JOURNAL OF CLINICAL MICROBIOLOGY, May 1989, p. 843-848 Vol. 27, No.

5
0095-1137/89/050843-06$02.0O/O
Copyright C 1989, American Society for Microbiology

Monitoring Human Immunodeficiency Virus Type 1-Infected

Patients by Ratio of Antibodies to gp4l and p24
G. SCHMIDT,'* K. AMIRAIAN,' H. FREY,2 J. WETHERS,' R. W. STEVENS,' AND D. S. BERNS'
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201,' and
St. John's Riverside Hospital, Yonkers, New York 107012
Received 24 October 1988/Accepted 23 January 1989

Antibody responses of 85 patients to human immunodeficiency virus type 1 antigens were quantitated by
densitometric analysis of Western blot (immunoblot) assays. All patients had been classified into the following
three clinical categories: asymptomatic (ASY), acquired immunodeficiency syndrome (AIDS)-related coniplex
(ARC), or AIDS. Fifty of the patients were monitored for 6 to 29 months. The gp4l/p24 antibody ratio was
examined in three studies. In the first study, initial specimens from each patient were analyzed. The mean
gp4l/p24 antibody ratios were 1.5 (ASY), 3.2 (ARC), and 5.4 (AIDS). Of ASY patients, 79% had antibody
ratios of <2.0. In contrast, 72% of patients with AIDS had ratios of .2.0. In the second study, serially obtained
specimens from ASY, ARC, and AIDS patients were analyzed. These patients were further grouped according
to progression of their clinical condition. Of ASY patients whose clinical condition progressed to ARC, 80%
consistently had ratios of .2.0. Of ARC patients whose clinical condition progressed to AIDS, 71 % consistently

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had ratios of .2.0. Of AIDS patients who died during the study, 100% consistently had ratios of .2.0. No
patients were treated with azidothymidine during the first two studies. In the third study, AIDS patients were
monitored before and during treatment with azidothymidine. During treatment, ratios stabilized or improved
transiently in five of seven patients. In these three studies, a gp4l/p24 antibody ratio of <2.0 correlated with
a benign clinical state and a ratio of .2.0 correlated with AIDS or progression to AIDS.

