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Clinical and Diagnostic Laboratory Immunology, January 2001, p. 203-205, Vol. 8, No. 1
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.1.203-205.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Correlation between Enzyme-Linked Immunosorbent
Assay and Immunofluorescence Assay with Lytic Antigens for Detection of
Antibodies to Human Herpesvirus 8
Simone
Topino,1
Laura
Vincenzi,2
Ivano
Mezzaroma,1
Emanuele
Nicastri,1
Massimo
Andreoni,3 and
Maria C.
Sirianni1,*
Department of Clinical Immunology, University
of Rome La Sapienza,1 IRCCS L. Spallanzani2 and Department of Infectious
Diseases, University of Tor Vergata,3 Rome,
Italy
Received 27 July 2000/Returned for modification 27 September
2000/Accepted 25 October 2000
 |
ABSTRACT |
The purpose of this study was to evaluate, in Kaposi's sarcoma
patients, the correlation between antibody titers to the lytic antigens
of human herpesvirus 8, as assessed by immunofluorescence assay, and
values obtained by an enzyme immunoassay. The methods showed a
stringent correlation, r = 0.625 (P < 0.001).
| |
TEXT |
DNA sequences from human herpesvirus
8 (HHV8) have been found in the lesions as well as in the peripheral
blood mononuclear cells (PBMC) of patients with all forms of
Kaposi's sarcoma (KS), including the AIDS-associated KS
(AIDS-KS) 6, classical or Mediterranean KS (C-KS),
African or endemic KS, and KS developing in organ transplant recipients
(T-KS) 3. The virus has subsequently been associated with
body cavity-based lymphomas (BCBL) 4, 5 and with some
variants of multicentric Castleman's disease 8. Serologic
evidence suggests that the virus is a necessary cofactor for KS, since
HHV8 seroconversion or an increase in the lytic antibody titer to HHV8
appears to be critical and highly predictive of KS development in human
immunodeficiency virus (HIV)-coinfected patients 10.
In addition, assessment of HHV8 serostatus is important in
monitoring organ transplant donors and recipients. Particularly, kidney
recipients infected by HHV8 prior to transplantation and receiving an
organ from a seropositive donor show an exceedingly high risk of KS
development, probably due to viral reactivation 15.
Several efforts have been made to develop serologic assays for the
detection of antibodies to HHV8, to be employed on a routine and
screening scale. Until now, no tests have been recommended for
diagnostic use, even if those already available and based on self-made
immunofluorescence assays (IFA) or on Western blotting confirmed a
stringent association of HHV8 seroprevalence with all forms of KS
1, 2, 9, 10, 12, 13, 14, 17, 19, 20. The majority of the
studies performed until now are, however, based on IFA, which is
time-consuming and not easy to use in large-scale studies to assess
disease reactivation, especially in countries where KS still has a high
incidence. There is only one commercially available system, based on an
enzyme-linked immunosorbent assay (ELISA), which detects antibodies to
the lytic antigens of HHV8 using whole virus as the substrate
7. The aim of our work was to study the antibody pattern
to the lytic antigens of HHV8 in KS patients using two different
methods, ELISA and IFA. Particularly, IFA antibody titers to lytic
antigens were compared with the optical densities (OD) obtained by
ELISA in order to establish a correlation between the two methods.
A total of 70 subjects were enrolled in the study. Seventeen AIDS-KS
patients were studied and staged according to the Krown classification
11. Eight of them were sampled at the time of first
clinical diagnosis and during protease inhibitor (PI)-containing highly
active antiretroviral therapy (HAART). In four AIDS-KS cases, diagnosis
was biopsy confirmed. Sera from the remaining patients were available
only during (two cases) or without (seven cases) PI treatment.
Thirty-one C-KS patients with a biopsy-confirmed diagnosis as well as
four T-KS patients were studied. The T-KS patients developed the
disease after a mean time of 8 months following renal transplantation
and subsequent immunosuppressive therapy, consisting of cyclosporin and
steroids. As a control group, 15 apparently healthy blood donors (BD)
born in Rome were studied. Three HIV-seropositive patients, including
the partner of an AIDS-KS patient, were also examined.
HHV8 ELISA.
Anti-HHV8 immunoglobulin G (IgG) antibodies
were detected by a commercially available assay (Advanced
Biotechnologies Incorporated, Columbia, Md.), according to the
manufacturer's instructions. Briefly, serum samples diluted
1:100 were incubated in the antigen-coated microtiter wells for 30 min
at 37°C. Antigen was represented by whole virus. The wells were then
washed to remove unbound sample components. Peroxidase-conjugated
anti-human IgG was then added to the wells and incubated for 30 min at
37°C. The wells were washed again to remove unreacted conjugate. The
microtiter wells containing immobilized peroxidase conjugate were
incubated with peroxidase substrate for a mean time of 15 min at room
temperature without light. Then the reaction was stopped, and the OD of
the solution was measured spectrophotometrically at 450 nm. The cutoff point was given at 0.023 OD unit.
IFA.
