared by solid-phase synthesis on an Applied Biosystems 430A peptide synthesizer. The peptide was prepared to greater than 99% purity (14). A 100-,u volume of a peptide solution containing 10 ,ug of the peptide per ml was used to coat 96-well flat-bottom microtiter plates (Nunc, Roskilde, Denmark). The plates were freeze-dried and stored at 4°C. After washing, 100-,Iu volumes of control sera or samples for the IgM assay, diluted to 1:20, were dispensed in duplicate. For the IgG assay, the control sera and samples were diluted 1:20 and 1:200, respectively. After incubation for 45 min at 37°C, the plates were washed. Alkaline phosphatase-labeled goat anti-human IgM or IgG (Sigma Chemical Co., St. Louis, Mo.) was added, and the plates were incubated for a further 45 min at 37°C. After a final wash, substrate solution (p-nitrophenylphosphate; Sigma) was added and A405 values were read. To investigate blocking of IgM by specific IgG, sera nonreactive for IgM and reactive for IgG in peptide and viral antigen assays were absorbed with rheumatoid factor (RF) absorbent (Behring, Marburg, Germany) and retested in the peptide antigen assay for IgM. The efficacy of RF absorption was controlled by using RF-positive sera (Department of Immunology, National Bacteriological Laboratory, Stockholm). The cutoff value for IgM was the mean value plus three times the standard deviation for the 158 blood donors. This

Corresponding author. 1376

VOL. 29, 1991

SYNTHElIC-PEPTIDE ELISA FOR IgM TO B19 PARVOVIRUS

value gives the best correlation between the viral 1gM assay and the peptide IgM antigen assay in the 281 serum samples. An IgG A405 value rise was defined as at least a doubling of the value between acute-phase and convalescent-phase serum samples, provided that the A405 value of the convalescent-phase serum sample was above 0.40. Viral antigen radioimmunoassays for B19 1gM and IgG. Viral antigen radioimmunoassays for B19 virus IgM and IgG were performed as described previously (6). In short, antihuman IgM was coated to the solid phase, 1gM in patient serum samples was captured, and B19 virus was bound. Monoclonal antibody to B19 was used to indicate the virus binding, and finally this reaction was detected by a radioactively labeled antibody directed against mouse IgG. In both the IgG and IgM assays, measurements in arbitrary units higher than 3 were regarded as positive, those between 1 and 3 were equivocal, and those below 1 were negative. Assays for other viral Igs. The assays for IgM(,) capture ELISAs (9) were performed by using antigens prepared in house (i.e., for cytomegalovirus, varicella-zoster virus [VZV], and measles virus) or commercially available (hemagglutination inhibition antigen for rubella; Wellcome Research Laboratories, Beckenham, England). The viral antigens were labeled with peroxidase by the periodate method (34). Indirect ELISAs for IgG and IgM (29, 30) were performed with an in-house-purified nucleocapsid antigen for herpes simplex virus and a purified whole viral antigen for Russian spring-summer encephalitis virus (received from C. Kunz, Vienna, Austria). For cytomegalovirus, VZV, rubella, and measles IgG, indirect ELISAs were performed with the same antigen as for IgM(,u) capture ELISAs. All antigens for complement fixation assays were prepared at the National Bacteriological Laboratory, Stockholm. Cells infected with the Sicilian strain of sandfly fever-Sicilian virus (10) were used in an immunofluorescence assay. EpsteinBarr virus viral capsid antigen (EBV-VCA) -producing cells or Epstein-Barr nuclear antigen -expressing cells fixed on slides were used as antigen for IgG EBV-VCA, IgM EBVVCA, and Epstein-Barr nuclear antigen (19). Serum samples. In total, the following 537 samples were included in the peptide antigen assay for IgM. (i) A total of 158 unselected volunteer blood donor samples were used that had been donated at Citytappen Blood Bank, Stockholm, on one day in May 1989 (a nonepidemic period for B19

A405 values

antigen

virus infection in Stockholm). (ii) A total of 281 stored serum samples submitted to the Virus Reference Laboratory, London, during January and February 1989, for B19 virus investigations were selected if there was sufficient material available for further testing. According to the viral antigen B19 virus IgM assay, 91 serum samples were positive, 189 were negative, and 1 was equivocal. Of the 281 samples, 206 had a known B19 IgG reactivity (117 positive, 81 negative, and 8 equivocal). (iii) A total of 93 serum samples trom 51 patients were selected because of the presence of ac;ute infection with the following agents: cytomegalovirus, VLV, measles virus, rubella virus, Russian sprnng-summer encephalitis virus, herpes simplex virus, influenza virus A or B, Mycoplasma pneumoniae, chlamydia, sandfly fever-Sicilian virus, or primary EBV. (iv) Five B19 IgG-reactive serum samples were selected which had high RF IgM directed against IgG. These last serum samples were artificially prepared by adding RF to known B19 IgG-positive, B19 IgM-negative serum samples.

1377

2,01

IgM

1,6 -

1,2 -

A

0,8 -

3% a

0,4 -

97% 0,0

Blood donor samples

HIG. 1. Peptude antigen donors.

assay

for B19

virus

lgM of 158 blood

RESULTS Blood donors. In the B19 1gM peptide antigen assay, 154 of 158 (97%) blood donors had A405 values below the cutoff of 0.40, with a mean value of 0.150 and a standard deviation of 0.(074 (Fig. 1) The A405 values for four additional donors in the IgM peptide antigen assay were 0.43, 0.45, 0.53, and 0.83, rhese four donors had an A405 value of less than 0.4 in the lgG peptide antigen assay. No clinical symptoms had been noted in these donors. They may still represent asymptomatic B19 infections, but serum samples to establish this were not available. Patients with suspected B19 virus disease. A total of 281 serum samples from persons with suspected B19 infection were studied in viral and peptide antigen for 1gM assays (Fig. 2), One hundred ninety (68%) were negative and 91(32%) were positive in the viral antigen assay, and 187 (67%) were negative and 94 (33%) were positive in the peptide antigen

peptide antigen

tb 0 VI

Cd

assay

175

15

(63%)

(50/°)

12 (4%)

79

rq

0

u "04

rq

co

09k

(28%)

"04

Oo

F1i. 2. Comparison of B19 IgM reactivities in peptide assay

and in viral antigen assay. Numbers of patient samples

category as

well

as percentages are given.

antigen in

each

1378

J. CLIN. MICROBIOL.

FRIDELL ET AL.

TABLE 1. Results of viral antigen assay with B19 1gM values of 0.4 IgG

IgM Patient

no.'

Viral antigen assay

10 11 12 13 14 15

(U)

Peptide Peptide

Viral aritigen antigen Viral

Peptide Peptide

Days

antigen assay (A405 values)

assay

onset

(U)

antigen assay (A405 values)

0.96 0.71 0.62 1.07 0.68 0.88

10

Evaluation of a synthetic-peptide enzyme-linked immunosorbent assay for immunoglobulin M to human parvovirus B19.

A synthetic peptide corresponding to a part of the virus protein 1-virus protein 2 overlapping region of human parvovirus B19 was used in an indirect ...
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