Enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus-specific IgM antibodies

Journa~of VirologicalMethods,4 Elsevier Biomedical Press

219

(1982) 219-227

ENZYME-LINKED IMM~OSORBENT

ASSAY (ELISA) FOR DETECTION OF

HERPES SIMPLEX VIRUS-SPECIFIC IgM ANTIBODIES

NINA KIMMEL, MAUREEN G. FRIEDMAN and ISRAEL SAROV* Virology Unit, Soroka University Hospital, FQCU& of Health Sciences, Ben Gution University of the Negev, Beer Sheva, Israel (Accepted 7 January 1982)

Herpes simplex virus (HSV)-specific IgM in human serum could be detected by a microplate enzyme-linked immunosorbent assay, using extracts of HSV-infected cells as antigen. Peroxidaseconjugated anti-human IgM was used to detect human IgM bound to viral antigen. Pretreatment of sera with protein A-bearing staphylococcus or with aggregated human IgG wasnecessary to eliminate false-positive results caused by the presence of rheumatoid factor. Specificity controls included sera of patients with other herpes group virus infections. ELISA

herpes simplex virus

IgM

INTRODUCTION

Herpes simplex virus infection and,

after

reactivated.

initial

infection,

is often asymptomatic,

enters

but the virusmay

cause stomatitis

into a latent

Severe forms of primary

state from which it may be periodically or secondary HSV infection include encephalitis

and keratitis (Nachmias and Norrild, 1979). Because it is now possible to treat some herpes virus infections, early confirmation of the etiologic agent is of great importance (Whitley et al., 1977). Diagnosis is usually based on virus isolation, which is readily carried out in the case of skin Iesions, but poses a problem in the case of enceph~itis. Falaky et al. (1977) used an ~munofluorescent technique to demonstrate HSV-speci~c serum IgM, and Kalimo et al. (1977) with a radio~munoa~ay @IA) showed that HSV-IgM can be detected in primary and severe secondary infections. An enzyme-linked immunosorbent assay (ELISA) has been shown to be sensitive and specific for detection of IgG against HSV (Denoyel et al., 1980). In the present study we describe an ELISA technique for the detection of HSV-specific IgM in serum and demonstrate its specificity.

* To whom correspondence 0166-0934/82/00~-~0~$02.75

should be addressed. @ 1982 Elsevier Biomedical Press

220

MATERIALS

AND METHODS

Antigen preparation

Vero cells were grown in RPMI-1640 (Roswell Park Memorial Institute Medium) (Bio-Lab, Jerusalem, Israel) supplemented with 10% fetal calf serum (FCS) (Gibco, Grand Island, NY), 2 mM L-glutamine, 100 units/ml penicillin and 200 pg/ml streptomycin at pH 7.2. Before infection, the Vero cultures were split and allowed to grow to confluency. One of the resulting sister cultures was infected and the other was the source of the control antigen. The virus used for infection consisted of a lysate of Vero cells infected with strain VR-3 of herpes simplex virus type 1 (HSV-I), and was used at a dilution of 1 : lO.‘The infected and uninfected cultures were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented as above, but with 2% FCS. Both cultures were incubated at 37°C overnight, when a strong cytopathic effect was observed in the infected cultures. The monolayers were washed with 10 ml of cold phosphatebuffered saline (PBS) (pH 7.3), removed by freezing and thawing, sonicated, and clarified (1400 X g, 15 min) as described previously (Friedman et al., 1979). Protein concentration was determined by the method of Lowry et al. (1951). A concentration of 200 pg/ml was found to be optimal for the study. Vero cells were periodically checked and found to be free of bacterial and mycoplasmal contaminants. Human sera

Sera were obtained from patients with clinical evidence of HSV infections, from healthy medical students and laboratory workers, and from otherwise healthy adults with clinically diagnosed recurrent herpes labialis. Twenty sera taken from nine patients with clinical diagnosis of other herpes group viral infections were also examined. All sera were stored at -2O’C until used. Before dilution the sera were absorbed with staphylococci or aggregated IgG, as described below. Serial fourfold dilutions were prepared for each serum using PBS-T (PBS containing 0.05% Tween 20), the dilution range being from 1 : 25 to 1 : 6400. IPAMA

