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Comparison of three immunoassays for the detection of anti-HHV6
Journal of Virological Methods, 38 (1992) 283-295 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
283
VIRMET 01349
Comparison of three immunoassays the detection of anti-HHV6 Peter V. Coyle”, Moya
Briggsb, Richard
S. Tedderb
for
and Julie D. Foxb
“Regional Virus Laboratory, Royal Victoria Hospital, Berfast (UK) and bDivision of Virology, Department of Medical Microbiology, University College and Middlesex School of Medicine, London (UK)
(Accepted 24 January
1992)
Summary Sera from 96 blood donors were tested for antibody to human herpesvirus 6 by indirect immunofluorescence (IF), circle immunoassay (CIA) and competitive radioimmunoassay (RIA). The correlation between the three assays was good but the CIA and competitive RIA were more sensitive for the detection of HHV6 antibody than indirect IF. The crossreaction of HHV6 antibody with that to the other human herpesviruses was also studied in this blood donor group. No correlation was found between antibody to human herpesvirus 6 by any of the methods described and antibody to any of the other human herpesviruses in these sera. Human herpesvirus 6; Competitive RIA; Circle immunoassay
Introduction A number of research groups have undertaken studies of the prevalence of antibody to human herpesvirus 6 (HHV6) in adults and children using immunofluorescence (IF) assays. Although seroprevalence rates quoted for HHV6 have varied greatly most people now agree that sera from the majority of adults are anti-HHV6 IgG positive and that specific antibody levels are generally low in the adult population. The subjective nature of IF makes variation in the results obtained between laboratories almost inevitable and Correspondence to: J. Fox, Division of Virology, University College and Middlesex School of Medicine, Windeyer Building, 46, Cleveland St., London, WIP 6DB, UK.
284
reported differences in HHV6 seroprevalence are probably due to problems in the determination of cut-off between positive and negative. Further sensitive serological assays are required for the detection of HHV6 antibody. In this study anti-HHV6 was measured in a group of 96 blood donors using competitive radioimmunoassay (RIA) and circle immunoassay (CIA). The latter is a modification of the line immunoassay published recently (Van der Groen et al., 1988). The results were compared with those previously reported for indirect IF. Serum samples from the blood donor group were also tested for antibody to the other human herpesviruses and the results compared with those for anti-HHV6 by competitive RIA and CIA.
Materials and Methods Virus
The AJ isolate of HHV6, obtained by culture of peripheral blood lymphocytes from a Gambian patient (Tedder et al., 1987) was used as source of antigen in the anti-HHV6 assays. Sera
Serum samples were available from 96 random blood donors attending the North London Blood Transfusion Centre, the gender of 93 of these blood donors was known. Indirect
immunofluorescence
The preparation of slides from HHV6 infected JJhan cells and the methods used for indirect IF were as previously described (Briggs et al., 1988, Fox et al., 1990). Competitive
radioimmunoassay
A globulin fraction was prepared by ammonium sulphate precipitation of serum from an individual with a high titre of anti-HHV6 (titre by indirect IF > 1 in 8000). Polystyrene remova-wells (Dynatech Ltd, Billingshurst, West Sussex, UK) were coated with 100 ~1 of a 1 in 1000 dilution of the globulin in 0.02 M Tris buffer (pH 7.5) containing 0.1% sodium azide (TAB) and left for 24 h at 4°C. The plates were washed in TAB, each well was filled to the rim with TAB containing 0.2% bovine serum albumin (BSA) and left for 1 h at room temperature in order to quench uncoated polystyrene. Excess buffer was removed and the plates either stored at 4°C or used immediately for detection of anti-HHV6. This solid-phase antibody was stable for at least 6 months at 4°C.
285
Antigen for the competitive RIA was prepared by freeze-thawing and sonication of HHV6-infected JJhan cells. Cellular debris was removed by centrifugation and the supernatant was stored at -20°C. Control antigen was prepared from uninfected JJhan cells in the same way. Antigen was stable for at least 6 months at - 20°C. Quenched plates were aspirated to dryness and 100 ~1 of optimally diluted antigen (1 in 10 to 1 in 30) in TAB + 0.5% BSA added. Incubation was for 24-48 h at room temperature and the plates were washed again but left moist. An IgG fraction of the high titre anti-HHV6 serum used for coating antibody was prepared by anion-exchange column chromatography using DE52 (Pharmacia, Central Milton Keynes, UK) equilibrated with 20 mM phosphate buffer (pH 8) as previously described (Tedder and Wilson-Croome, 1980). The IgG was labelled with ‘25I and the unlabelled antibody removed using the conditions described by Sutherland and Briggs (1983). Labelled IgG was stable for at least 1 month at 4°C in TAB + 10% BSA. Just prior to use, the antigen-coated wells were aspirated to dryness and 20 ~1 of TAB + 2% BSA added to each. A fixed volume of 5 ~1 of test serum followed by 75 ~1 TAB + 2% BSA containing 100 000 cpm of ‘251-labelled IgG were added to the well. Each serum was tested in duplicate wells. The plates were sealed and left in a moist chamber at room temperature overnight, washed in TAB to remove unbound label and the wells counted in a 16-channel gamma counter (NE 1600, N. E. Technology, Reading, Berkshire, UK). The maximum labelled antibody bound to the solid phase was calculated for 4 wells containing 5 ~1 foetal calf serum (FCS) instead of the test serum. The average % inhibition of the maximum label bound for each test serum was calculated. Sera from adults giving > 40% inhibition of label bound were considered to contain anti-HHV6, those giving 3140% inhibition were designated weakly reactive (&-) and those giving < 30% inhibition were considered not to contain detectable anti-HHV6. All preparations of first phase IgG, antigen and radiolabel were diluted and assayed prior to use in the competitive format to confirm a binding ratio of > 1O:l (HHV6 antigen: control antigen) and to ensure reproducibility of the assay. Circle immunoassay
Virus and control antigens were prepared together. Briefly 6 x lo6 JJhan cells were incubated with either 1 ml of stock, cell-free virus solution, stored at - 70°C and having a titre of lo3 infectious particles/ml, or 1 ml of RPM1 1640 medium with 10% foetal calf serum (RPMI), for 1 h at 37°C. They were then cultured in 30 ml of RPM1 for 7 days at 37°C in 10% CO*, clarified of cellular debris by centrifugation and then ultracentrifuged at 26000 rpm (SW28 swing out rotor, Beckman, High Wycombe, Bucks, UK) for 1 h. The supernatant was discarded and the pellet resuspended in 0.5 ml of phosphate buffered saline. This was stored at -70°C until needed and was stable for at least 3 months. Prior to use all antigen preparations were tested for optimal reactivity.
