95

Journal of‘ Virological Methods, 40 (1992) 95-106 0 1992 Elsevier Science Publishers B.V. i All rights reserved / 0166-0934/92/$05.00

VIRMET 01400

An M-antibody capture radioimmunoassay (MACRIA).for detection of JC virus-specific Wendy

A. Knowles,

Virus Rtference

IgM

Patricia E. Gibson, Julian F. Hand and David W.G. Brown

Division, Central Public Health Lahorutory.

London (UK)

(Accepted 20 May 1992)

Summary A solid-phase M-antibody capture radioimmunoassay (MACRIA) for detecting JC-specific IgM is described. The assay is based on a JC-specific monoclonal antibody (17.7.6) and Nonidet P40-treated, glycine-extracted antigen. MACRIA is more sensitive for JC IgM detection than haemagglutination inhibition (HI) following serum fractionation on a sucrose density gradient, and can be applied to large numbers of sera. The specificity of the assay was confirmed by examining sera from several acute virus infections and also those containing rheumatoid factor. Sera collected from renal transplant recipients with known active JC virus infection were found to contain more than 5 units of JC IgM. In this group of patients JC IgM represents either primary or reactivated JC infection. JC IgM was detected by MACRIA in 15 of 100 unselected blood donors, indicating that JC IgM is frequently produced in healthy seropositive individuals. Thirteen of the 15 sera positive from blood donors contained only low levels of JC IgM (< 5 units), but the specificity of all these results was confirmed in a blocking assay. It is suggested that these low levels of JC IgM may occur in up to 28% of seropositive individuals and result from active JC antigenic stimulation in healthy immunocompetent adults. JC-specific

IgM;

MACRIA; Renal transplant recipient; Healthy adult

Correspondence to: W.A. Colindale Avenue, London

Knowles, Virus Reference NW9 5HT, UK.

Division,

Central

Public

Health

Laboratory,

61

96

Introduction The human polyomavirus, JC, was first isolated in 1971 from post-mortem brain material of a patient with progressive multifocal leucoencephalopathy (PML) (Padgett et al., 1971). An initial antibody study, using the haemagglutination inhibition (HI) test, showed that in North America primary JC infections occurred throughout childhood. Antibody prevalence reached 65% by fourteen years of age, with a further rise to more than 75% in adults over fifty years of age (Padgett and Walker, 1973). Subsequent studies suggested that. in England, JC antibody is acquired somewhat later in adoIescence than in North America, with the maximum antibody prevalence of 50~60% being reached by 18-25 years of age (McCance and Gardner. 1987). By contrast, in Japan (Taguchi et al.. 1982) and in an isolated Indian population in Mexico (Golubjatniko~/, 1983), >60% and 320/o, respectively, of young children were seropositive for JC. After primary infection JC virus remains latent, possibly in the kidney (Chesters et al., 19X3), and can become JC viruria has been identified reactivated during immunosuppression. following renal (Hogan et al., 1980; Becktnann et al., 1982; Gardner et al., 1984) and bone marrow transplantation (O’Reilly et al., 1981; McCance and Gardner, 1987; Arthur et al., 1988). and during pregnancy (Coleman et al., 1980). JC virus has been established as the cause of PML? which occurs as a complication in imm~lnocompromised patients (Walker and Padgett, 1983). It is not known, however, whether PML is the result of a primary JC infection while immunity is impaired, or of reactivation of latent virus. As yet no other clinical features have been associated with primary or reactivated JC virus infection. Detection of JC-specific IgM antibody may identify patients experiencing a current JC infection, and so help to define other conditions associated with this infection. The presence of JC-specific IgM antibody has already been reported in PML patients (Padgett and Walker, 1983; McCance and Gardner, 1987; Knight et al., 1988), renal transplant recipients (Gardner et al., 1984) and pregnant women (Gibson et al., 1981). The techniques used were laborious and not suitable for large-scale screening of patients. In this report we describe the develop~lent and evaluation of a solid-phase assay (MACRIA) for the detection of JC-specific IgM antibody.