Infection with human immunodeficiency virus (HIV) type
1 results in chronic infection which, after a variable period of
latency, progresses to acquired immunodeficiency syndrome
(AIDS). It is not clear what factors or events trigger progres-
sion of infection. Identification of markers to follow or
predict the progression of infection would be useful (i) to
physicians evaluating and monitoring infected individuals,
(ii) for identifying infected individuals at risk of imminent
progression to AIDS, and (iii) for monitoring the efficacy of
anti-HIV therapy. Various cellular immune responses (12,
13, 18, 26, 28) and humoral immune responses (15, 18, 30;
R. B. Bhalla, B. Safai, R. Mertelsmann, and M. K.
Schwartz, Letter, Clin. Chem. 29:1560, 1983; P. Francioli,
F. Clément, and C. H. U. Vaudois, Letter, N. Engl. J. Med.
307:1402-1403, 1982) appear to correlate with stage of HIV
infection. Several of these correlations have been proposed
as staging or prognostic schemes (10, 17, 23, 27; R. B.
Bhalla, B. Safai, S. Pahwa, and M. K. Schwartz, Letter,
Clin. Chem. 31:1411-1412, 1985). More recently, antibody
responses to HIV antigens have been shown to correlate
with clinical state and may aid in prognosis (4, 5, 7, 8, 19-22,
24, 25, 29, 31). In many of these studies, decline in p24
antibody correlated with progression of disease. This has
been documented quantitatively by using competitive en-
zyme-linked immunosorbent assay for p24 antibody (31) or
densitometry of Western blot (immunoblot) assays (6, 16, 24;
G. Schmidt, K. Amiraian, J. Wethers, H. Frey, R. W.
Stevens, and D. S. Berns, Abstr. Annu. Meet. Am. Soc.
Microbiol. 1988, V4, p. 418).
We previously reported the prevalence of antibodies to
each of seven HIV antigens on Western blot assays of 430
seropositive individuals in the clinical states designated
asymptomatic (ASY), AIDS-related complex (ARC), or
AIDS (24). Prevalence of antibodies to six. of seven viral
*
Corresponding author.
843
proteins decreased with progression of clinical status, while
prevalence of antibody to the transmembrane protein, gp4l,
increased. The following markers correlated with progres-
sion of clinical status: gp4l/p24 antibody ratio, gp4l/total
HIV antibody ratio, and total HIV antibody. These markers
appeared in preliminary studies to have prognostic value in
ASY individuals who subsequently developed ARC and
AIDS.
In this report we describe the use of reflectance densitom-
etry to quantitate Western blot assays for HIV type 1
antibodies in order to obtain a ratio of antibodies to gp4l and
p24. Each of 85 patients was classified by the physician into
the clinical state ASY, ARC, or AIDS. Of these patients, 50
were monitored clinically and by ratio for 6 to 29 months.
The gp4l/p24 antibody ratio was examined in three studies.
First, initial specimens from patients in each clinical state
were examined for antibody ratio. Second, patients who
were not treated with 3'-azido-3'-deoxythymidine (azidothy-
midine [AZT]) during the study were monitored for up to 29
months. These patients were grouped into ASY, ARC, and
AIDS patients whose clinical condition did or did not pro-
gress. Third, seven AIDS patients were monitored before
and during treatment with AZT. Antibody ratio (gp4l to p24)
was evaluated as a tool for identifying the stage of HIV
infection (first study), for predicting clinical outcome
(second study), and for monitoring AIDS patients treated
with AZT (third study).
MATERIALS AND METHODS
Study population. Sera from the patients in the study
population were initially submitted to the Laboratories for
Diagnostic Immunology, Wadsworth Center for Laborato-
ries and Research, New York State Department of Health,
Albany, by their private physician (H.F.) to determine the
presence of antibodies to HIV. Each patient was seen by
that physician and underwent a complete physical examina-
844 SCHMIDT ET AL.
tion and routine laboratory testing at initial evaluation and at
each follow-up visit. Clinical status was assigned by the
physician as ASY, ARC (ARC-lymphadenopathy-preac-
quired immunodeficiency), or AIDS, by using definitions
established by the Centers for Disease Control (9). All
specimens from these patients were reactive by enzyme-
linked immunosorbent assay (Electro-Nucleonics, Inc., Co-
lumbia, Md.) and confirmed reactive by Western blot assay.
Specimens from 85 patients were examined for gp4l/p24
antibody ratio. Fifty of these patients were monitored for up
to 29 months. Three studies were conducted. First, initial
specimens from 81 patients in one of the three clinical states
were examined for antibody ratio. Second, 46 of those
patients were monitored by antibody ratio at intervals of at
least 1 month for periods of 6 to 29 months. These patients
were not treated with AZT during that period. Third, seven
patients with AIDS were monitored before and during treat-
ment with AZT. Four of the seven patients in the AZT study
were not included in the first or second studies. Thus, the 85
patients examined include 81 from the first study plus 4 from
the third study; the 50 patients monitored include 46 from the
second study plus 4 from the third study.
Western blot assays. Western blot assays were conducted
as described previously (24) on Immobilon-P (Millipore
Corp., Bedford, Mass.) strips prepared with antigen from
Organon Teknika (Malvern, Pa.). HIV proteins were sepa-
rated by preparative electrophoresis of detergent-treated,
heat-inactivated (100°C for 2 min) virus on sodium dodecyl
sulfate-polyacrylamide (10%) gels in a discontinuous buffer
system and electrophoretically transferred to Immobilon
sheets. The sheets were cut into strips, and enzyme-linked