286
Antigen solutions were added as 5 ~1 drops onto Zetaprobe (Bio-Rad, Watford, Hertfordshire, UK) membrane. The drop spread rapidly to form a circle, the centre of which was pierced with a needle to allow pin-pointing of the antigen when dry. Doubling dilutions of the antigen where dried onto the membrane and, to avoid any non-specific binding of antibody, the membrane was blocked in PBS containing 0.05% Tween 20 and 1% skimmed milk (PBSTM). The membrane was then incubated for 30 min at 37°C in an ascites preparation of an HHV6 specific monoclonal antibody (H68H3) made by one of us (PVC), diluted 1 in 100 in PBSTM. A 20 min wash in PBSTM was followed by incubation for 30 min in a goat anti-mouse IgG horseradish peroxidase conjugate (Bio-Rad), diluted 1 in 3000 in PBSTM. After a final 30 min wash in PBST, the reaction was developed in a 0.05% solution of 3,3’-diaminobenzadine tetrahydrochloride in 20 mM Tris-HCl, pH 7.5 (DAB), before transferring the membrane to distilled water. The highest dilution of virus antigen reacting strongly (+ + +) and the corresponding dilutions of control antigen were employed in the test. The CIA was similar to the technique for antigen assessment. Optimal dilutions (1 in 4 to 1 in 8) of virus and control antigen were applied as pairs (5 ,LJ each) for each serum assayed with an additional pair to act as a conjugate control. After blocking in PBSTM, the membrane was transferred to a sheet of Whatman’s No. 1 paper soaked in PBS, spread on a ceramic tile, and the excess surface moisture allowed to soak through. Ten microlitres of each serum were then pipetted onto a pair of antigen containing circles and PBSTM was added to the conjugate control pair. The tile and membrane were then placed in a humidified box and incubated for 1 h at 37°C. Subsequent steps were as for the antigen test procedure above, with the replacement of the mouse conjugate with a human counterpart supplied by Sigma (Poole, Dorset, UK) used at a dilution of 1 in 3000. A positive result, i.e. the detection of specific IgG antibody was recorded when the reaction in the circle containing viral antigen was greater than in the control antigen circle. Negative reactivity was assigned to sera giving no reaction or reaction of equal intensity in virus and control antigen. Weakly reactive sera were accorded an equivocal score of +, while positive reactivity was graded +, + + or + + + in ascending order of strength of reaction, respectively. Other anti-herpesvirus immunoassays Antibody to cytomegalovirus (CMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV) was measured by the complement fixation test. Antibody to Epstein-Barr virus (EBV) VCA IgG was measured by indirect IF at a fixed dilution, as previously described (Fox et al., 1990).
287
Statistics The x2 test incorporating the Yate’s correction was used to assess differences in HHV6 antibody between male and female blood donors. Spearman rank correlation coefficients were calculated for comparison of antLHHV6 assays and for the assessment of HHV6 serological crossreaction with the other human herpesviruses.
Results Indirect IF Anti-HHV6 IgG results by indirect IF for this group of blood donors have been published previously (Briggs etal., 1988; Fox et al., 1990). 52% of samples from blood donors were anti-HHV6 IgG positive by this assay. There was no correlation between the presence or level of HHV6 antibody by indirect IF and presence or level of antibody to any of the other herpesviruses in these individuals. The number of strong positive sera by indirect IF was significantly higher in female than in male donors.
20 -
r B S -0 8 s *
15-
lo-
0
B -E z’
5-
0
-r
O-10 11.20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100
Anti-HHV6 by competitive RIA (% inhibition ) Fig. 1. AntGHHV6
by competitive RIA in sera from 96 blood donors. The average maximum expected label bound for each test serum is given.
% inhibition
of the
288
+
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*
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++
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+
Anti-HHV6 by RIA (% inhibition)
Anti-HHV6 by RIA (% inhibition)
Anti-HHV6 by RIA (96 inhibition)
Anti-HHV6 by RIA (95 inhibit~n)
+
Fig. 2. Comparison of anti-HHV6 by competitive RIA and antibody to the other human herpesviruses in sera from 96 blood donors. There was no significant correlation between HHV6 antibody levels by competitive RIA and antibody levels to any of the other human herpesviruses in this population; rank correlation coefficients -0.13 (HSV), -0.01 (CMV), 0.09 (VZV) and 0.04 (EBV) (P >O.l).
Foetal calf serum served as a ‘negative’ control in the competitive RIA to give a value for maximum binding of label in the absence of specific antibody for HHV6. Approximately 2% (2000 cpm) of the labelled IgG bound to the solid phase when there was no competing antibody present compared with 0.05% (50 cpm) bound in the presence of high titre anti-HHV6.
289
I
O-10
l l ea 0
0 . . .
.
I
I
11-20 21.30
0
. . .
