Aids assay

An assay which is responsive to the levels of anti-p24 antibodies in serum samples is provided which comprises the steps of: (a) forming a test mixture comprising the sample and an antigen solution containing free p24 antigen within a predetermined concentration range: (b) incubating the test mixture under conditions whereby anti-p24 antibodies from the sample, if any, can react with the free p24 antigen to form antibody-antigen complexes; (c) determining the concentration of free p24 antigen remaining in the test mixture after the incubation; (d) determining the concentration of free p24 antigen in the antigen solution; and (e) calculating the difference between the concentration of free p24 antigen in the antigen solution and the concentration of freee p24 antigen in the test mixture after the incubation. The difference calculated in step (e) represents the "binding capacity" of the serum sample for p24 antigen. This parameter is shown to serve as an accurate and convenient measure of a patient's level of anti-p24 antibodies. In certain preferred embodiments, the anti-p24 antibody assay is integrated with a p24 antigen assay so that declining anti-p24 antibody levels in combination with rising p24 antigen levels can be readily monitored. This combination has been found to generally indicate a poor prognosis for HIV-infected patients.

BACKGROUND OF THE INVENTION 
This invention relates to the acquired immune deficiency syndrome (AIDS) 
and, in particular, to assays which function as prognostic indicators of 
the development of AIDS by people infected with the human immunodeficiency 
virus (HIV). 
The human immunodeficiency virus has been established as the etiologic 
agent of AIDS, chronically infecting and eventually killing cells critical 
to normal immunologic function. In addition to producing end-stage AIDS, 
HIV directly or indirectly contributes to immunologic dysfunction 
characterized clinically by the development of persistent generalized 
lymphadenopathy and AIDS-related complex (ARC). HIV-infected patients are 
usually staged by clinical findings and the numbers of CD4 positive 
lymphocytes (helper cells). 
Patients infected with HIV can remain essentially symptom free for years, 
e.g., three years or longer, and not all HIV-infected people actually 
progress to AIDS. See, for example, Weber et al., "Human immunodeficiency 
virus infection in two cohorts of homosexual men: neutralizing sera and 
association of anti-gag antibody with prognosis," Lancet, Jan. 17, 1987, 
pages 119-121. As a result, extensive efforts have been made to develop 
laboratory tests which are prognostic indicators of the development of 
AIDS, ARC and other AIDS-related conditions by persons infected with HIV. 
See, for example, Spira, et al., "Human Immunodeficiency Virus Viremia as 
a Prognostic Indicator in Homosexual Men with Lymphadenopathy Syndrome," 
The New England Journal of Medicine, Vol. 317, No. 17, Oct. 22, 1987, 
pages 1093-1094. The need for predictive tests has become increasingly 
important as anti-HIV therapies directed at suppressing HIV replication 
have been developed. See, for example, Forster et al., "Decline of 
Anti-p24 Antibody Precedes Antigenaemia as Correlate of Prognosis in HIV-1 
Infection," AIDS, 1987, Vol. 1, No. 4, pages 235-240. 
One indicator which has been considered is the level of p24 antigen in a 
patient's serum, i.e., p24 antigenemia. See, for example, Wolf, et al., 
"Risk of AIDS Related Complex and AIDS in Homosexual Men with Persistent 
HIV Antigenaemia," British Medical Journal, Vol. 295, 1987, pages 569-572. 
In particular, an increase in the level of p24 antigen has been associated 
with progression to AIDS. Thus, Forster et al., supra, report that in 40% 
of the AIDS patients in their study, detectable levels of p24 antigen were 
seen 2 years prior to the actual development of AIDS. 
The level of anti-p24 antibodies in a patient's serum has also been 
considered for use as a prognostic indicator. In this case, a decrease in 
the level of anti-p24 antibodies has been associated with progression to 
AIDS. See Biggar, et. al., "Variation in Human T Lymphotropic Virus III 
(HTLV-III) Antibodies in Homosexual Men: Decline Before Onset of Illness 
Related to Acquired Immune Deficiency Syndrome (AIDS)," British Medical 
Journal, Volume 291, Oct. 12, 1985, pages 997-998; Lange, et al., 
"Distinct IgG Recognition Patterns During Progression of Subclinical and 
Clinical Infection with Lymphadenopathy Associated Virus/Human T 
Lymphotropic Virus," British Medical Journal, Vol. 292, Jan. 25, 1986, 
pages 228-230; Pan et. al., "Patterns of Antibody Response in Individuals 
Infected with the Human Immunodeficiency Virus," Journal of Infectious 
Diseases, Vol. 155, No. 4, Apr. 1987, pages 626-632: Weber, et al., supra. 