immunosorbent assays were conducted. Sera were diluted
100-fold in buffer containing 5% nonfat dry milk and were
incubated with strips. Reactivity of antibodies with antigen
was detected by adding peroxidase-conjugated goat immu-
noglobulin G against human immunoglobulin G (heavy-chain
specific) (Organon Teknika), further washing, and adding
peroxidase substrate, 3,3'-diaminobenzidine tetrahydrochlo-
ride dihydrate (Aldrich Chemical Co., Milwaukee, Wis.).
Densitometry. Western blot strips were examined as pre-
viously described (24) by reflectance densitometry with a
video densitometer (model 620; Bio-Rad Laboratories, Rich-
mond, Calif.) serially linked to an integrator (model 3392A;
Hewlett-Packard Co., Palo Alto, Calif.). Settings used for
both instruments were as recommended by Bio-Rad. The
full-scale ordinate value was set at 1.0 absorbance unit. HIV
antigens were identified by comparison with HIV-reactive
human reference antiserum and by extrapolation of plots of
relative mobilities of prestained proteins of known molecular
weights (Bethesda Research Laboratories, Inc., Gaithers-
burg, Md.). The tracing generated for each strip was cor-
rected for background by subtracting a tracing of the HIV-
nonreactive human control serum for that blot. Each
antibody response on Western blot assay strip was quanti-
tated by integrating the area under the corresponding peak of
the tracing of that strip. The integrator delineated each peak
by tick marks and identified peaks (i.e., antibody bound to
individual HIV antigens) by their distance from the begin-
ning of the tracing. Area was measured in increments of
0.125 ,uV. s. We have defined 1 signal unit as 1.0 x 106
increments.
Reproducibility of measurements. Reproducibility of band
intensity depends on standardized reagents and protocols.
Use of the ratio of antibody concentrations minimizes the
effect of variation in band intensity. The ratio of antibody
responses to gp4l and p24 was determined for the HIV-
J. CLIN. MICROBIOL.
reactive human control serum on each blot. Reactive control
sera on more than 200 blots were analyzed in this and a
previous study (24). Mean antibody ratios were calculated.
In 90% (191 of 214) ofthese assays, the antibody ratios ofthe
reactive control sera fell within one standard deviation of the
means established from 150 assays. Only data obtained from
blots with reference reactive-serum ratios within one stan-
dard deviation of the mean were included in this study.
Tabulation and analysis of data. A positive antibody re-
sponse was defined as-1.0 signal unit. The ratio of gp4l
antibody to p24 antibody was calculated. Any ratio of .10.0
was valued at 10.0. The beginning of the study for each
patient was defined as the date of collection of the initial
specimen from that patient. Data were organized by clinical
status (ASY, ARC, or AIDS), date of collection of sera, and
clinical outcome. Data were examined for correlation with
clinical state or clinical outcome of patients who were or
were not treated with AZT`.
The gp41/p24 antibody ratio which correlated with disease
or more rapid progression to AIDS was established as -2.0
for this in-house Western blot assay. This was a value (i)
below which at least 75% of the ASY population fell, (ii)
above the mean and median values of the ASY population,
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and (iii) below the mean and median values of the AIDS
population (see Fig. 2). These criteria have been successfully
applied to establish a predictive cutoff for a commercially
available immunoblot assay (unpublished results) which was
effective in staging populations of ASY, ARC, or AIDS
patients qualitatively by gp41/p24 antibody ratio (R. W.
Stevens, J. A. Wethers, D. S. Berns, and A. Polito, 4th Int.
Conf. AIDS, Stockholm, Sweden, 1988, abstr. no. 1160, p.
152).
Treatment with AZT. Seven patients with AIDS were
monitored for at least 6 months before and during treatment
six times daily with 200 mg of AZT (Burroughs Wellcome,
Research Triangle Park, N.C.). Despite transfusion, no
patient was able to tolerate full prescribed treatment, and all
experienced periods of no treatment or treatment at reduced
dosage.
RESULTS
Densitometric tracings of Western blots. Examples of
Western blot assays and corresponding densitometric trac-
ings are illustrated in Fig. 1. The first example (Fig. 1A) is of
serum from an ASY individual, and the second example
(Fig. 1B) is of serum from an AIDS patient. A Western blot
from a seronegative individual is illustrated for comparison
(Fig. 1C). The antibody responses evident are to the glycos-
ylated envelope (env) gene products (envelope gpl20 and
transmembrane gp4l), polymerase (pol) gene products (re-
verse transcriptase p66/p51 and endonuclease p30), 3' open
reading frame gene product (p28), and core (gag) gene
products (precursor p53 and core p24 and p18). Further
details of Fig. 1 are provided in the Discussion.
Antibody ratios of initial specimens from ASY, ARC, or
AIDS patients. The ratio of gp4l antibody to p24 antibody in
clinical populations is illustrated in Fig. 2. Ratios are of
initial specimens from 81 patients classified as ASY, ARC,
or AIDS. Although there was a wide range of values within
each clinical category, a pattern is evident. The mean ratios
were 1.5 (ASY), 3.2 (ARC), and 5.4 (AIDS). Corresponding
median ratios were 1.2, 1.8, and 4.2. The percentage of each
population with a ratio of -2.0 was 21.4 (ASY), 41.7 (ARC),
and 72.4 (AIDS).
Patients monitored by antibody ratio. Fifty patients were
monitored for 6 to 29 months by analysis of serially obtained
VOL. 27, 1989 MONITORING HIV TYPE 1 INFECTION BY ANTIBODY RATIO 845