0.0
0 I
31.40 41.50
I
I
I
51-60 61-70 71-80
1
I
81-90 91-100
Anti-HHV6 by competitive RIA (% inhibition) Fig. 3. Correlation
between competitive RIA and indirect IF for anti-HHV6 in samples donors; rank correlation coefficient 0.78 (P
from 96 blood
The discrimination between positive and negative by competitive RIA was not clear (Fig. 1) and it was difficult to establish true negatives in this group (21 samples gave ~40% inhibition). It is possible that as all samples gave some inhibition in the competitive RIA when compared with FCS that all blood donor sera contained some anti-HHV6. In other experiments, sera taken from children with roseola infantum prior to seroconversion for HHV6 gave inhibitions varying from 16-27% in the competitive RIA. Using these data, serum samples from adults giving ~40% inhibition of label bound were considered to contain anti-HHV6, those giving 3140% inhibition were weakly reactive (+) and those giving < 30% inhibition were considered not to contain antLHHV6. Using this scoring system 76% of sera from the blood donors were classified as HHV6 antibody positive in this assay and a further 9% were designated weakly reactive. There was no significant difference in the proportion of HHV6 antibody positives when comparing male and female blood donors; 38/50 (76%) of males were reactive by RIA compared with 35/43 (81%) of females. The distribution of high level reactivity by RIA was also not significantly different when comparing males and females; 24/50 (48%) sera from male donors gave > 60% inhibition of label binding compared with 22/43 (51 Oh) sera from female donors. Antibody to HHV6 by competitive RIA did not correlate with the presence or level of antibody to any of the other human herpesviruses (Fig. 2). The distribution of reactivity in this assay was bimodal (Fig. 1). This was not investigated further.
290
Correlation between competitive
RIA and indirect IF
A comparison of antibody levels to HHV6 by indirect IF and competitive RIA was made using the results for the blood donor group (Fig. 3). There was good correlation between score by IF and % inhibition by competitive RIA (rank correlation coefficient 0.78, P 40% inhibition of label bound in the competitive RIA, the IF positive sample that was designated equivocal by RIA gave 40% inhibition. Half of the samples designated negative or f by IF were reactive by RIA suggesting that the latter method is more sensitive for the detection of anti-HHV6. Circle immunoassay The results for anti-HHV6 by CIA in the blood donor group are given in Fig. 4. Again a proportion of sera (20%) was designated equivocal in this assay but 71% of the blood donors were scored + , + + or + + + . There was no significant difference in the prevalence of HHV6 antibody positive sera when comparing male and female blood donors; 3 l/50 (62%) of sera from males were anti-HHV6 IgG positive by CIA compared with 34/43 (79%) from females. The distribution of HHV6 high level reactivity by CIA was also not significantly different when comparing sera from males and females; 1 l/50 (22%) sera from male donors were scored + + or + + + compared with 16143 50 1
40
30
20
0 2
+
++
+++
Anti-HHV6 by CIA (1 in 50) Fig. 4. Anti-HHV6
IgG by CIA in sera from 96 blood donors.
291
(37%) from female donors. Antibody by CIA did not correlate with the presence or level of antibody to any of the other human herpesviruses (Fig. 5).
There was good correlation between score by IF and CIA (rank correlation coefficient 0.73, P
128 64 32 16 8
+
c+*
+t +
4
+
+*+
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+++
2
+
<2
f:
+
++
+++
f
+
l
l
l
tt:s ++
lt**
+++
AntLHHV6 IgG by CIA (1 in 50)
Anti-HHV6 IgG by CIA (1 in 50)
++++
e .s
64
++
32
8
16
g :, ..!# 2
8 4 2 <2
***
+
l
1:
+
++
Anti-HHV6 IgG by CIA (1 in 50)
t +++
2
+
++
+++
Anti-HHV6 IgG by CIA (1 in 50)
Fig. 5. Comparison of anti-HHV6 IgG by CIA and antibody to the other human herpesviruses in 96 blood donors. There was no significant correlation between HHV6 antibody levels by CIA and antibody levels to the other human herpesviruses in this population; rank corretation coeffZents -0.04 (HSV), 0.03 (CMV), 0.17 (VZV) and -0.01 (EBV) (P >O.l).
292
+++
l
l
ee ee
*e*
l l
e ee
.**
l
.
++
e e*
ee
l
*e* a
l
l
l
*** me** l **e l *e* l
l
+
eee e
l
.
e?
.*
.
l l **
. b
‘.
l
.
.
.** l
l **
f
l-
ee
l .e 0.0
0.0 l eo
l
.‘.
*e I
1
I
++
+
t
+++
Anti-HHV6 IgG by CIA (1 in 50) Fig. 6. Correlation
between
CIA and indirect IF for anti-HHV6 (correlation coeffkient 0.73, P
le
O-10
l l oe
l l ee l ee
le
l ee
le
le
11-20
21-30
31.40
41.50
IgG in samples
from 96 blood
donors
e
l
l
we
eee
l ee
l ee
l ee l ee
l e*
51.60
61-70
71-80
81-90
91-100
AntLHHV6 by competitive RIA (% inhibition) Fig. 7. Correlation
between
competitive RIA and CIA for antLHHV6 in samples (correlation coeffkient 0.72, P
from 96 blood donors
293
that the latter method was more sensitive than IF for the detection of HHV6 specific IgG. Six samples scored ) by CIA were positive by IF, one of these samples was also equivocal by RIA. Correlation between competitive
RIA and CIA
There was a good correlation between reactivity in RIA and CIA (Fig. 7, rank correlation coefficient 0.72, P < 0.001). Half of the sera scored negative or f by CIA (15/30) gave > 40% inhibition of label binding in the competitive RIA. Twenty-one sera gave 40% or less inhibition in the competitive RIA, 6 of these were positive by CIA. The two assays would seem to give comparable overall sensitivity for the detection of anti-HHV6 but different sera give variable results around the cut-off for each assay. When comparing all 3 assays, 64/96 (67%) sera from blood donors had concordant results (50 antibody positive and 14 antibody negative). Of the 33 discordant results, 15 sera were concordant in two. Ten were positive by RIA and CIA and 5 were positive by RIA and IF. There were 18 samples which were only positive by 1 anti-HHV6 assay; 9 were positive by RIA, 8 were positive by CIA and 1 was positive by indirect IF.