The immunoperoxidase antibody-membrane antigen (IPAMA) technique for the determination of HSV-specific IgG antibody was carried out as described by Haikin and Sarov (1980). ELBA

The procedure is a modification of that described by Voller and Bidwell (1976) and has been reported previously by Kahane et al. (1979). Briefly, 25 ~1 drops of Vero-HSV or Vero antigen at the same protein concentration were dried overnight at room temperature on Nunc polystyrene microtest plates 96-U 1182- 1 (Nunc, Denmark). The plates could be stored at -70°C or used the following day, Prior to use, antigen-coated plates

221

were washed with PBS-T. The diluted sera to be tested were added as described. After 1 h incubation (37°C) the plates were rinsed and incubated for 1 h at 37°C with 25 ~1 of peroxidase-linked rabbit anti-human IgM (specific for p-chain, obtained from Dakopatts, Copenhagen,

cat. No. P21.5) diluted

1 : 100 in PBS-T. The dilution

of IgM to be used

was determined for optimal colour development on standard positive and negative sera. After the plates were rinsed vigorously in PBS-T, enzyme substrate, consisting of 0.05% Hz02 with 0.08% S-aminosalicylic acid, was added. The reaction took place at room temperature and was easily detected visually. The reaction was stopped with 0.1 N NaOH, and contents of each well were transferred immediately into test tubes and diluted with 1 ml of distilled water. The absorption was determined at a wavelength of 450 nm (Gilford microsample spectrophotometer, known positive and negative sera were included,

model Stasar II). In each experiment as well as 12 wells containing serum

diluent only. An average background absorption level was determined from those 12 wells. The serum titer was defined as the intersection between the titration curve using viral antigen and the corresponding curve obtained with control antigen as previously described (Goldberg and Sarov, 1980; Sarov et al., 1980). Negative sera gave similar absorbance readings with both antigens. Representative titration curves of a positive and a negative serum appear in Fig. 1, lowest frames. Rheumatoid factor The sera were tested for the presence of rheumatoid factor (RF) by agglutination of the Rheumaton reagent (Warnpole Laboratories, Dist., Div. of Carter-Wallace, Inc., Cranbury, NY). Staphylococcal protein A absorption Preparation of bacteria and absorption of sera were performed essentially according to the method described by Skaug and Tjotta (1974). The optimal conditions of absorption, as determined

by preliminary

0.3 ml of the bacterial

tests, were 0.1 ml of serum diluted

suspension

(10% w/v). Absorption

1 : 10 in PBS plus

was performed

at 37°C for 45

min. The bacteria were then sedimented by centrifugation for 15 min at 1500 X g and 0.07 ml of the supernatant fluid was carefully harvested with a microdispenser (Drummund Scientific Co., U.S.A.). Absorption with aggregated IgG Aggregated IgG was prepared as described by McCarthy et al. (1981). Briefly, 100 1.11 of IgG (Miles Laboratories Ltd., code No. 64-145-1) at a concentration of 30 mg/ml in PBS was placed in a cellulose propionate tube (cat. No. 341288, Beckman), which was incubated at 63°C for 14 min and immediately transferred onto ice. Within a few minutes 100 1.11of serum, diluted 1 : 5 in PBS, was added to the tube, and the tube was incubated for 1 h at 37°C. The aggregated IgG, complexed with rheumatoid factor, if present, was sedimented by centrifugation in a Beckman Airfuge at 20 lb/in* for 25 min. 70 ~1 of the supernatant fluid was used in the test.

222

RESULTS

Several variables in the ELISA system were examined conditions

for the detection

of IgM antibodies

in order to find the optimal

to HSV.

Optimal antigen concentration Preliminary experiments were carried out in order to determine the optimal antigen concentration to discriminate between HSV-I~-positive and IgM-negative sera. Viral and control antigen concentrations of 50, 100, 200, 300 and 400 pg protein/ml were tested with known positive and negative sera. No significant difference in reactivity was found when antigen concentration varied between 200 and 400 pg protein/ml, while concentrations of 50 and 100 /Ig proteinlml considerably diminished the reactivity. On the basis of the equal sensitivity of antigen concentrations of 200, 300 and 400 fig protein/ml, an antigen concentration of 200 pg protein/ml was chosen for further work. The suitability of this concentration was confirmed by the above method for each new batch of antigen. Eiimi~tion of false positives associated with the presence of rheu~toid

factor (RF)

Two procedures were tested for overcoming false-positive IgM determinations caused by the presence of rheumatoid factor. One was absorption with Staphylococcus aureus, strain Cowen I, which reduced greatly the IgG content of the serum to be tested; and the second was absorption with aggregated human IgG, which removed rheumatoid factor directly. Fig. 1A shows the results of treatment of a serum positive for HSV-specific IgM by the two methods IgM false-positive

and Fig. 1B shows the results of treatment

serum by the two methods.