Antibodies to lytic antigens of HHV8 were detected using
an IFA based on the BCBL-1 cell line (obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID, NIH,
from M. McGrath and D. Ganem). The BCBL-1 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS),
antibiotics (100 IU of penicillin and 100 µg of streptomycin per ml),
and 5 × 105 M 2-mercaptoethanol. For IFA to antilytic
antibodies, smears were prepared by sedimenting BCBL-1 cells after
treatment with 20 ng of tetradecanoyl phorbol ester acetate (Sigma) per
ml for 48 h. Ten microliters of a 4 × 106-cell/ml cell suspension was smeared on slides, air
dried at room temperature, and fixed with a methanol-acetone solution
(1:1; vol/vol) at 
20°C for 10 min. For IFA, fixed smears were
preblocked by incubation with phosphate-buffered saline (PBS)
supplemented with 3% FCS for 30 min in a humidified chamber and
then incubated in two steps of 45 min each at 37°C with the
test serum diluted 1:10 (in PBS supplemented with 1% glycerine and 2%
FCS) and with goat fluorescein isothiocyanate-conjugated anti-human Ig
antibodies. Titrations were done with twofold serial dilutions. Samples
reactive at >1:10 dilution in the antilytic test with at least two
fluorescent cells for each microscopic field were considered positive.
All microscopic examinations were done under code in a blinded fashion.
Linear correlation was used to study the correlation between IFA
antibody titrations and ELISA OD in all groups of patients. Student's
t test in paired analysis was used to test HHV8 titer variations in AIDS-KS patients at baseline and during HAART. Group means were compared using analysis of variance. If the corresponding F test was statistically significant, individual means were
compared using the Bonferroni additive inequality.
The demographic features of the study groups are reported in Table
1. Eight AIDS-KS patients were examined
for HHV8 serology
at baseline and during HAART, whereas for two
patients blood samples
were only available during HAART
(4 months of therapy). One patient
was treated with
nucleoside reverse transcriptase inhibitors (NRTI)
plus chemotherapy,
which was based on bleomycin and vincristine,
and never underwent
HAART.
Figure
1 reports the values of the
antibody titers obtained by IFA compared to the OD obtained by ELISA.
No BD was seropositive
for HHV8 infection, whereas all except two KS
patients had detectable
antibody titers. Those two patients were
affected by AIDS-KS and
were sampled during the PI-sparing regimen.
They showed low antibody
production, as detected by IFA (titer 1:20),
but ELISA failed
to reveal this, detecting OD values below the cutoff
point (0.015
and 0.009, respectively). All the remaining patients had
antibodies
with titers ranging from 1:20 to 1:1,240. A stringent
correlation
between the two methods was found (
r = 0.625,
P < 0.001) (Fig.
1). The significant
correlation was maintained at all different
stratifications of antibody
titration; particularly, the strongest
correlation was observed in
patients with absent or low titers
(
1:20) of antibody production. A
significant correlation between
IFA and ELISA results was also
maintained between AIDS-KS and
C-KS patients (
r = 0.548,
P = 0.004, and
r = 0.597,
P < 0.001,
respectively). None of the HIV-seropositive
patients without KS
or the AIDS-KS partner had detectable antibodies by
both methods
(data not shown).
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FIG. 1.
Correlation between values obtained by ELISA and IFA
applied to the detection of antibodies to HHV8 in the populations
studied. ELISA values are expressed as OD, whereas IFA values are given
as log10 antibody titers.
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|
Table
2 reports the distribution of the
IFA antibody titers to lytic antigens as well as the ELISA OD values in
HHV8-seropositive
KS patients as well as in BD. In AIDS-KS patients, a
strong increase
in antibody titers was found during HAART (4-month
follow-up),
in comparison to pretherapy values. These data were
confirmed
by both methods. Particularly, the mean IFA titer increased
significantly
from the baseline value of 1:95 to 1:360 (
P = 0.01), whereas the
OD reached a value of 0.719 from a baseline
level of 0.393 (
P = 0.28). The T-KS subjects showed the
highest titers to HHV8 with
both methods (mean IFA titers of 1:480 and
mean OD of 1.05). The
analysis of variance did not show any significant
difference among
the groups of patients. Probably the small number of
T-KS patients
enrolled in the study (four subjects) did not allow
statistical
significance for these apparently intriguing data. BD had
undetectable
antibodies to HHV8, as shown by both methods (IFA less
than 1:10,
OD < 0.023).
Several efforts have been made to develop assays aimed at the
serological survey of HHV8 infection
1,
2,
10,
12,
14,
17,
20. Interassay studies have shown a relatively poor
concordance
and low sensitivity of the available serologic assays,
especially in
the normal population
16. Conversely, among KS
patients a
relatively good concordance was found
9,
13,
16,
even if
in these patients the role played by the antibody pattern
to HHV8 lytic
and latent antigen still remains to be fully clarified.
However, data
from our patients showed a high correlation between
the two methods
employed to assess the antibody status to lytic
antigens. No BD had
anti-HHV8 antibodies with both methods and,
conversely, all but three
KS patients were seropositive (one C-KS
and two AIDS-KS). However, the
last (AIDS-KS), with OD levels
lower than the cutoff value, showed a
weakly positive IFA titer
of 1:20. The remaining HHV8-seronegative C-KS
patient was under
chemotherapy at the sampling
time.
Herpesvirus replication may be inhibited under chemotherapy. As our
previous report has in fact shown, by the use of PCR,
that C-KS
patients undergoing chemotherapy can clear the virus
from peripheral
blood mononuclear cells
18. The ELISA alternative
to the
IFA assay, although less sensitive, could be particularly
useful for
monitoring viral reactivation, considering that IFA
is largely operator
and training dependent. The data obtained
must be always validated by
other assays and compared with the
clinical and therapeutic findings
for the subjects being
tested.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from Italian Istituto Superiore
di Sanità and MURST to M.C.S.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Immunology, University of Rome "La Sapienza," Viale dell'
Università 37, 00185 Rome, Italy. Phone: 39 06 49972037. Fax: 39 06 4466209. E-mail: sirianni{at}uniromal.it.
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Clinical and Diagnostic Laboratory Immunology, January 2001, p. 203-205, Vol. 8, No. 1
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.1.203-205.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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