Discussion Most studies of HHV6 antibody prevalence in healthy adults have utilised indirect or anticomplementary IF for the detection of anti-HHV6 IgG. The subjective nature of IF has led to difficulties in the interpretation of results and published HHV6 seroprevalence rates have varied greatly. Where anti-HHV6 specific ELISAs have been developed (Saxinger et al., 1988, Dahl et al., 1990) the sensitivity seems to be better than that of IF but there is still a problem in the interpretation of sera giving reactions around the cut-off. We have reported two further assays for the detection of HHV6 antibody which are more sensitive than indirect IF. The correlation between IF, RIA and CIA was very good although the sera reacting at the cut-off level for each assay were different. The RIA gives an absolute value (Oh inhibition) for HHV6 antibody in serum samples and although the CIA was read by eye in this study the colour reaction for each serum could be measured using a densitometer. In both RIA and CIA the distribution of reactivity was broad, with no clear differentiation between reactive and unreactive sera. This was particularly so for the RIA where sera showed in addition a bimodal distribution identifying a subpopulation of individuals with high competitive reactivity. All human sera gave some inhibition in the RIA when compared with FCS. This observation implies that all human sera contain some level of anti-HHV6. Were the RIA sensitivity to be increased by the addition of 25 or even 50 ~1 of test serum rather than 5 ~1 as at present, greater levels of competitive reactivity in human sera would confirm this hypothesis and perhaps reduce the number of sera
294
designated equivocal by RIA. Such a modification would be unlikely to alter the specificity of a competitive RIA. Indirect IF, competitive RIA and CIA seem to be specific for the detection of anti-~HV6 in this group of blood donors. There was no correlation between presence or level of anti-HHVG and presence or level of antibody to any of the other human herpesviruses in these individuals, Similar cross-correlation studies have been carried out by another group, with similar results (Saxinger et al., 1988). There have been a number of reports of HHV6 primary infection and reactivation in adults, often associated with a concomitant infection with one of the other human herpesviruses. Active HHV6 infection in transplant recipients has been demonstrated by a number of research groups (Irving et al., 1988; Morris et al., 1988; Asano et al., 1989; Ward et al., 1989; Chou and Scott, 1990; Okuno et al., 1990; Sutherland et al., 1991) and recently Carrigan et al. (1991) reported association of HHVG infection with interstitial pneumonitis in two bone marrow transplant recipients. Whilst the significance of an equivocal result for RIA or CIA is unclear, the number of sera reacting at the cut-off level for these assays was less than for indirect IF. Sensitive assays, such as the competitive RIA and CIA, will be useful in the determination of the HHV6 immune status of transplant patients who may be at risk from active HHV6 infection. Acknowledgements
The authors would like to thank Dr. M. Contreras and Dr. W, Irving for providing sera. This work was funded, in part, by a grant from The Wellcome Trust. References Asano, Y., Yoshikawa, T., Suga, S., Yazaki, T., Hirabayashi, S., Ono, Y., Tsuzuki, K. and Oshima, S. (1989) Human herpesvirus 6 harbouring in kidney. Lancet ii, 1391. Briggs, M., Fox, J. and Tedder, R. (1988) Age prevalence of antibody to human herpesvirus 6. Lancet i, 1058-1059. Carrigan, D., Drobyski, W., Russler, S., Tapper, M., Knox, K. and Ash, R. (1991) Interstitial pneumonitis associated with human herpesvirusinfection after marrow transplantation. Lancet 338, 147-149. Chou, S. and Scott, K. (1990) Rises in antibody to human herpesvirus 6 detected by enzyme immunoassay in transplant recipients with primary cytomegalovirus infection. J. Clin. Microbial. 28, 851-854. Dahl, H., Linde, A., Sundqvist, V.-A. and Wahren, B. (1990) An enzyme-linked immunosorbent assay for IgG antibodies to human herpesvirus 6. J. Virol. Methods 29, 313-324. Fox, J., Ward, P., Briggs, M., Irving, W., Stammers, T. and Tedder, R. (1990) Production of IgM antibody to HHV6 in reactivation and primary infection. Epidemiol. Infect. 104, 289-296. Irving, W., Cunningham, A., Keogh, A. and Chapman, J. (1988) Antibody to both human herpesvirus 6 and cytomegalovirus. Lancet ii, 63%63 1.