The false-positive

of an RF-positive

HSV

serum became negative

after either treatment. Both treatments also reduced non-specific serum reactivity (reactivity with control antigen). Staphylococcal absorption of the true positive serum did lower its titer somewhat (~400-32~). Such effects have been noted previously. All sera to be tested for HSV-specific IgM were routinely treated by one of the two methods. Reproducibility of the test The reproduc~b~ty of ELISA was quite satisfactory. One serum, which was tested in experiments, using two batches of antigen, had a mean titer of 1700

eight different 5 300. Specific@

The specificity of the test was examined with regard to several parameters.

223

A 05 No Treatment

25

1 /SERUM

IO0

400

I600

DILUTION

Fig. 1. Effect of staphylococcal (A) or aggregated IgG (0 absorption on detection of IgM in HSVIgM-positive (A) or false-positive (B) serum samples. 0, serum without treatment; -, serum tested on HSV antigen; ------, serum tested on control antigen.

Control healthy adults. Fifty-two sera of medical students were tested for antibody levels to HSV. Of these, 21 were positive; IgG titers in the range of 8-64 were found by IPAMA. All these sera were tested for HSV-specific IgM antibodies by ELISA and proved to be negative (ELISA titer <25). Cross-reactivity with other herpes viruses. Twenty paired sera of nine patients hospitalized with herpes group virus infections were tested for the presence of HSV-specific IgM; each had IgM antibodies against the virus causing his illness: one patient had an Epstein-Barr virus (EBV) infection; four had varicella-zoster virus (VW) infections; and four had human cytomegalovirus (CMV) infections. VZV- and CMV-specific IgM were detected by ELISA techniques previously described (Hacham et al., 1980; Levy and Sarov, 1980) and EBV-specific IgM was detected by the IPAMA test (Haikin and Sarov, 1980). All these patients were HSV-&M-negative (titer (25).

224

NSV ~~~ie~ts. Sixty-three sera of 20 HSV patients were tested by ELBA for HSV-IgM antibodies and by IPAMA for HSV-IgG antibodies. Six patients, presumably with primary HSV infections,

had HSV-specific IgM antibodies

and 4) suffered from encephalitis. had significant

Cerebrospinal

(Table 1). Two of these patients (Nos. 1 fluid (CSF) samples from these patients

IgG titer rises to HSV when tested by the IPAMA method.

In relatively

late CSF samples of patient No. 1, HSV-specific IgM was detected, while it was not detected in any of the CSF samples of patient No . 2 (Table 1). However, forboth patients, HSV-specific serum IgM was detected already in the first serum sample. (Serum/CSF ratios of HSV-specific IgG antibodies for these patients are also noted in Table 1.) Sera absorbed with staphylococci or aggregated human IgG and which contained IgM directed against HSV antigens were further tested for the specificity of the IgM detected. They were tested on VZV and CMV antigens, prepared as described previously (Hacham et al., 1980; Levy and Sarov, 1980). Although the unabsorbed sera were all positive for IgG directed against CMV and VZV (by IPAMA), the absorbed sera were all negative (titer (2.5) for CMV- or VZV-specific IgM. Six patients

hospitalized

with HSV-related

illness of a recurrent

nature had rising

TABLE I HSV antibody titers from patients with HSV-associated infection as determined by IPAMA (IgG) and ELISA (IgM) Patient NO.

