Vol. 29, No. 7JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1991, P. 1376-13810095-1137/91/071376-06$02.00/0Copyright ©D 1991, American Society for Microbiology

Evaluation of a Synthetic-Peptide Enzyme-Linked ImmunosorbentAssay for Immunoglobulin M to Human Parvovirus B19

E. FRIDELL,l.2* B. J. COHEN,3 AND B. WAHREN"2Department 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 ofhuman parvovirus B19 was used in an indirect enzyme-linked immunosorbent assay. Antibodies of theimmunoglobulin (Ig) M class were measured in serum samples from patients with erythema infectiosum andcontrols. 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 viraldiseases. Specific IgM reactivities to short synthetic viral peptides have previously been reported only withEpstein-Barr virus. Since other sources of viral antigen are limited, the peptide antigen assay may be a usefulalternative for the diagnosis of B19-associated disease in human beings.

The human parvovirus B19 was found by chance in serafrom blood donors in 1975 (7). About 5 years later, two menwith mild fever were shown to have B19 virus in their blood(27). In 1982, the virus appeared to be the main cause ofaplastic crisis in patients with sickle cell anemia (24) andlater in patients with other chronic hemolytic diseases (36).When cared for in the hospital, these patients have transmit-ted the B19 virus to other patients (3, 11). In the spring of1983, an outbreak of erythema infectiosum occurred in aLondon school and B19 virus was identified as the etiologicalagent (1). The virus causes arthritis or arthralgia in about50% of infected adult women (26). Some of these womenexperience the arthralgia for years, and rheumatoid arthritishas been a common differential diagnosis. During wide-spread outbreaks of B19 infection, nonimmune women ofchildbearing age are at some risk of acquiring an intrauterineinfection. Hydrops fetalis and fetal loss may ensue duringany part of the pregnancy (23). However, intrauterine B19infection rarely if ever causes fetal anomalies (31, 33).During 1988 and 1989, persistent (i.e., lasting months toyears) infections with B19 virus were described as causingintermittent anemia in patients with damaged or weakenedimmunity, e.g., leukemic children, bone marrow recipients,or patients with congenital immunodeficiency or AIDS (12,17, 18, 32). These patients have intermittent or chronic virusshedding for long periods and may constitute a risk for otherimmunodeficient patients. Since B19 is a stable virus and isdifficult to inactivate, it has also been spread through trans-fusion with factor VIII concentrates (2, 21).

Investigations of erythema infectiosum outbreaks willconfirm the diagnosis of B19 virus infection and identifypeople at risk ofbecoming infected through contact. The B19virus can only be cultivated in limited amounts, and there-fore antibody assays have been based on whole virionsprepared from the plasma of viremic blood donors (viralantigen assay). These donors are rarely found, and theassays have been limited to a few laboratories. An assay forB19 immunoglobulin (Ig) M and IgG using a synthetic

* Corresponding author.

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

MATERIALS AND METHODS

Peptide ELISAs for B19 virus IgM and IgG. The peptideenzyme-linked immunosorbent assays (ELISAs) for IgMand IgG were performed with a synthetic peptide. Thepeptide was identified by systematically synthesizing pep-tides, 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 majorcapsid protein of B19 virus. The amino acid sequences ofneighboring reactive peptides included two cysteines. A24-amino-acid-long peptide (FSPAASSCHNASGKEAKVCTISPI), containing the initially identified reactive peptide(underlined) and stabilized through a sulfur bridge betweenthe cysteines, was prepared by solid-phase synthesis on anApplied Biosystems 430A peptide synthesizer. The peptidewas prepared to greater than 99% purity (14). A 100-,uvolume of a peptide solution containing 10 ,ug of the peptideper ml was used to coat 96-well flat-bottom microtiter plates(Nunc, Roskilde, Denmark). The plates were freeze-driedand stored at 4°C. After washing, 100-,Iu volumes of controlsera or samples for the IgM assay, diluted to 1:20, weredispensed in duplicate. For the IgG assay, the control seraand samples were diluted 1:20 and 1:200, respectively. Afterincubation 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 theplates were incubated for a further 45 min at 37°C. After afinal wash, substrate solution (p-nitrophenylphosphate;Sigma) was added and A405 values were read. To investigateblocking of IgM by specific IgG, sera nonreactive for IgMand reactive for IgG in peptide and viral antigen assays wereabsorbed with rheumatoid factor (RF) absorbent (Behring,Marburg, Germany) and retested in the peptide antigenassay for IgM. The efficacy of RF absorption was controlledby 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