295 Morris, D., Littler, E., Jordan, D. and Arrand, J. (1988) Antibody responses to human herpesvirus 6 and other herpes viruses. Lancet ii, 142551426. Okuno, T., Higashi, K., Shiraki, K., Yamanishi, K., Takahashi, M., Kokado, J., Ishibashi, M., Takahara, S., Sonoda, T., Tanaka, K., Baba, K., Yabuuchi, H. and Kurata, T. (1990) Human herpesvirus 6 infection in renal transplantation. Transplantation 49, 519522. Saxinger, C., Polesky, H., Eby, N., Grufferman, S., Murphy, R., Tegtmeir, G., Parekh, V., Memon, S. and Hung, C. (1988) Antibody reactivity with HBLV (HHV-6) in US populations. J. Virol. Methods 21, 199-208. Sutherland, S. and Briggs, D. (1983) The detection of antibodies to cytomegalovirus in the sera of renal transplant patients by an IgM antibody capture assay. J. Med. Viral II, 147-159. Sutherland, S., Christofinis, G., O’Grady, J. and Williams, R. (1991) A serological investigation of human herpesvirus 6 infections in liver transplant recipients and the detection of cross-reacting antibodies to cytomegalovirus. J. Med. Virol. 33, 172-176. Tedder, R. and Wilson-Croome, R. (1980) Detection by radioimmunoassay of IgM class antibody to hepatitis B core antigen: a comparison of two methods. J. Med. Virol. 6, 235-247. Tedder, R., Briggs, M., Cameron, C., Honess, R., Robertson, D. and Whittle, H. (1987) A novel lymphotropic herpesvirus. Lancet ii, 39&393. Van der Groen, G., Beelaert, G., Demets, P., Kestens, L., Van Overmeir, C. and Wery, M. (1988) Line immunoass iew serologic test for malaria. Ann. Sot. Belg. Med. Trop. 68, 3741. fstathiou, S. (1989) Primary human herpesvirus 6 infection in a patient Ward, K., Gray, J. following liver transplantation from a seropositive donor. J. Med. Virol. 28, 69-72.
283
VIRMET 01349
Comparison of three immunoassays the detection of anti-HHV6 Peter V. Coyle”, Moya
Briggsb, Richard
S. Tedderb
for
and Julie D. Foxb
“Regional Virus Laboratory, Royal Victoria Hospital, Berfast (UK) and bDivision of Virology, Department of Medical Microbiology, University College and Middlesex School of Medicine, London (UK)
(Accepted 24 January
1992)
Summary Sera from 96 blood donors were tested for antibody to human herpesvirus 6 by indirect immunofluorescence (IF), circle immunoassay (CIA) and competitive radioimmunoassay (RIA). The correlation between the three assays was good but the CIA and competitive RIA were more sensitive for the detection of HHV6 antibody than indirect IF. The crossreaction of HHV6 antibody with that to the other human herpesviruses was also studied in this blood donor group. No correlation was found between antibody to human herpesvirus 6 by any of the methods described and antibody to any of the other human herpesviruses in these sera. Human herpesvirus 6; Competitive RIA; Circle immunoassay
Introduction A number of research groups have undertaken studies of the prevalence of antibody to human herpesvirus 6 (HHV6) in adults and children using immunofluorescence (IF) assays. Although seroprevalence rates quoted for HHV6 have varied greatly most people now agree that sera from the majority of adults are anti-HHV6 IgG positive and that specific antibody levels are generally low in the adult population. The subjective nature of IF makes variation in the results obtained between laboratories almost inevitable and Correspondence to: J. Fox, Division of Virology, University College and Middlesex School of Medicine, Windeyer Building, 46, Cleveland St., London, WIP 6DB, UK.
284
reported differences in HHV6 seroprevalence are probably due to problems in the determination of cut-off between positive and negative. Further sensitive serological assays are required for the detection of HHV6 antibody. In this study anti-HHV6 was measured in a group of 96 blood donors using competitive radioimmunoassay (RIA) and circle immunoassay (CIA). The latter is a modification of the line immunoassay published recently (Van der Groen et al., 1988). The results were compared with those previously reported for indirect IF. Serum samples from the blood donor group were also tested for antibody to the other human herpesviruses and the results compared with those for anti-HHV6 by competitive RIA and CIA.
Materials and Methods Virus
The AJ isolate of HHV6, obtained by culture of peripheral blood lymphocytes from a Gambian patient (Tedder et al., 1987) was used as source of antigen in the anti-HHV6 assays. Sera
Serum samples were available from 96 random blood donors attending the North London Blood Transfusion Centre, the gender of 93 of these blood donors was known. Indirect
immunofluorescence
The preparation of slides from HHV6 infected JJhan cells and the methods used for indirect IF were as previously described (Briggs et al., 1988, Fox et al., 1990). Competitive
radioimmunoassay
A globulin fraction was prepared by ammonium sulphate precipitation of serum from an individual with a high titre of anti-HHV6 (titre by indirect IF > 1 in 8000). Polystyrene remova-wells (Dynatech Ltd, Billingshurst, West Sussex, UK) were coated with 100 ~1 of a 1 in 1000 dilution of the globulin in 0.02 M Tris buffer (pH 7.5) containing 0.1% sodium azide (TAB) and left for 24 h at 4°C. The plates were washed in TAB, each well was filled to the rim with TAB containing 0.2% bovine serum albumin (BSA) and left for 1 h at room temperature in order to quench uncoated polystyrene. Excess buffer was removed and the plates either stored at 4°C or used immediately for detection of anti-HHV6. This solid-phase antibody was stable for at least 6 months at 4°C.