1

Age (Ye

‘I,*

Diagnosis

Encephalitis

2

13/,

Stomatitis

3

l”fl2

Stomatitis

4

l’%,

Enceph~itisb

.5

11

Facialis

6

20

Stomatitis

Illness

IgG

day

IPAMA

2 10 15 24 12 23 8 20 1 5 13 25 51 5 18

Ratio serum/CSF

Sera

CSF

10 80 160 1280 80 80 40 640 40 1280 2560 32 32 20 320

<2 8 l@ 16a

10 10 80

5

8

20

128

IgM ELISA serum

100 1600 1600 1200 <25 1600 400 400 400 <25 <25 <25 400 <25 6400

a These CSF samples, when tested by the ELISA, contained HSV-specific IgM at titers of 10 and 80 respectively. b The patient’s CSF samples did not contain IgG antibodies to CMV (titer <2 by IPAMA), although the patient had a serum IPAMA titer of 80 to CMV.

225

titers of HSV-specific

IgG, but no HSV-specific

IgM (titer <25). The clinical features of

these patients were as follows: two had characteristic

herpes keratitis; two had recurrent

body lesions (HSV-1 isolated);

stomatitis

and two had recurrent

(one severe, with HSV- 1

isolation, and one with accompanying measles infection). Serial serum samples from eight persons with recurrent herpes labialis were examined for HSV-specific IgM. Three or more serial samples were drawn at weekly intervals after the appearance of the current lesions. HSV-specific IgG in these samples did not fluctuate significantly. No HSV-specific IgM (titer <25) was detected in any of these sera. DISCUSSION

Three points are of concern with regard to tests for HSV-specific IgM antibody: I) the method should be virus- and immunoglobulin class-specific; 2) it should provide for avoidance of false-positive results caused by the presence of IgM class rheumatoid factor (RF); and 3) it should be available for use in all diagnostic laboratories. The ELISA test described is virus-specific; no cross-reactivity with heterologous herpes viruses was found; neither was HSV-IgM detected in 31 seropositive medical students tested, demonstrating the immunoglobulin class specificity of the test. In the present study it was possible to detect HSV-specific IgM antibody by ELISA in sera of six patients with presumably primary HSV infections; these six patients were for the most part quite young and did not have a history of previous HSV-associated illness {Table 1). A serious obstacle in specific IgM dete~ination appears to be ~terference by RF through its binding to complexes of the test antigen and specific IgG, thus giving rise to false-positive results. Two methods for overcoming this problem are protein A absorption of sera and absorption with aggregated IgG. Staphylococcal absorption depletes the sera of IgG (except subclass IgG,) and has been successfully reaction

used to eliminate

the false-positive

in sera tested

(Skaug and Tjotta, 1980; Sarov et al., which can then be in cases of rubella,

for IgM directed against HSV, rubella, CMV and VZV viruses 1974; Leinikki et al., 1978; Levy and Sarov, 1980; Hacham et al., 1981). Aggregated IgG works by complexing directly with the RF, removed by sedimentation; the technique has been recently used measles and CMV infections (Leinikki et al., 1978; Meurman and

Zioh, 1978, Vuorimaa et al., 1978; Krishna et al., 1980). Although a slight decrease in HSV-specific IgM titer was found using staphylococcal absorption, as has been reported in other virus systems as well (Skaug and Tjotta, 1974; Hacham et al., 1980), both methods of eliminating RF interference appear to be suitable for ELISA HSV-IgM determination (Fig. 1). The ELBA technique described is not difficult to adopt in diagnostic laboratories. The antigen, a crude, clarified lysate of HSV-infected cells, may be frozen for longterm storage, and the labeled antibody is commercially available. Since the test is completed within a few hours, sameday results can be obtained. Using the ELISA test described, we did not detect IgM antibodies in six patients with recurrent infection, or in eight persons with herpes labialis. These results are in agree-

ment with those of Schmidt et al. (1975) who did not detect HSV-IgM antibody in recurrent HSV infections by the neutralization technique. Recently, Kalimo et al. (1977) by a sensitive solid-phase RIA, have shown a significant level of HSV-IgM antibody against viral envelope antigens in patients with severe secondary infections. With regard to zoster, which represents a reactivation of latent VZV, specific IgM was also detected in only some patients (Ross and McDaid, 1972; Schmidt et al., 1975; Arvin et al., 1980; Hacham et al., 1980; Levy and Sarov, 1981). Development of serological tests for the detection of non-primary herpes-group virus infections is of major importance. Recently, Sarov et al. (1981) and Levy and Sarov (1981) have shown that specific IgA antibodies are produced in recurrent CMV and VZV infections. The possibility that specific IgA antibody