Materials and Methods JC untigm

and control

untigen

JC antigen was prepared from the COL 5 strain of JC virus (Knowles et al.l 1991). Cultures of primary human foetal glial cells (PHFG) were inoculated with a lo-- ’ dilution of JC virus, pass 6, and harvested at 7 weeks when there

was extensive cytopathic effect (cpe) and the haemagglutinin (HA) titre of the culture fluid was 1: 128. Two antigen preparations were made: (1) cells and fluid were frozen and thawed 3 times and the cells disrupted by ultrasonic disintegration for 30 sec. The antigen was then stored in aliquots at -30°C; (2) culture fluid was removed from the cells and replaced with a third of the volume of 0.2 M glycine-NaOH buffer, pH 9. The cells were then disrupted and the antigen stored as for preparation (1). Control antigen preparations were made in the same way from uninoculated PHFG cell cultures of the batches used for the JC antigens. Antigens were treated with Nonidet P40 (NP40) as follows: 1 vol of 2.5% NP40 in phosphate-buffered saline (PBS) was added to 9 vol of antigen and the mixture was warmed at 37°C for 30 min. Monoclonal

antibody

(MAh)

A J&specific MAb 17.7.6 (Knowles et al., 1991) was used in the MACRIA. IgG was purified from clarified ascitic fluid using a protein G Sepharose column (Pharmacia MAb Trap G). The final preparation, which represented a 1 in 5 dilution of the ascitic fluid, contained 0.95 mg/ml IgG. ““I-Lahelled

anti-mouse

antiserum

‘251-labelled sheep anti-mouse Amersham International plc. Anti-JC

whole antibody

(IM 13 1) was obtained

from

negative ~~~~an serum

Packs of whole, titrated human blood not suitable for transfusion were obtained from the North London Blood Transfusion Centre and screened for HI antibody to JC virus. A pack without detectable JC antibody (titre < 1:5) was chosen for use in the MACRIA, and serum was separated from it. Cells were removed by centrifugation and 0.5 ml of 30% aqueous calcium chloride solution was added per 100 ml of plasma. The clot which formed during 1.5 h at 36°C was loosened from the sides of the bottle, and serum was removed from the shrunken clot after freezing overnight at -30°C and thawing.

Etched polystyrene beads, 6.5 mm diameter (Northumbria Biologicals) were suspended in distilled water, treated in a sonicator for 20 s, washed again in distilled water and coated with a 1:3000 dilution of goat anti-human IgM (Tago) in 0.05 M carbonate/bicarbonate buffer, pH 9.6. After incubation on a shaker at room temperature for 2 h, the beads were stored in coating solution at 4°C for 1 to 6 days before use. The coated beads were washed three times in PBS/Tween buffer (PBST; PBSA pH, 7.3 with 0.1% Tween 20) and placed in the wells of an Abbott

9x

reaction tray, which contained 200 ~1 of test serum diluted in test diluent (PBST with 10% foetal calf serum, FCS). Following incubation at 37’C for 2 h, the beads were washed four times with PBST. The beads were then incubated, with intervening washes, in 200 1.~1of diluted JC antigen at 4°C for 16 h. 200 ,~l of diluted .IC MAb at 37°C for 2 11.and, for 2 h at 37°C in a dilution in 5% antianti-mouse antiserum JC negative human serum, 200 ,~l of ““I-labelled containing 75 000 cpm. The beads were then transferred to tubes and the bound radioactivity was counted for 5 min in a NE 1600 gamma counter (Nuclear Enterprises). The JC-specific IgM was quantified, using a strongly positive serum and a negative serum, as described for BK-specific IgM (Brown et al., 1984). The strongly positive serum was arbitrarily taken to contain 100 units of JC-specific IgM and was diluted in negative serum to give 30, 10. 3, 1 and 0.3 units. A standard curve was thus obtained against which test sera were evaluated.

These tests were performed

This test was performed

as previously

as previously

described

described

(Knowles

et al., 1989).

(Brown et al., 1984).

&Z-specific IgM antibody was detected in sera by WI, following serum fractionation on a sucrose density gradient, as previously described (Gibson et al.? 1981). Serum fractions were tested undiluted and at doubling dilutions up to 1 in 1024.