As reported by Forster et al., supra, this indicator can provide 
predictive information up to 18 months earlier than the antigenemia 
indicator. (Note that Groopman, et al., "Serological Characterization of 
HTLV-III Infection in AIDS and Related Disorders," Journal of Infectious 
Diseases, Vol. 153, No. 4, Apr. 1986, pages 736-742, have found anti-p24 
antibodies in both symptomatic and asymptomatic seropositive individuals.) 
A number of recent studies have concluded that the combination of an 
increasing p24 antigen level and a declining anti-p24 antibody level is 
predictive of HIV-related clinical complications, including AIDS. See 
Allain, et al., "Long-Term Evaluation of HIV Antigen and Antibodies to p24 
and gp41 in Patients with Hemophilia," The New England Journal of 
Medicine, Vol. 317, No. 18, Oct. 29, 1987, pages 1114-1121: Forster, et 
al., supra: Goudsmit, et al., "Antigenemia and Antibody Titers to Core and 
Envelope Antigens in AIDS, AIDS-Related Complex, and Subclinical Human 
Immunodeficiency Virus Infection," The Journal of Infectious Diseases, 
Vol. 155, No. 3, Mar. 1987, pages 558-560; Pedersen, et al., "Temporal 
Relation of Antigenaemia and Loss of Antibodies to Core Antigens to 
Development of Clinical Disease in HIV Infection," Britisn Medical 
Journal, Vol. 295, 1987, pages 567-569. 
Various highly-sensitive assays for p24 antigen levels have been developed 
and are available from such companies as Abbott Laboratories (North 
Chicago, Ill.) and E. I. Du Pont de Nemours & Co. (Biomedical Products 
Department, Boston, Mass.). See, for example, Allain et al., supra: 
Forster, et al., supra; Goudsmit, et al., supra; Wolf, et al., supra. 
These assays employ a sandwich assay format in which serum samples are 
incubated with bound and enzyme-labelled anti-p24 antibodies to form an 
antibody/p24-antigen/antibody sandwich. Concentrations of p24 antigen are 
determined from standard curves obtained by performing the assay on a 
series of standards containing known amounts of p24 antigen. With these 
assays, p24 antigen levels on the order of 50 picograms/milliliter can be 
detected. See also Pedersen, et al., supra. 
Assays for anti-p24 antibodies have also been developed. These assays 
suffer from a number of problems. The early assays, which continue to be 
used, employed Western blot techniques in which HIV virus proteins were 
separated by SDS-PAGE, transferred to a nitrocellulose sheet, incubated 
with the test serum, and developed using an antihuman IgG from an 
appropriate animal and a suitable enzyme-labeling system. See Biggar, et 
al., supra: Lange, et al., supra: Groopman, et al., supra: Pan, et al., 
supra. These assays were obviously difficult to perform, time consuming, 
and not well-suited for use in a clinical laboratory setting. See Pan, et 
al., supra. Also, the assays did not give reliable quantitative measures 
of anti-p24 antibodies in sera being tested. Radioimmunoprecipitation 
assays (RIPA) using radiolabeled antigens, immobilized test sera, and 
SDS-PAGE separation, have also been employed. See, for example, Weber, et 
al., supra. These assays suffer from the same problems as the Western blot 
assays and are not suitable for wide spread clinical use. 
More recently, enzyme immunoassays for anti-p24 antibodies have been 
developed. See, for example, Forster, et al., supra. In particular, Abbott 
Laboratories has developed an assay in which beads coated with recombinant 
HIV core antigen, including p24 antigen, are incubated with serial 
dilutions of the serum being analyzed and with enzyme-labelled antibody to 
HIV core antigen. After a 16-22 hour incubation, the beads are washed and 
developed using a suitable substrate for the enzyme label. Since this is a 
competitive assay, the amount of antibody in the sample is inversely 
related to the intensity of the color generated during the development 
step. Positive and negative controls are run to establish a cutoff level 
for the presence of anti-p24 antibody, i.e., the midpoint between the 
controls. Titers are defined as the highest dilution which produces a 
color level below the cutoff. See Goudsmit, et al., supra; Pedersen, et 
al., supra. A similar competitive assay has been described by Weber, et 
al., supra. Significantly, Weber, et al., found that the classical 
radioimmunoprecipitation procedure was more sensitive than the competitive 
assay procedure. In addition to these competitive assays, direct ELISA 
assays for anti-p24 antibodies employing immobilized antigen and 
enzyme-labelled anti-human globulin antibodies have also been considered. 