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SYNTHElIC-PEPTIDE ELISA FOR IgM TO B19 PARVOVIRUS 1377

value gives the best correlation between the viral 1gMantigen assay and the peptide IgM antigen assay in the 281serum samples. An IgG A405 value rise was defined as atleast a doubling of the value between acute-phase andconvalescent-phase serum samples, provided that the A405value of the convalescent-phase serum sample was above0.40.

Viral antigen radioimmunoassays for B19 1gM and IgG.Viral antigen radioimmunoassays for B19 virus IgM and IgGwere performed as described previously (6). In short, anti-human IgM was coated to the solid phase, 1gM in patientserum 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 radioac-tively labeled antibody directed against mouse IgG. In boththe IgG and IgM assays, measurements in arbitrary unitshigher than 3 were regarded as positive, those between 1 and3 were equivocal, and those below 1 were negative.

Assays for other viral Igs. The assays for IgM(,) captureELISAs (9) were performed by using antigens prepared in

house (i.e., for cytomegalovirus, varicella-zoster virus

[VZV], and measles virus) or commercially available (hem-agglutination inhibition antigen for rubella; Wellcome Re-search Laboratories, Beckenham, England). The viral anti-gens were labeled with peroxidase by the periodate method(34). Indirect ELISAs for IgG and IgM (29, 30) were per-

formed with an in-house-purified nucleocapsid antigen forherpes simplex virus and a purified whole viral antigen forRussian spring-summer encephalitis virus (received from C.Kunz, Vienna, Austria). For cytomegalovirus, VZV, ru-

bella, and measles IgG, indirect ELISAs were performedwith the same antigen as for IgM(,u) capture ELISAs. Allantigens for complement fixation assays were prepared atthe National Bacteriological Laboratory, Stockholm. Cellsinfected with the Sicilian strain of sandfly fever-Sicilian virus(10) were used in an immunofluorescence assay. Epstein-Barr virus viral capsid antigen (EBV-VCA) -producing cellsor Epstein-Barr nuclear antigen -expressing cells fixed onslides were used as antigen for IgG EBV-VCA, IgM EBV-VCA, 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 of158 unselected volunteer blood donor samples were usedthat had been donated at Citytappen Blood Bank, Stock-holm, on one day in May 1989 (a nonepidemic period for B19virus infection in Stockholm). (ii) A total of 281 stored serum

samples submitted to the Virus Reference Laboratory, Lon-don, during January and February 1989, for B19 virusinvestigations were selected if there was sufficient materialavailable for further testing. According to the viral antigenB19 virus IgM assay, 91 serum samples were positive, 189were negative, and 1 was equivocal. Of the 281 samples, 206had a known B19 IgG reactivity (117 positive, 81 negative,and 8 equivocal). (iii) A total of 93 serum samples trom 51patients were selected because of the presence of ac;uteinfection with the following agents: cytomegalovirus, VLV,measles virus, rubella virus, Russian sprnng-summer enceph-alitis virus, herpes simplex virus, influenza virus A or B,Mycoplasma pneumoniae, chlamydia, sandfly fever-Sicilianvirus, or primary EBV. (iv) Five B19 IgG-reactive serum

samples were selected which had high RF IgM directedagainst IgG. These last serum samples were artificiallyprepared by adding RF to known B19 IgG-positive, B19IgM-negative serum samples.

A405 values2,01

1,6 -

1,2 -

0,8 -

0,4 -

0,0

IgM

A

3%a

97%

Blood donor samples

HIG. 1. Peptude antigen assay for B19 virusdonors.

lgM of 158 blood

RESULTSBlood donors. In the B19 1gM peptide antigen assay, 154 of

158 (97%) blood donors had A405 values below the cutoff of0.40, with a mean value of 0.150 and a standard deviation of0.(074 (Fig. 1) The A405 values for four additional donors inthe IgM peptide antigen assay were 0.43, 0.45, 0.53, and0.83, rhese four donors had an A405 value of less than 0.4 inthe lgG peptide antigen assay. No clinical symptoms hadbeen noted in these donors. They may still represent asymp-tomatic B19 infections, but serum samples to establish thiswere not available.