may be a marker of HSV reactivation

is presently

being investigated

in this

laboratory. With regard to herpes encephalitis, it may be noted that two of our patients (Nos. 1 and 4, Table 1) had HSV-specific IgM in the first serum sample drawn. Levine et al. (1978) suggest that it may be possible to diagnose HSV encephalitis by serum/CSF antibody ratios of <20, so that brain biopsy would not be necessary to prove the etiology of the encephalitis. However, such ratios may not be reached in some patients until 7-10 days after onset of illness (Levine et al., 1978). The ELISA test for the detection of HSVspecific IgM may be a useful adjunct to other non-invasive diagnostic techniques (McCallum et al., 1974; Levine et al., 1978) especially for early indication of HSV involvement. The method should be tested further, and ELISA studies in brain-biopsy proven cases of primary and recurrent HSV encephalitis should be carried out. REFERENCES Arvin, A.H. and C.M. Koropchack, Denoyel,

G.A., A. Gaspar

Falaky,

1980, J. Clin. Microbial.

and C. Nouyrigat,

LH., B.F. Vestergaard

12, 374.

1980, J. Clin. Microbial.

and A. Hornsleth,

1977, Stand.

Friedman,

M.G., S. Leventon-Kriss

Goldberg,

R.D. and I. Sarov, 1980, Isr. J. Med. Sci. 16, 111.

and I. Sarov, 1979, J. Clin. Microbial.

Hacham, M., S. Leventon-Krissand I. Sarov, 1980, Intervirology Haikin, H. and I. Sarov, 1980, Intervirology 14,155. Kahane,

S., V. Goldstein

Kalimo,

K.O.K.,

and I. Sarov,

B.R. Ziola,

11, 114.

J. Infect.

1979, Intervirology

M.K. Viljanen,

K. Granfors

Dis. 9, 79. 9,1.

13,214.

12, 39. and P. Toivanen,

1977, J. Immunol.

14,183. Krishna,

R.V., O.H. Meurman,

Leinikki,

P.O., I. Shekarchi,

Levine, D.P., C.B. lamer

T. Ziegler and U.H. Krech,

1980, J. Clin. Microbial.

12,46.

P. Drseft and J.L. Sever, 1978, J. Lab. Med. 92,849.

and A.M. Lerner,

1978, J. Am. Med. Assoc. 240, 356.

Levy, E. and I. Sarov, 1980, J. Med. Virol. 6, 249. Levy, E. and I. Sarov, 1981, Intervirology Lowry,

O.H., N.J. Rosebrough,

McCaBum,

15, 103.

A.L. Farr and R.J. Randall,

F.O., I.J. Chinn and J.V.T. Gostling,

McCarthy,

D., D.H. Goddard,

Meurman,

D.H. and B.R. Ziola,

Nachmias,

J. and B. Norrild,

1951, J. Biol. Chem.

1974, J. Med. Microbial.

B.K. Pell and E.J. Holborow, 1978, J. Clin. Pathol.

1979, Disease-a-Month,

1981, J. Immunol.

31,483. Vol. 25, No. 10.

193,265.

7, 325. Methods

41,63.

Methods

221

Ross, Ch. C. and R. McDaid, 1972, Br. Med. J. iv, 522. Sarov, I., P. Andersen and H.K. Andersen, 1980, Acta Pathol. Microbial. Stand. Sect. B 88, 1. Sarov, I., M. Siqueira-Linhares, Y. Chardonnet, E. Levy, M. Aymard, S. Bosshard, E. Nord and J.P. Revillard, 1981, Intervirology (in press). Schmidt, N.J., B. Forghani and E.M. Lennette, 1975, Infect. Immun. 12,728. Skaug, K. and E. Tjotta, 1974, Acta Pathol. Microbial. Stand. Sect. B 82, 323. Voller, A. and D.E. Bidwell, 1976, Br. J. Exp.Pathol. 57,243. Vuorimaa, T.O., P.P. Arstila, B.R. Ziola, A.A. Salmi, P.T. Hanninen and P.E. Halonen, 1978, J. Med. Virol. 2,271. Whitley, R.J., S.J. Soong, R. Dolin, G. Galasso et al., 1977, N. Engl. J. Med. 297,289.