The primary disadvantage of these assay systems is that they employ serial 
dilutions of the patient's serum. Accordingly, in essence, numerous 
individual assays and measurements must be made to arrive at an antibody 
titer. This makes the assays difficult to use, labor intensive, and 
expensive. Indeed, Pedersen, et al., supra, after specifically pointing 
out that prognostic tests must be simple to do and interpret, recommended 
using a p24 antigen test for selecting patients for antiviral treatment, 
but significantly did not recommend using the Abbott competitive enzyme 
immunoassay for anti-p24 antibodies for this purpose. 
Recently, a variation of the Abbott assay has been reported by Allain, et 
al., supra, in which titers are calculated by comparison with a reference 
curve obtained by serial dilutions of a sample containing standard 
antibody. The range of titers which can be measured by the modified assay 
is only between 1:2 and 1:256, and the assay requires the determination of 
a cutoff point for each series of assays so that the recorded optical 
densities can be transformed to titers using the standard curve. 
Significantly, with regard to the enzyme immunoassays for anti-p24 
antibodies, as well as all of the other assays for these antibodies, there 
is no integration between the assay for antibody levels and an assay for 
p24 antigen levels. As discussed above, it is the combination of a falling 
antibody level and a rising antigen level which appears to be most 
predictive of the clinical course of an HIV infection. Yet, as presently 
practiced, completely separate assays are being performed for these two 
important prognostic indicators. 
SUMMARY OF THE INVENTION 
In view of the foregoing state of the art, it is an object of this 
invention to provide an assay which is responsive to the levels of 
anti-p24 antibodies in a biological fluid, e.g., a serum sample, and which 
is easier to perform, less expensive, and more suitable for wide spread 
clinical use than existing anti-p24 antibody assays. It is a further 
object of this invention to provide an assay for anti-p24 antibodies which 
can be readily integrated with a p24 antigen assay so that both assays can 
be conveniently performed simultaneously. In this way, the combination of 
a falling level of anti-p24 antibodies and a rising level of p24 antigen 
can be readily detected. As discussed above, this combination has been 
found to be indicative of a poor prognosis for HIV-infected patients. 
To achieve the foregoing and other objects, the invention, in accordance 
with certain of its aspects, provides an assay which is responsive to the 
levels of anti-p24 antibodies in a sample of a biological fluid comprising 
the steps of: 
(a) forming a test mixture comprising: 1) the sample; and 2) a solution 
(hereinafter referred to as the "p24 antigen solution" or simply the 
"antigen solution") containing free p24 antigen within a predetermined 
concentration range, the relative amounts of the sample and the antigen 
solution being such that the concentration of free p24 antigen in the 
antigen solution is not substantially diluted by the formation of the test 
mixture: 
(b) incubating the test mixture under conditions whereby anti-p24 
antibodies from the sample, if any, can react with the free p24 antigen to 
form antibody-antigen complexes; 
(c) determining the concentration of free p24 antigen remaining in the test 
mixture after the incubation; 
(d) determining the concentration of free p24 antigen in the antigen 
solution; and 
(e) calculating the difference between the concentration of free p24 
antigen in the antigen solution and the concentration of free p24 antigen 
in the test mixture after the incubation. 
The difference calculated in step (e) is referred to herein as the sample's 
"p24 binding capacity" or simply the sample's "binding capacity". This 
parameter has been found to be an accurate and convenient measure of the 
level of anti-p24 antibodies in patient samples. 
Specifically, the higher the level of anti-p24 antibodies in a sample, the 
more antibody-antigen complexes which are formed in step (b); the more 
antibody-antigen complexes which are formed in step (b), the lower the 
concentration of free p24 antigen determined in step (c); the lower the 
concentration of free p24 antigen determined in step (c), the larger the 
binding capacity value calculated in step (e). Accordingly, the higher the 
level of anti-p24 antibodies, the larger the calculated binding capacity. 
This straightforward relation between binding capacity and the level of 
anti-p24 antibodies is easily understood by both patient and physician, 
which is an important advantage of the assay. 