Patients with suspected B19 virus disease. A total of 281serum samples from persons with suspected B19 infectionwere 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%) werenegative and 94 (33%) were positive in the peptide antigen

peptide antigen assay

tb0VICd

rq0u"04

rqco

09k"04Oo

F1i. 2. Comparison of B19IgM reactivities in peptide antigenassay and in viral antigen assay. Numbers of patient samples in eachcategory as well as percentages are given.

175 15(63%) (50/°)

12 79(4%) (28%)

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1378 FRIDELL ET AL.

TABLE 1. Results of viral antigen assay with B19 1gM values of <1 U and of peptide antigen assay with 819 IgM A405 values of >0.4

IgM IgGPatient Peptide Viral antigen Peptide Days Patient age Sbno.' Viral antigen Peptide Viral aritigen Peptide after Clinical data (yr)

Comment

assay (U) antigen assay assay antigen assay onset(A405 values) (U) (A405 values)

10 <1 0.96 10 0.79 NKb Sickle cell anemnia NK RF neg11 <1 0.71 <1 0.64 9 Rash, aplastic anemia 7 RF neg12 <1 0.62 <1 1.45 NK Arthralgia 15 RF neg13 <1 1.07 <1 0.70 8 Hemolytic anemia 62 RF neg14 <1 0.68 >100 <0O.40 >90 Arthralgia 44 RF pos15 <1 0.88 <1 <0.40 14 Thrombocytopenia 47 RF neg

a Samples 1 to 9 had equivocal B19 IgM A405 values of 0.41 to 0.52 in the peptide antigen assay and are therefore not included.b neg, negative; pos, positive.c NK, not known.

assay. Viral antigen assay values ranged from <1 to >100 assay but were positive for IgM in the viral antigen assayarbitrary units, and peptide antigen assay A405 values ranged (Table 2). In addition, they were negative in the IgM peptidefrom 0 to 2.00 arbitrary units. Two hundred fifty-four serum antigen assay after RF absorption. IgG was detectable in allsamples (79 positive and 175 negative) gave concordant 12 seruni samples by one or both assays.results in the two assays. When all values were considered, Patients with other viral and related diseases. Sera withsimple curve fits of results from the two assays gave r values antiviral 1gM reactivity of other specificities as well as serabetter than 0.85. with high levels of B19-specitic IgG and RF might giveWe compared the sensitivities and specificities of the two false-positive results in indirect IgM ELISAs. We therefore

assays for IgM in two ways. First, we regarded the viral studied 51 patients with other current infections and fiveantigen assay as giving the true values. This gave a sensitiv- selected RF-positive serum samples (Table 3). Three of theity of 92% and a specificity of 87% for the peptide antigen 14 patients with acute VZV infection had, in their convales-assay. We then regarded the peptide assay as giving the true cent-phase sera, elevated A405 values for IgM B19 by thevalues. This gave a sensitivity of 84% and a specificity of peptide antigen assay; values are shown in detail in Table 4.91% for the viral antigen assay. They had stable levels of or no 819 IgG. In the viral antigenTwenty-seven serum samples gave discrepant results (Fig. assay, 2 of the 14 patients had IgM values slightly above the

2). Nine of 15 of these serum samples had equivocal values cutoff, and all three of them had IgG, one with a high IgGin the IgM peptide antigen assay and were negative in the value (83 U) and the other two with low positive IgG values.viral antigen assay. They are not discussed further here. Six All VZV convalescent-phase serum samples had IgM VZVserum samples had a clear reactivity in the B19 IgM peptide reactivity, and IgG VZV seroconversion or titer rises wereassay (Table 1). Four of these (samiiples 10 to 13) also had a measured in paired sera. These patients, therefore, clearlypeptide antigen IgG response, and two had a viral antigen were infected with VZV as judged by serology.IgG response. One serum was RF positive and gave an A405 One of the rubella-positive serum samples had a slightlyvalue of 0.68 in the peptide antigen assay befote absorption elevated IgM B19 value (A405, 0.44) in the peptide antigenand a value of 0.69 after absorption with RF absorbent (14). assay but a negative IgM value and a positive IgG B19 valueThe viral antigen assay for this serumi also gave the same in the vital antigen assay. Aniother patient with acute rubellavalues before and after absorption with RF. had, in the peptide antigen assay, a rise in the B19 IgG A405Twelve serum samples were negative in the peptide IgM from 0.32 to 0.63 and a negative IgM value (Table 3). In the