A B c
.85
.22 (gp 120) 25
.36
.54

.83 p66
p53
p51
1.42 gp4I 1-.48 _u

p30 p28
.83 p28- 1.84
4
_ p24
2.22 '2.23

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.86

FIG. 1. HIV Western blot assays and corresponding densitometric tracings of sera from individuals classified as ASY (A), AIDS (B), or
seronegative (C). Bands and corresponding peaks represent antibody to viral antigens (molecular mass indicated in kilodaltons). Numbers
beside tracings indicate distance from the origin and aid in identifying peaks. Peak boundaries are indicated by tick marks.

specimens. Four patients were treated with AZT after less whether disease did or did not progress. Figure 3 illustrates
than 6 months, and their cases are discussed below. The the gp4l/p24 antibody ratio in serially obtained specimens of
remaining 46 were classified at initial presentation as ASY, ASY patients whose clinical condition did or did not prog-
ARC, or AIDS and were further grouped according to ress to ARC or AIDS. Of those whose condition did prog-
ress, 80% (4 of 5) had ratios of .2.0. Of those whose clinical
condition did not progress, 72% (10 of 14) had ratios remain-
ing at <2.0. Those whose condition progressed were three
MEAN O
times as likely to have ratios of -2.0 as those whose
condition did not progress. The use of antibody ratio to
- MEDIAN a O O O
>10- a I 0 00

A: NO PROGRESSION (n=14)

10 >10
It
C\%j
8- * O 10
"- o
cmJ
C5) o
6- e O
'
6.
4--4it o
I I 4.
2- .__.......
p.
2.
O &-
fARC
a@ o i
ASY AIDS O _
o 8 16 0 24
(n=28) (n=24) (n=29) MONTHIS
CLINICAL STATUS FIG. 3. Ratio of gp4l antibody to p24 antibody in ASY patients
FIG. 2. Ratio of gp4l antibody to p24 antibody in initial speci- whose clinical condition did not (A) or did (B) progress to ARC or
mens from patients classified as ASY, ARC, or AIDS. AIDS.
846 SCHMIDT ET AL. J. CLIN. MICROBIOL.

Antibody ratios in AIDS patients treated with AZT. The

ratio of gp41/p24 antibody was determined in serially ob-
tained specimens of patients with severe symptoms of AIDS
before and during at least 6 months of treatment with AZT.
Of these patients, 86% (6 of 7) had early antibody ratios of
.2.0. In three patients the ratio was .10.0 before or at start
of therapy. Despite their advanced clinical state, during
therapy ratios stabilized or improved transiently in the five
patients with lower ratios. Ultimately, the ratios were not
improved by AZT, and mortality during the study was not
4
improved over that of untreated AIDS patients (57% [4 of 7]
2- ---- -----
versus 62% [8 of 13]).

DISCUSSION
0 8 16 24 0 8 16 24
Western blot assay is the standard confirmatory test for
MONTHS detecting antibodies to HIV type 1. The patterns of antibody
FIG. 4. Ratio of gp4l antibody to p24 antibody in patients with responses evident on these assays are varied and have been
ARC whose clinical condition did not (A) or did (B) progress to observed to correlate with clinical status qualitatively (3, 4,
AIDS. 14, 19) or semiquantitatively (7). Western blot assays can
productively be further analyzed by using densitometry to
quantitate antibody responses (6, 16, 24).
predict clinical outcome would have been successful in 70 to We chose to examine the ratio of gp4l antibody to p24