Serum samples The following sera were tested: (1) 63 sera from 16 renal transplant patients who had previously been tested for JC-specific IgM antibody by HI, following sucrose density gradient ~entrifugation; (2) 52 sera collected from patients aged 55-90 years with dementia or memory loss, in which JC HI antibody was undetectable at a dilution of 1 in 10; (3) 12 sera, mainly from renal transplant patients, containing between 5.8 and > 100 units of BK specific IgM antibody; (4) 43 sera containing IgM antibody specific for parvovirus B19, hepatitis B virus core antigen, hepatitis A virus, mumps, rubella or measles virus; (5) 15 sera positive for rheumatoid factor by a latex agglutination test (Rapi Tex RF, Hoechst - Behring); (6) Sera collected from 100 unselected blood donors in North London in 1990.

99

Results Standardisation

of’ the MACRIA

procedure

The MAb to be used in the assay was selected from seven ascitic fluids containing antibody to JC virus (Knowles et al., 1991). Each fluid was tested at dilutions of IO-’ and low5 in the third stage of the MACRIA. One ascitic fluid (from clone 17.7.6), which gave a much higher test/negative (T/N) ratio at 10m5 than at 10p3, was chosen for use in the test, and the IgG fraction was purified from it. The results using this purified MAb in the MACRIA at dilutions from lop2 to 10K8 are shown in Table 1. At high MAb concentrations a prozone effect was seen, but a very high T]iV ratio (196) was obtained at a dilution of 10K5 (9.5 ng/ml of IgG), which was, therefore, chosen as the working dilution. Sera from renal transplant patients with high titres of HI antibody to JC virus were screened by MACRIA, and the serum giving the strongest label binding was chosen as the positive control. Sera with no detectable HI antibody to JC were similarly screened, and the serum giving the weakest binding was used as the negative control. To determine the optinlum dilution at which to test sera, i.e., the dilution at which the T/N ratio was highest, the positive and ne ative control sera were B tested in MACRIA at dilutions between 10-l and lo- . A dilution of 10m2 was chosen for screening test sera because it gave the best discrimination between positive and negative. JC antigen preparations 1 and 2, as described in Materials and Methods, were tested in a MACRIA, both untreated (la and 2a) and after treatment with Nonidet P40, final concentration 0.25% (lb and 2b). Each antigen was diluted between 1O-o.5 and lo-“.“. The results are shown in Fig. 1. Antigen 2b, at a dilution of 1500, was chosen for use in the MACRIA because a sufficiently high T/N ratio was obtained at this high antigen dilution. Addition of 5% anti-JC negative human serum to the label diluent was found to greatly reduce label binding by the negative control serum relative to TABLE I The effect of varying the dilution of purified mAb in the JC MACRIA MAb dilution

Label binding (5 min count)

T/M ratio

Positive serum (10m2) Negative serum (IO ‘)

lo-’ IO-' 1O-4 10

-5

lO-6 10-7 1O-s No MAb T/N = Test/negative

5306 7082 1I426 19006 5643 661 114 70 control

7.51 185 ;; 98 65 67 91

1.1 38.3 150.3 195.9 51.6 10.2 1.7 0.8

100

loo90807060T/N

50403020lo0'

I

I

I

I

I

104.5

10-l

IO-I.5

10-2

10-2.5

I

10-3

I

10-3.5

Antigen dilution Fig. 1. The effect of varying the concentration of four antigen preparations in the JC MACRIA. I(a) disrupted cell antigen; l(b) NP40-treated disrupted cell antigen: 2(a) glycine-extracted disrupted cell antigen; 2(b) NP40-treated glycine-extracted disrupted cell antigen. T/IV test to ncgatlve control ratm.

the positive control Determination

serum, leading to a higher T/N ratio.