TABLE 2. Results of viral antigen assay with 819 IgM values of >1 U and of peptide antigen assay with B19 IgM A405 values of 50.4'

IgM IgGPatientLDays Patient

PatVent Peptide V t Pcpade after Cliniial data ageno. Viral antigen antigen assay alltgen antigen asy onset (yr)

assay (U) (A405 values) assay (U) (A4.5 values)

16 6 .-0.40 > 100 1.09 60 Polyarthritis 2917 7 s0.40 51 0.54 14 Rash, respiratory tract infection 1018 9 s0.40 92 0.94 5 Rash, arthropathy 919 10 -0.40 NDb 1.41 51 Rash 2320 13 s0.40 > 100 0.89 19 Rash 921 17 .0.40 ND 0.99 1 Rash, conjunctivitis 622 18 s0.40 >100 1.25 31 Rash 2823 28 s0.40 >100 1.32 62 Polyarthritis 3824 38 s0.40 86 1.50 16 Rash 1025 3 s0.40 ND 1.09 7 Rash 726 50 s0.40 >100 0.95 Late Fetal death 3327 82 s0.40 73 1.79 33 Aplastic crisis 45

a Negative in peptide antigen assay before and after absorption of IgG.b ND, Not done.

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TABLE 3. Patients with other viral and related diseases reactivein B19 peptide antigen assay

Patients with B19 reactivity

Primary acute Positive IgM IgG titer rise (no.infection (no. positive/ with titer rise/no.

total no. of of patients withpatients)a paired sera)

VZV 3/14 0/14Rubella 1/9 1/4EBV 0/10 0/1Measles 0/4 0/4Other viruses 0/11 0/11Other infections (chlamydia, 0/3 1/3Mycoplasma pneumoniae)a Zero of five patients were RF positive.

viral antigeii assay, this patient had no IgM and a stable levelof IgG to B19 virus.Of 10 patients with primary EBV infection, 4 with mea-

sles, and 11 with other current viral infections, none had IgMagainst the B19 peptide. In the group of patients with otherinfections, one patient with a current mycoplasma infectionhad an IgG titer rise against the B19 peptide (the A405 valuerose from 0.20 to 0.46) (Table 3) but lacked IgG to B19 in theviral antigen assay. The complement fixation assay titeragainst Mycoplasma pneumoniae for this patient showed arise from 20 to 640.The five RF-positive serum samples were all negative in

the peptide antigen assay for IgM both before and after RFabsorption. -They were not examined by the viral antigenassay for B19 IgM since they were prepared for the purposeof examining RF interference in the peptide assay (seeMaterials and Methods).

DISCUSSION

The peptide antigen assay measures IgM early in currentB19 infections. A comparison with the viral antigen assayshowed specificities and sensitivities for the peptide antigenassay of around 90%, regardless of which assay was re-garded as the true assay. Among blood donors, 97% had A405values lower than the chosen cutoff value. One of the donorshad a high IgM value but no detectable IgG, indicating eitheran early infection or a nonspecific reaction. It is interestingto note that frequently occurring specific IgM reactivities toshort synthetic viral peptides have previously been reportedonly with EBV (28).Of the blood donors, 53% (n = 158) had B19 virus IgG A405

TABLE 4. Patients with acute VZV infection reactivein B19 peptide assay

VZV antibody value B19 antibodyPaired sera value

IgM IgG IgM IgG

Acute phase Negative <100 0.06 <0.40Convalescent phase Positive 4,500 0.84 <0.40

Acute phase Negative <100 0.40 0.68Convalescent phase Positive 1,000 0.61 0.67

Acute phase Positive 4,400 0.15 <0.40Convalescent phase Positive 91,000 0.55 <0.40

values of >0.4; 76% had values above 0.2 (data not shown)with the peptides used here. The peptides in the peptideantigen assay represent B19 VP2 and thus the part of VP1that overlaps VP2. It is conceivable that other IgG-reactivesites may be discovered in the part of the VP1 sequence notinvestigated here.