285
Antigen for the competitive RIA was prepared by freeze-thawing and sonication of HHV6-infected JJhan cells. Cellular debris was removed by centrifugation and the supernatant was stored at -20°C. Control antigen was prepared from uninfected JJhan cells in the same way. Antigen was stable for at least 6 months at - 20°C. Quenched plates were aspirated to dryness and 100 ~1 of optimally diluted antigen (1 in 10 to 1 in 30) in TAB + 0.5% BSA added. Incubation was for 24-48 h at room temperature and the plates were washed again but left moist. An IgG fraction of the high titre anti-HHV6 serum used for coating antibody was prepared by anion-exchange column chromatography using DE52 (Pharmacia, Central Milton Keynes, UK) equilibrated with 20 mM phosphate buffer (pH 8) as previously described (Tedder and Wilson-Croome, 1980). The IgG was labelled with ‘25I and the unlabelled antibody removed using the conditions described by Sutherland and Briggs (1983). Labelled IgG was stable for at least 1 month at 4°C in TAB + 10% BSA. Just prior to use, the antigen-coated wells were aspirated to dryness and 20 ~1 of TAB + 2% BSA added to each. A fixed volume of 5 ~1 of test serum followed by 75 ~1 TAB + 2% BSA containing 100 000 cpm of ‘251-labelled IgG were added to the well. Each serum was tested in duplicate wells. The plates were sealed and left in a moist chamber at room temperature overnight, washed in TAB to remove unbound label and the wells counted in a 16-channel gamma counter (NE 1600, N. E. Technology, Reading, Berkshire, UK). The maximum labelled antibody bound to the solid phase was calculated for 4 wells containing 5 ~1 foetal calf serum (FCS) instead of the test serum. The average % inhibition of the maximum label bound for each test serum was calculated. Sera from adults giving > 40% inhibition of label bound were considered to contain anti-HHV6, those giving 3140% inhibition were designated weakly reactive (&-) and those giving < 30% inhibition were considered not to contain detectable anti-HHV6. All preparations of first phase IgG, antigen and radiolabel were diluted and assayed prior to use in the competitive format to confirm a binding ratio of > 1O:l (HHV6 antigen: control antigen) and to ensure reproducibility of the assay. Circle immunoassay
Virus and control antigens were prepared together. Briefly 6 x lo6 JJhan cells were incubated with either 1 ml of stock, cell-free virus solution, stored at - 70°C and having a titre of lo3 infectious particles/ml, or 1 ml of RPM1 1640 medium with 10% foetal calf serum (RPMI), for 1 h at 37°C. They were then cultured in 30 ml of RPM1 for 7 days at 37°C in 10% CO*, clarified of cellular debris by centrifugation and then ultracentrifuged at 26000 rpm (SW28 swing out rotor, Beckman, High Wycombe, Bucks, UK) for 1 h. The supernatant was discarded and the pellet resuspended in 0.5 ml of phosphate buffered saline. This was stored at -70°C until needed and was stable for at least 3 months. Prior to use all antigen preparations were tested for optimal reactivity.
286
Antigen solutions were added as 5 ~1 drops onto Zetaprobe (Bio-Rad, Watford, Hertfordshire, UK) membrane. The drop spread rapidly to form a circle, the centre of which was pierced with a needle to allow pin-pointing of the antigen when dry. Doubling dilutions of the antigen where dried onto the membrane and, to avoid any non-specific binding of antibody, the membrane was blocked in PBS containing 0.05% Tween 20 and 1% skimmed milk (PBSTM). The membrane was then incubated for 30 min at 37°C in an ascites preparation of an HHV6 specific monoclonal antibody (H68H3) made by one of us (PVC), diluted 1 in 100 in PBSTM. A 20 min wash in PBSTM was followed by incubation for 30 min in a goat anti-mouse IgG horseradish peroxidase conjugate (Bio-Rad), diluted 1 in 3000 in PBSTM. After a final 30 min wash in PBST, the reaction was developed in a 0.05% solution of 3,3’-diaminobenzadine tetrahydrochloride in 20 mM Tris-HCl, pH 7.5 (DAB), before transferring the membrane to distilled water. The highest dilution of virus antigen reacting strongly (+ + +) and the corresponding dilutions of control antigen were employed in the test. The CIA was similar to the technique for antigen assessment. Optimal dilutions (1 in 4 to 1 in 8) of virus and control antigen were applied as pairs (5 ,LJ each) for each serum assayed with an additional pair to act as a conjugate control. After blocking in PBSTM, the membrane was transferred to a sheet of Whatman’s No. 1 paper soaked in PBS, spread on a ceramic tile, and the excess surface moisture allowed to soak through. Ten microlitres of each serum were then pipetted onto a pair of antigen containing circles and PBSTM was added to the conjugate control pair. The tile and membrane were then placed in a humidified box and incubated for 1 h at 37°C. Subsequent steps were as for the antigen test procedure above, with the replacement of the mouse conjugate with a human counterpart supplied by Sigma (Poole, Dorset, UK) used at a dilution of 1 in 3000. A positive result, i.e. the detection of specific IgG antibody was recorded when the reaction in the circle containing viral antigen was greater than in the control antigen circle. Negative reactivity was assigned to sera giving no reaction or reaction of equal intensity in virus and control antigen. Weakly reactive sera were accorded an equivocal score of +, while positive reactivity was graded +, + + or + + + in ascending order of strength of reaction, respectively. Other anti-herpesvirus immunoassays Antibody to cytomegalovirus (CMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV) was measured by the complement fixation test. Antibody to Epstein-Barr virus (EBV) VCA IgG was measured by indirect IF at a fixed dilution, as previously described (Fox et al., 1990).
287
Statistics The x2 test incorporating the Yate’s correction was used to assess differences in HHV6 antibody between male and female blood donors. Spearman rank correlation coefficients were calculated for comparison of antLHHV6 assays and for the assessment of HHV6 serological crossreaction with the other human herpesviruses.
Results Indirect IF Anti-HHV6 IgG results by indirect IF for this group of blood donors have been published previously (Briggs etal., 1988; Fox et al., 1990). 52% of samples from blood donors were anti-HHV6 IgG positive by this assay. There was no correlation between the presence or level of HHV6 antibody by indirect IF and presence or level of antibody to any of the other herpesviruses in these individuals. The number of strong positive sera by indirect IF was significantly higher in female than in male donors.
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O-10 11.20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100
Anti-HHV6 by competitive RIA (% inhibition ) Fig. 1. AntGHHV6
by competitive RIA in sera from 96 blood donors. The average maximum expected label bound for each test serum is given.