of the cut-off

Fifty-two sera, which were either negative (titre < 1:5) or equivocal (titre 1:5) by HI were tested in the MACRIA, and the results were expressed in units as shown in Table 2. Fifty-one sera gave results ranging from 0.13-0.8 1 units, and one serum gave 1.7 units. The population mean for these sera was 0.305 units and three standard deviations were calculated to be 0.75 units. A value of 1 unit was chosen to represent the cut-off between positive and negative. Sensitivity

of the MACRIA

for detecting JC-specific

IgM antibody

Sixty-three sera collected from 16 renal transplant patients, which had previously been tested for JC-specific IgM by HI following serum fractionation on a sucrose density gradient (SDG/HI), were re-tested in the MACRIA. Twenty sera, negative by SDG/HI, gave a MACRIA result of < 1 unit and thirty-eight sera, positive by SDG/HI, gave MACRIA results of between 1.2

101

TABLE 2 JC IgM reactions of various groups of sera Sera tested

No. tested

JC HI 1:640 and contained high levels of JC-specific IgM. The levels of JC IgM in the 63 sera from renal transplant patients are shown in Table 2. Specificity

of the MACRIA

for detecting JC-spec@-

IgM antibody

To examine any cross-reactivity between JC IgM and IgM to the related human polyomavirus BK, 12 sera containing between 5.8 and > 100 units of BK IgM were tested in the JC MACRIA. The results are shown in Table 2. Nine of the 12 sera gave results of < 1 unit and three gave a result of 32 units. A further 43 sera, containing IgM antibody specific for one of six other viruses, were also tested in the JC MACRIA and results are shown in Table 2. Thirty-three sera gave a result of < 1 unit, but 10 sera, taken during the acute phase of infection with mumps, rubella, parvovirus B19 or HBV, gave results of between 1.0 and 9.9 units. Fifteen sera containing rheumatoid factor, were tested in the JC MACRIA and the results are shown in Table 2; three of the 15 sera gave a result of 3 1 unit.

102

Prewlence

qj’ fC-spectfi’c

IgM antibody in unselected blood donors

In addition to samples taken from patients with clinical symptoms, samples from 100 healthy, unselected blood donors were tested. The results are shown in Table 2. Fifteen sera were positive, with results ranging from 1.2 to 12.5 units. The mean value was 0.721 units and three standard deviations was calculated to be 4.38 units. All 15 positive sera contained JC HI antibody at titres of 1:lO to 1:1280. Co@zation

of the specificit?* of’ lveak positive results

Further tests were carried out on 28 sera which had given a result of between 1 and 5 units, to confirm their specificity. Sera were re-tested in a MACRIA using JC antigen and control antigen in parallel, and all gave a result of < 1 unit with the control antigen. The IgM specificity of the reaction was also tested in a blocking assay. Each serum was incubated for 1 h at room temperature with 5% goat anti-human IgM (Tago) or 5% goat anti-human IgG (Tago) before addition of the coated beads, and the test was completed in the usual way. In 25 of the 28 sera the count obtained after incubation with the anti-p antiserum was reduced by >50% compared to that with the anti-;> antiserum; the count for the positive control serum was reduced by 97.5%. The three sera which were not blocked in this test were re-tested using 5% rabbit anti-human IgM (Dako) and 5% rabbit anti-human IgG (Dako). In this further test the count of all three sera was reduced by > 50% by the anti-p antiserum compared to the anti-7 antiserum, the positive control serum count being reduced by 99.2%. It was concluded that all 28 sera did contain JC IgM. When tested for JC HI antibody 27 were positive with titres ranging from 1:5 to 1:1280.

Discussion This is the first description of a solid-phase immunoassay for JC IgM. Detection methods for JC-specific IgM antibody have previously been reported by three laboratories. The methods used were indirect immunofluorescence on JC-infected primary human foetal glial cells (Padgett and Walker, 1983) or human amnion cells (Daniel et al., 1981) and sucrose density gradient fractionation of sera followed by HI or immune electron microscopy on the fractions (Gibson et al., 1981). As well as being time consuming and/or difficult to interpret, these techniques are not suitable for large-scale screening of sera. These problems are overcome by the MACRIA described here. Once the assay is optimised large numbers of sera can be accurately and objectively tested. The results of testing 63 sera from renal transplant patients showed MACRIA to be slightly more sensitive than SDG/HI. Three of the four sera positive by MACRIA but not by SDG/HI, were collected during or after a rise