Downloaded from jcm.asm.org by on June 17, 2009

80% of these ASY patients.
Figure 4 illustrates the gp4l/p24 antibody ratio in serially
obtained specimens of patients with ARC whose clinical
condition did or did not progress to AIDS. Of those whose
condition did progress, 72% (5 of 7) had ratios of .2.0. Of
those whose condition did not progress, 67% (4 of 6) had
ratios remaining at <2.0. Those whose condition progressed
were twice as likely to have ratios of .2.0 as those whose
condition did not progress. The use of antibody ratio to
predict clinical outcome would have been successful in 70%
of these patients with ARC.
Figure 5 illustrates the gp4l/p24 antibody ratios in serially
obtained specimens of patients with AIDS who were or were
not still living at the time of completion of the study. Of
those who were no longer living, 100% (11 of 11) had ratios
of .2.0. Of those who were still living, 60% (3 of 5) had
ratios remaining at <2.0. Those who were no longer living
were three times as likely to have ratios of -2.0 as those who
were still living. The use of antibody ratio to predict clinical
outcome would have been successful in 100% and 60%,
respectively, of AIDS patients who were not and were living
at the completion of the study.
A: LIVING (n=5) B: NO LONGER LIVING
>10
C%1
0~
0 8 16 24 0 8 16
MONTHS
FIG. 5. Ratio of gp4l antibody to p24 antibody in patients with
AIDS who were still living (A) or no longer living (B) at the time of
completion of the study.
antibody because in a data base derived from Western blot
assays of 430 seropositive individuals (24), (i) antibodies to
gp4l and p24 were more prevalent than any other antibodies,
(ii) antibody to gp4l was the only antibody response found to
be more closely associated with AIDS than with ARC or
ASY, (iii) antibody to p24 was less prevalent with more
severe disease, and (iv) the ratio of gp4l antibody to p24
antibody increased with more severe disease. The first three
observations were later confirmed by DeVico et al. (11).
Antibodies to envelope and core proteins have also been
quantitated by densitometry of autoradiograms of gel elec-
trophoresis of immunoprecipitates (20) and by commercially
available second-generation enzyme-linked immunosorbent
assays prepared from recombinant proteins (2, 21, 31). We
have used Western blots to obtain ratios for the following
reasons. First, radioimmunoprecipitation is not in common
use, and antibody to gp4l is not well represented in the assay
(20). Second, genetically engineered p24 antigen in the
assays we have examined and in studies by others (2, 21) is
not as reactive as the p24 in denatured native virus used for
Western blot; consequently, use of these assays to predict
clinical outcome results in incorrect identification of some
individuals as being at increased risk of disease (unpublished
results).
(n=11) We have used reflectance densitometry of Western blot
assays of specimens from 85 seropositive individuals with
clinical status ASY, ARC, or AIDS to calculate an antibody
ratio of gp4l to p24. Fifty of these patients were monitored
by antibody ratio for up to 29 months. The results demon-
strate that this ratio correlates with clinical status (Fig. 2),
correlates with clinical outcome (Fig. 3 to 5), and can be
used to monitor patients undergoing anti-HIV therapy.
The first study examined gp4l/p24 antibody ratios of
patients with clinical status ASY, ARC, or AIDS. The mean
and median values of antibody ratio increased with progres-
sion of clinical status. A total of 79% of ASY patients had
antibody ratios of <2.0, while 72% of AIDS patients had
ratios of .2.0.
24 Figure 1 illustrates Western blots and corresponding den-
sitometric tracings of sera from an ASY patient and a patient
with AIDS. The ASY patient had a gp4l/p24 antibody ratio
of 1.0 (Fig. 1A). The patient remained ASY, with qualita-
tively and quantitatively similar antibody responses through-
VOL. 27, 1989 MONITORING HIV TYPE 1 INFECTION BY ANTIBODY RATIO
out the 8 months the patient was monitored. The AIDS
patient had a gp41/p24 antibody ratio of 7.2 (Fig. 1B). She
had AIDS (cryptococcal meningitis), with antibody re-
sponses remaining qualitatively and quantitatively similar
throughout the 14 months before she died. Antibody to p24
was present on a Western blot assay of serum drawn when
the patient was last monitored, 2 weeks before death. She
died with Staphylococcus aureus, arthritis, sepsis, and cen-
tral nervous system toxoplasmosis.
The second study examined antibody ratios of serially
obtained specimens of ASY, ARC, and AIDS patients who
were monitored for 6 to 29 months. Patients were grouped
according to whether their clinical conditions did or did not
progress. During this period, 26% (5 of 19) of ASY patients
progressed to ARC or AIDS, 54% (7 of 13) of ARC patients