Another important advantage of the assay is that it can be readily 
integrated with existing assays for p24 antigen levels. Specifically, in 
accordance with this further aspect of the invention, an assay system 
responsive to both anti-p24 antibody levels and p24 antigen levels is 
provided which comprises the steps of: 
(a) forming the test mixture of the anti-p24 antibody assay using 1) a 
first sample from the biological fluid to be tested, and 2) the p24 
antigen solution; 
(b) incubating the test mixture; 
(c) assaying the test mixture for p24 antigen concentration; and 
(d) assaying a second sample from the biological fluid for p24 antigen 
concentration; 
the assays of step (c) (for the level of anti-p24 antibodies) and step (d) 
(for the level of p24 antigen) being performed substantially 
simultaneously using the same assay technique. 
This assay system is readily incorporated in a clinical laboratory setting. 
Indeed, the assay for the level of anti-p24 antibodies in essence boils 
down to simply running the p24 antigen assay on additional samples, i.e., 
samples which have been mixed with the antigen solution. Accordingly, the 
integration of the anti-p24 antibody assay with the p24 antigen assay 
involves a minimum of additional steps and new procedures. This 
straightforward integration is in direct contrast to the existing anti-p24 
antibody assays discussed above which essentially have no commonality with 
p24 antigen assays. 
In certain preferred embodiments of the invention, p24 antigen levels are 
measured by a sandwich assay technique using anti-p24 antibodies, e.g., a 
bound (immobilized) anti-p24 antibody and a soluble anti-p24 antibody 
which carries a label or is labelled as part of the assay procedure. In 
other preferred embodiments, the same assay procedure used to measure p24 
antigen levels in the test mixture (and in the second sample when the 
assay system is employed) is used to determine the concentration of p24 
antigen in the antigen solution. This concentration can be determined at 
the same time the p24 antigen level in the test mixture is determined or 
can be determined separately either before or after the test mixture is 
assayed. In a clinical laboratory setting, the p24 antigen level in the 
antigen solution will generally be, and preferably is, determined at the 
same time the p24 antigen level in the test mixture is determined both as 
a control for the assay and so as to minimize assay-to-assay variations in 
the calculated binding capacity. 
The description of the preferred embodiments set forth below further 
describes the principles and advantages of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As discussed above, the present invention relates to 1) an assay which is 
responsive to the level of anti-p24 antibodies in a biological fluid, and 
2) the integration of that assay with p24 antigen assays. 
The biological fluid to be tested will usually be human serum. Other 
biological fluids which can be tested include whole blood, plasma, 
cerebrospinal fluid (CSF), saliva, and the like. Although to date there 
has been only limited success in developing animal and laboratory models 
for use in studying AIDS, the present invention can be used in testing 
biological fluids from the models which presently exist as well as those 
that may be developed in the future. 
The assay can be performed on both diluted and undiluted samples of the 
biological fluid. In the case of human serum, undiluted samples are 
preferred. If dilution is desired, a suitable dilution medium comprises 
phosphate buffered saline containing 0.5% or 1% normal human sera. When 
diluted samples are used, the binding capacity for the biological fluid is 
calculated by multiplying the binding capacity calculated for the diluted 
sample by the dilution factor, e.g., by 2 in the case of a sample which 
has been diluted by 1:2. 
To perform the assay, the sample of the biological fluid is mixed with a 
measured quantity of a p24 antigen solution which contains p24 antigen 
within a predetermined concentration range. The p24 antigen can be 
obtained in various ways including the lysis of whole virus grown in 
culture and through the use of recombinant DNA techniques. p24 antigen 
from viral lysates has been found preferable for use with the invention 
because of the low solubility in physiological saline of existing 
recombinant p24 proteins. The entire p24 molecule need not be used, but 
rather only those parts of the molecule which will bind to anti-p24 
antibodies in patient samples and which can be detected by p24 antigen 
assays. 
A viral lysate was used in the examples presented below. This lysate was 
prepared from an HIV isolate (HIV.sub.hs) recovered from a homosexual 
patient with 
Kaposi's sarcoma (KS) as follows. HIV.sub.hs was passaged twice (P2) in 
normal PHA-stimulated peripheral blood lymphocytes cultured in RPMI 1640 
media (M.A. Bioproducts/Whittacker) with 20% fetal calf serum (Whittacker) 
and 10% T cell growth factor (Electro Nucleonics, Inc). The cell culture 
supernatant was clarified by centrifugation at 600.times.g for 20 minutes 
(twice), diluted in phosphate buffered saline with 0.5% (v/v) Triton X-100 
(Sigma), and stored in aliquots at -70.degree. C. until used. Just prior 
to use, normal human serum was added at a concentration of 0.5%. The final 
p24 antigen concentration in the antigen solution was between 350 ng/l 
(nanograms/liter) and 450 ng/1. 