103

in JC HI antibody in whole serum, and so these results are likely to represent specific JC IgM antibody. Thirteen of 55 sera containing IgM antibody to viruses other than JC, including BK, gave JC IgM results of 3 1 unit. However, there was no association with any one particular virus so antigenic cross-reaction seems unlikely. Furthermore, 15 out of 100 sera from healthy blood donors also gave a result of 3 1 unit, indicating that specific JC IgM activity is quite frequent. Although the presence of rheumatoid factor can lead to false positive results in this type of assay when specific IgG antibody is also present (Meurman, 1983) only three of 15 sera known to contain rheumatoid factor gave results of 3 1 unit. Rheumatoid factor did not interfere significantly in analogous Mantibody capture assays described for BK virus (Brown et al., 1984; Flaegstad and Traavik, 1985; Mahony et al., 1989). The JC- and IgM-specificity of all the low level MACRIA results (between 1 and 5 units) was confirmed, either by substituting control antigen for JC antigen or in a p blocking assay. All but one of the 28 sera containing this level of JC IgM was positive for JC antibody, as measured by HI. Therefore, these 27 patients had been infected with JC at some time and were probably harbouring the virus. As no symptoms have yet been associated with primary JC virus infection it is not easy to identify primary infection in healthy individuals. However, JC IgM levels ranging from five units to greater than 100 units were found, following primary or reactivated JC infection in immunocompromised renal transplant recipients. These levels of JC IgM subsequently decreased and in several cases had fallen to below five units in further sera collected several months later. The higher prevalence of JC IgM found in unselected blood donors than in patients with no previous exposure to JC (P= ~0.02) suggests that active JC infection may be occurring in many healthy seropositive adults. As the overall prevalence of JC HI antibody in adults over 18 years of age in England was reported to be 54% (McCance and Gardner, 1987) and sera collected from 15 of 100 unselected blood donors contained JC IgM, it can be calculated that at any one time reactivation may be occurring in 15 of 54 (28%) seropositive adults. The level of JC IgM detected in the blood donors was generally lower than in the immunocompromised patients. Indeed, in only two of the 15 positive blood donor sera was the JC IgM level greater than five units (5.4 and 12.5 units, respectively). This may represent the normal background prevalence of JC IgM in the population and so account for the low levels of JC IgM detected in sera from other acute viral infections. It is interesting to speculate whether the JC IgM detected represents sporadic reactivation in most or all seropositive adults or a long-term inability to suppress JC virus infection in some individuals. Flower et al. (1977) reported persistent low level BK IgM in healthy control subjects. Whilst JC viruria has been demonstrated in patients whose immunity is compromised (Hogan et al., 1980; O’Reilly et al., 1981; Beckmann et al., 1982;

104

Gardner et al., 1984; McCance and Gardner, 1987; Arthur et al., 1988) and presumably follows virus reactivation in most cases, it is not known whether reactivation of JC in immunocompetent individuals would also lead to viral shedding. Very few JC excretors were found amongst healthy control groups in studies based on urinary cytology and virus isolation. However Cobb et al. (1987), using a hybridot assay, reported finding JC DNA in the urine of six of 50 (12%) outpatients at a genito-urinary clinic in London: all urines were negative by cytology. Recently Kitamura et al. (1990) also studying urine samples by a blot hybridisation technique from outpatients at a urology clinic in Japan, reported JC DNA in 35 of 120 (29.2%) of patients. None of the patients in either of these studies was receiving immunosuppressive treatment. These findings are consistent with our interpretation of the present IgM results which is that active JC infection often occurs in immunocompetent individuals. Studies on serial serum and urine specimens, incorporating both JC IgM measurement and a sensitive method of virus detection in urine, would confirm this.

Acknowledgements We thank Dr E.F.D Mackenzie, Southmead General Hospital, Bristol foi providing the sera from renal transplant recipients, Dr J.B. Kurtz, John Radcliffe Hospital, Oxford for the sera from patients with dementia, and Dr J. Barbara, North London Blood Transfusion Centre for the blood donor sera. We also thank Dr B.J. Cohen for the rheumatoid factor results and Dr P.P. Mortimer for helpful discussion.

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