% inhibition
of the
288
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Anti-HHV6 by RIA (% inhibition)
Anti-HHV6 by RIA (% inhibition)
Anti-HHV6 by RIA (96 inhibition)
Anti-HHV6 by RIA (95 inhibit~n)
+
Fig. 2. Comparison of anti-HHV6 by competitive RIA and antibody to the other human herpesviruses in sera from 96 blood donors. There was no significant correlation between HHV6 antibody levels by competitive RIA and antibody levels to any of the other human herpesviruses in this population; rank correlation coefficients -0.13 (HSV), -0.01 (CMV), 0.09 (VZV) and 0.04 (EBV) (P >O.l).
Foetal calf serum served as a ‘negative’ control in the competitive RIA to give a value for maximum binding of label in the absence of specific antibody for HHV6. Approximately 2% (2000 cpm) of the labelled IgG bound to the solid phase when there was no competing antibody present compared with 0.05% (50 cpm) bound in the presence of high titre anti-HHV6.
289
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51-60 61-70 71-80
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81-90 91-100
Anti-HHV6 by competitive RIA (% inhibition) Fig. 3. Correlation
between competitive RIA and indirect IF for anti-HHV6 in samples donors; rank correlation coefficient 0.78 (P
from 96 blood
The discrimination between positive and negative by competitive RIA was not clear (Fig. 1) and it was difficult to establish true negatives in this group (21 samples gave ~40% inhibition). It is possible that as all samples gave some inhibition in the competitive RIA when compared with FCS that all blood donor sera contained some anti-HHV6. In other experiments, sera taken from children with roseola infantum prior to seroconversion for HHV6 gave inhibitions varying from 16-27% in the competitive RIA. Using these data, serum samples from adults giving ~40% inhibition of label bound were considered to contain anti-HHV6, those giving 3140% inhibition were weakly reactive (+) and those giving < 30% inhibition were considered not to contain antLHHV6. Using this scoring system 76% of sera from the blood donors were classified as HHV6 antibody positive in this assay and a further 9% were designated weakly reactive. There was no significant difference in the proportion of HHV6 antibody positives when comparing male and female blood donors; 38/50 (76%) of males were reactive by RIA compared with 35/43 (81%) of females. The distribution of high level reactivity by RIA was also not significantly different when comparing males and females; 24/50 (48%) sera from male donors gave > 60% inhibition of label binding compared with 22/43 (51 Oh) sera from female donors. Antibody to HHV6 by competitive RIA did not correlate with the presence or level of antibody to any of the other human herpesviruses (Fig. 2). The distribution of reactivity in this assay was bimodal (Fig. 1). This was not investigated further.
290
Correlation between competitive
RIA and indirect IF
A comparison of antibody levels to HHV6 by indirect IF and competitive RIA was made using the results for the blood donor group (Fig. 3). There was good correlation between score by IF and % inhibition by competitive RIA (rank correlation coefficient 0.78, P
40
30
20
0 2
+
++
+++
Anti-HHV6 by CIA (1 in 50) Fig. 4. Anti-HHV6
IgG by CIA in sera from 96 blood donors.
291
(37%) from female donors. Antibody by CIA did not correlate with the presence or level of antibody to any of the other human herpesviruses (Fig. 5).
There was good correlation between score by IF and CIA (rank correlation coefficient 0.73, P
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Anti-HHV6 IgG by CIA (1 in 50)
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Anti-HHV6 IgG by CIA (1 in 50)
Fig. 5. Comparison of anti-HHV6 IgG by CIA and antibody to the other human herpesviruses in 96 blood donors. There was no significant correlation between HHV6 antibody levels by CIA and antibody levels to the other human herpesviruses in this population; rank corretation coeffZents -0.04 (HSV), 0.03 (CMV), 0.17 (VZV) and -0.01 (EBV) (P >O.l).
292
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Anti-HHV6 IgG by CIA (1 in 50) Fig. 6. Correlation
between
CIA and indirect IF for anti-HHV6 (correlation coeffkient 0.73, P
le
O-10
l l oe
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21-30
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61-70
71-80
81-90
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AntLHHV6 by competitive RIA (% inhibition) Fig. 7. Correlation
between
competitive RIA and CIA for antLHHV6 in samples (correlation coeffkient 0.72, P
from 96 blood donors
293
that the latter method was more sensitive than IF for the detection of HHV6 specific IgG. Six samples scored ) by CIA were positive by IF, one of these samples was also equivocal by RIA. Correlation between competitive
RIA and CIA
There was a good correlation between reactivity in RIA and CIA (Fig. 7, rank correlation coefficient 0.72, P < 0.001). Half of the sera scored negative or f by CIA (15/30) gave > 40% inhibition of label binding in the competitive RIA. Twenty-one sera gave 40% or less inhibition in the competitive RIA, 6 of these were positive by CIA. The two assays would seem to give comparable overall sensitivity for the detection of anti-HHV6 but different sera give variable results around the cut-off for each assay. When comparing all 3 assays, 64/96 (67%) sera from blood donors had concordant results (50 antibody positive and 14 antibody negative). Of the 33 discordant results, 15 sera were concordant in two. Ten were positive by RIA and CIA and 5 were positive by RIA and IF. There were 18 samples which were only positive by 1 anti-HHV6 assay; 9 were positive by RIA, 8 were positive by CIA and 1 was positive by indirect IF.