progressed to AIDS, and 69% (11 of 16) of AIDS patients
died (Fig. 3-5). Of ASY patients whose clinical condition
progressed to ARC, 80% had antibody ratios of .2.0. Of
patients with ARC whose clinical condition progressed to
AIDS, 71% had ratios of .2.0. Of patients with AIDS who
died during the study, 100% had ratios of .2.0. Thus, a ratio
of -2.0 in ASY patients predicted progression to ARC or
AIDS, in patients with ARC predicted progression to AIDS,
and in patients with AIDS predicted that patients had less
time to live.
The correlation ofthe gp4l/p24 antibody ratio of -2.0 with
disease or risk of more rapid progression to AIDS was
approximately 75% overall and thus was not always a
definitive criterion. However, this ratio compares favorably
with other correlates (1, 3, 8, 18, 21, 26, 28, 31) and is a very
useful tool. The predictive (cutoff) value of 2.0 established
for the in-house Western blot might vary somewhat with the
test system, but cutoff can be readily established for other
test systems. We have established such a cutoff in a com-
mercially available immunoblot assay (see Materials and
Methods). The gp41/p24 antibody ratio may prove even
more valuable in combination with other laboratory data
(e.g., T4/T8 and p24 antigen). Refinement of the technique of
quantification of individual antibody responses (e.g., by
antibody isotype or subclass to a specific epitope of an
individual viral protein) may yet yield a fully definitive
criterion for disease.
Results of our first and second studies are compatible with
those of others attempting to correlate stage of HIV infection
with either decline ofp24 antibody (1, 3-8, 20, 24, 25, 29, 31)
or increase in gp4l/p24 antibody ratio (6, 7, 24). Quantitation
of antibody responses to HIV antigens has been accom-
plished by enzyme-linked immunosorbent assay (21, 31) and,
more recently, by densitometry of Western blot assays (6,
16, 24) or of radioimmunoprecipitation (20).
In the third study, patients with AIDS were monitored
before and during treatment with AZT. These patients were
not able to tolerate AZT treatment well; despite this, during
therapy ratios stabilized or improved transiently in the five
patients who had the lowest antibody ratios. Ultimately,
however, neither ratios nor percent mortality was improved
by treatment. Because patients treated earlier in infection
are better able to tolerate the drug and consequently there
may be a better chance of forestalling disease progression,
we are currently undertaking a study to monitor patients
treated with AZT earlier in infection.
Clinical assessment and staging of HIV-infected patients
can be difficult. Our patient population reveals some of these
difficulties. The data were plotted according to the history
submitted with the test specimen, using a progressive clas-
sification scheme (i.e., a patient once classified as ARC or
847
AIDS would not later be classified as ASY, even if symp-
toms disappeared). Retrospective classification sometimes
differs from contemporary classification. Had we applied
retrospective classification to our data, it would have
strengthened somewhat the correlation of antibody ratio
with clinical status. However, and more importantly, a
discrepancy between clinical evaluation and ratio suggests to
the clinician the need for reevaluation of patients who do not
have ARC or AIDS and yet have antibody ratios of .2.0.
Among the 50 patients monitored were several whose anti-
body ratio appeared inconsistent with the initial clinical
classification. Clinical reevaluation led to reclassification of
six patients. Two patients classified as ASY would have
been classified as ARC if neurological symptoms or dissem-
inated tuberculosis had been initially diagnosed. Similarly, a
patient classified as ASY had nodules in the lungs; the
etiology of the nodules was found subsequently to be Myco-
bacterium avium intracellulare, requiring reclassification as
AIDS. Three patients classified as ARC would have been
reclassified as ASY: one had neurological symptoms initially
ascribed to HIV but which were subsequently attributed to
varicella-zoster virus, and two had constitutional symptoms
which resolved when intravenous drug abuse was discontin-
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ued.
In these studies, a gp4l/p24 antibody ratio of <2.0 corre-
lated with a benign clinical state. A ratio of 22.0 correlated
with AIDS or progression to AIDS. A ratio of 22.0 in a
patient with clinical status of ASY or ARC could serve to
alert the clinician that the patient is at risk of more rapid
progression to AIDS. Such a patient could be reevaluated for
clinical symptoms, monitored more closely in anticipation of
progression of disease, or identified as a candidate for early
therapeutic intervention.
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