Six serum samples that were nonreactive in the IgM viralantigen assay had a clear reactivity in the IgM peptideantigen assay. Since five of them also had IgG reactivities inone or both assays, the results might be explained by thedifferent antigenic regions and exposures of the assays or bythe different indicator systems. It is also possible that thesesix reactions in the peptide assay were false-positive results,seemingly not due to the presence of RF.Twelve serum samples were positive in the viral antigen

IgM assay but were negative in the peptide antigen assay(Table 2). All had IgG reactivities against the peptide anti-gen, and it is possible that these antibodies competed withthe specific IgM in the peptide assay. Most of these 12 serumsamples had low levels of B19 IgM in the viral antigen assay,and half of them were collected more than 4 weeks after theonset of symptoms. This suggests that the viral antigenassay, which is based on the antibody capture technique, canmeasure specific IgM for a longer period than the peptideassay.Most of the patients we investigated who had infections

other than B19 were nonreactive in the peptide antigen assayfor IgM, but three patients with current VZV infection, onewith rubella, and one with mycoplasma infection had IgMagainst the B19 peptide or a titer rise in the IgG peptideassay. Two of the serum samples from VZV-positive pa-tients also had IgM values slightly above the cutoff value inthe viral antigen assay, but the other four patients werenonreactive in the viral antigen assay for IgM. Five serumsamples with RF and B19 IgG did not display any IgMreactivity in the peptide antigen assay. The double reactivityin the B19 antigen assay was thus seen in infections com-monly spread in crowded groups of children. Children inkindergarten or school are often exposed to many diseaseagents, and double infections are not uncommon. Thisexplanation is not satisfactory for the patients we studiedwho had other infections, because four of them showed noevidence of current B19 infection and two showed onlytraces of IgM to the viral B19 antigen. Other causes ofcross-reactivity are common antigenic regions as have beendescribed for VZV and herpes simplex virus, cytomegalovi-rus and EBV, and also paramyxoviruses, flaviviruses, andenteroviruses. A study of the B19 peptide sequence, how-ever, did not reveal significant homology to any other knownpeptide (13). Many DNA viruses remain latent in the cellsafter infection, and reactivation is frequent. This may beanother cause of cross-reactivity, but it is not knownwhether B19 virus remains latent and reactivates or whetherreinfection occurs.

Recently, assays for B19 virus 1gM have been describedthat use viral proteins expressed in bacteria (20), Chinesehamster ovary cells (16), human erythroid progenitor cells(35), or insect cells using recombinant baculovirus vectors(4). A fifth system uses native virus but amplifies thedetecting signal (5). Most of these assays have not yet beenextensively used in clinical practice. Although specific IgMdetection is often sufficient to diagnose recent B19 virusinfection, not all patients can produce IgM or IgG. Fetusesstart to produce antibodies only at about week 18 of gesta-tion (15) and then in limited amounts because the immunesystem is immature. Patients with congenital or acquired

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1380 FRIDELL ET AL.

immunodeficiency also have impaired immune functions. Inaddition, some patients (especially those with chronic hemo-lytic anemia) become symptomatic during the first days ofviremia before any antibody production is evident. Samplesfrom such patients must therefore be investigated directlyfor the presence of B19 virus by electron microscopy or

DNA detection techniques or follow-up specimens must becollected and tested for IgM antibody.An IgM assay is the test of choice for B19 virus infections

in otherwise healthy persons, since specific IgM is usuallypresent at the onset of symptoms such as rash and jointpains. The peptide assay described here could detect B19IgM in the first serum samples collected from such patients.The peptide IgM assay should be adequate for diagnosis ofB19 infection in acute-phase serum samples, but furtherevaluation in prospective surveys is required. These studiesshould include immunocompromised patients with B19 in-fection who may develop antibodies very late. The peptideassay for B19 IgM may help to overcome the problem ofproviding laboratory diagnosis for B19 infection which has

been limited because of the shortage of native viral antigen.

ACKNOWLEDGMENTS

We would like to thank M. M. Buckley for technical assistancewith the viral antigen assay, A.-C. Malmsten for assistance with thepeptide antigen assay, and P. P. Mortimer for critical reading of themanuscript.

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