Discussion Most studies of HHV6 antibody prevalence in healthy adults have utilised indirect or anticomplementary IF for the detection of anti-HHV6 IgG. The subjective nature of IF has led to difficulties in the interpretation of results and published HHV6 seroprevalence rates have varied greatly. Where anti-HHV6 specific ELISAs have been developed (Saxinger et al., 1988, Dahl et al., 1990) the sensitivity seems to be better than that of IF but there is still a problem in the interpretation of sera giving reactions around the cut-off. We have reported two further assays for the detection of HHV6 antibody which are more sensitive than indirect IF. The correlation between IF, RIA and CIA was very good although the sera reacting at the cut-off level for each assay were different. The RIA gives an absolute value (Oh inhibition) for HHV6 antibody in serum samples and although the CIA was read by eye in this study the colour reaction for each serum could be measured using a densitometer. In both RIA and CIA the distribution of reactivity was broad, with no clear differentiation between reactive and unreactive sera. This was particularly so for the RIA where sera showed in addition a bimodal distribution identifying a subpopulation of individuals with high competitive reactivity. All human sera gave some inhibition in the RIA when compared with FCS. This observation implies that all human sera contain some level of anti-HHV6. Were the RIA sensitivity to be increased by the addition of 25 or even 50 ~1 of test serum rather than 5 ~1 as at present, greater levels of competitive reactivity in human sera would confirm this hypothesis and perhaps reduce the number of sera
294
designated equivocal by RIA. Such a modification would be unlikely to alter the specificity of a competitive RIA. Indirect IF, competitive RIA and CIA seem to be specific for the detection of anti-~HV6 in this group of blood donors. There was no correlation between presence or level of anti-HHVG and presence or level of antibody to any of the other human herpesviruses in these individuals, Similar cross-correlation studies have been carried out by another group, with similar results (Saxinger et al., 1988). There have been a number of reports of HHV6 primary infection and reactivation in adults, often associated with a concomitant infection with one of the other human herpesviruses. Active HHV6 infection in transplant recipients has been demonstrated by a number of research groups (Irving et al., 1988; Morris et al., 1988; Asano et al., 1989; Ward et al., 1989; Chou and Scott, 1990; Okuno et al., 1990; Sutherland et al., 1991) and recently Carrigan et al. (1991) reported association of HHVG infection with interstitial pneumonitis in two bone marrow transplant recipients. Whilst the significance of an equivocal result for RIA or CIA is unclear, the number of sera reacting at the cut-off level for these assays was less than for indirect IF. Sensitive assays, such as the competitive RIA and CIA, will be useful in the determination of the HHV6 immune status of transplant patients who may be at risk from active HHV6 infection. Acknowledgements
The authors would like to thank Dr. M. Contreras and Dr. W, Irving for providing sera. This work was funded, in part, by a grant from The Wellcome Trust. References Asano, Y., Yoshikawa, T., Suga, S., Yazaki, T., Hirabayashi, S., Ono, Y., Tsuzuki, K. and Oshima, S. (1989) Human herpesvirus 6 harbouring in kidney. Lancet ii, 1391. Briggs, M., Fox, J. and Tedder, R. (1988) Age prevalence of antibody to human herpesvirus 6. Lancet i, 1058-1059. Carrigan, D., Drobyski, W., Russler, S., Tapper, M., Knox, K. and Ash, R. (1991) Interstitial pneumonitis associated with human herpesvirusinfection after marrow transplantation. Lancet 338, 147-149. Chou, S. and Scott, K. (1990) Rises in antibody to human herpesvirus 6 detected by enzyme immunoassay in transplant recipients with primary cytomegalovirus infection. J. Clin. Microbial. 28, 851-854. Dahl, H., Linde, A., Sundqvist, V.-A. and Wahren, B. (1990) An enzyme-linked immunosorbent assay for IgG antibodies to human herpesvirus 6. J. Virol. Methods 29, 313-324. Fox, J., Ward, P., Briggs, M., Irving, W., Stammers, T. and Tedder, R. (1990) Production of IgM antibody to HHV6 in reactivation and primary infection. Epidemiol. Infect. 104, 289-296. Irving, W., Cunningham, A., Keogh, A. and Chapman, J. (1988) Antibody to both human herpesvirus 6 and cytomegalovirus. Lancet ii, 63%63 1.
295 Morris, D., Littler, E., Jordan, D. and Arrand, J. (1988) Antibody responses to human herpesvirus 6 and other herpes viruses. Lancet ii, 142551426. Okuno, T., Higashi, K., Shiraki, K., Yamanishi, K., Takahashi, M., Kokado, J., Ishibashi, M., Takahara, S., Sonoda, T., Tanaka, K., Baba, K., Yabuuchi, H. and Kurata, T. (1990) Human herpesvirus 6 infection in renal transplantation. Transplantation 49, 519522. Saxinger, C., Polesky, H., Eby, N., Grufferman, S., Murphy, R., Tegtmeir, G., Parekh, V., Memon, S. and Hung, C. (1988) Antibody reactivity with HBLV (HHV-6) in US populations. J. Virol. Methods 21, 199-208. Sutherland, S. and Briggs, D. (1983) The detection of antibodies to cytomegalovirus in the sera of renal transplant patients by an IgM antibody capture assay. J. Med. Viral II, 147-159. Sutherland, S., Christofinis, G., O’Grady, J. and Williams, R. (1991) A serological investigation of human herpesvirus 6 infections in liver transplant recipients and the detection of cross-reacting antibodies to cytomegalovirus. J. Med. Virol. 33, 172-176. Tedder, R. and Wilson-Croome, R. (1980) Detection by radioimmunoassay of IgM class antibody to hepatitis B core antigen: a comparison of two methods. J. Med. Virol. 6, 235-247. Tedder, R., Briggs, M., Cameron, C., Honess, R., Robertson, D. and Whittle, H. (1987) A novel lymphotropic herpesvirus. Lancet ii, 39&393. Van der Groen, G., Beelaert, G., Demets, P., Kestens, L., Van Overmeir, C. and Wery, M. (1988) Line immunoass iew serologic test for malaria. Ann. Sot. Belg. Med. Trop. 68, 3741. fstathiou, S. (1989) Primary human herpesvirus 6 infection in a patient Ward, K., Gray, J. following liver transplantation from a seropositive donor. J. Med. Virol. 28, 69-72.