The concentration of p24 antigen in the antigen solution as well as the 
relative amounts of sample and antigen solution used in performing the 
assay are critical to the assay's success. Specifically, these quantities 
must be selected so that the assay is responsive to the levels of anti-p24 
antibodies which exist at difference stages of HIV infection. The 
quantities must also be chosen to take account of the variability in 
anti-p24 antibody levels among patients at the same stage of infection. 
In practice, the quantities are determined by testing a bank of sera 
samples taken from various patients through the course of their infection. 
Using this technique for the viral lysate described above and for the 
assay protocol described below, it was determined that: 1) the 
concentration of p24 antigen in the antigen solution should be between 
about 350 ng/l and about 450 ng/l, and preferably between about 380 ng/l 
and about 425 ng/l, where the antigen concentrations are measured using 
the Du Pont p24 assay described below; and 2) the antigen solution to 
sample ratio for undiluted sera should be approximately 250 parts solution 
to 1 part sample (in particular, in the examples presented below, 250 mcl 
(microliters) of antigen solution were combined with 1 mcl of sample). As 
will be evident to persons of ordinary skill in the art, the same 
technique can be used to determine the corresponding concentrations and 
solution/sample ratios for other assay protocols, sera dilutions, antigen 
sources and p24 assay systems. 
In performing the assay, the sample to be tested and the antigen solution 
are mixed together to form a test mixture, and the test mixture is 
incubated under conditions whereby anti-p24 antibodies from the sample can 
react with free p24 antigen in the antigen solution. Various incubation 
times and temperatures can be used. For example, in the examples presented 
below, the antigen solution and the samples were incubated for 30 minutes 
at 37.degree. C. in a shaking water bath. Essentially identical results 
were achieved with an incubation period of a few minutes at 37.degree. C. 
At least some further reaction between the free p24 antigen from the 
antigen solution and the anti-p24 antibodies from the sample takes place 
during the incubation portion of the p24 antigen assay, e.g., for a 
forward sandwich assay during the incubation of the sample with the 
immobilized antibody. If desired, the assay of the present invention can 
be formatted without a separate incubation of the test mixture prior to 
the commencement of the p24 antigen assay, the incubation portion of the 
p24 antigen assay serving as the incubation step of the present assay for 
this format. Formats of this kind wherein the incubation portion of the 
p24 antigen assay is conducted at a temperature below 37.degree. C., e.g., 
at room temperature, will in general produce somewhat different values for 
p24 binding capacity than formats which include a separate incubation step 
at 37.degree. C. 
After the sample and the antigen solution have been combined and the p24 
antigen/anti-p24 antibody complexes have formed, the concentration of free 
p24 antigen remaining in the test mixture is determined. This 
concentration can be determined by a variety of methods now known or 
subsequently developed. For example, as discussed above, commercial 
sandwich assays having sensitivities on the order of 50 
picograms/milliliter (pg/ml) are available from Abbott and Du Pont. In 
particular, the Du Pont assay discussed below has a low limit of 31 pg/ml. 
These as well as other assays can be used in the practice of the 
invention. 
The examples presented below used an antigen capture enzyme immunoassay 
sold by Biotechnology Systems, E. I. Du Pont, Billerica, Mass. 
(hereinafter referred to as the "Du Pont assay"). See McDougal, et al., 
"Immunoassay for the detection and quantitation of infectious human 
retrovirus, lymphadenopathy-associated virus (LAV)", J. Immunol. Methods, 
1985, Vol. 76, pages 171-183. Specifically, 200 mcl of the test mixture 
were transferred to a well of a polystyrene plate coated with polyclonal 
rabbit anti-HIV p24. Following overnight antigen capture at room 
temperature, the plates were washed with phosphate buffered saline 
containing Tween-20 (PBST). Detection of the antibody-antigen complex was 
accomplished with biotinylated rabbit anti-p24 in a 2 hour incubation at 
37.degree. C. Following washes with PBST, the 
antibody-antigen-antibody-biotin complex was further complexed with 
streptavidin-horseradish peroxidase for 15 minutes at room temperature, 
which thereafter was reacted with an o-phenylalanine diamine (OPD) 
substrate producing a colored product. Absorbance of the product was 
measured at 490 nm using a spectrophotometer (Bio-Tek Instruments). A 
standard curve (range 31 to 1000 ng/l) was established utilizing a whole 
virus lysate whose p24 protein concentration was compared with 
affinity-purified p24 antigen. The protein concentration of this purified 
antigen was measured by the Burnett method. Standard curve and sample 
performance was evaluated by quadrantic analysis utilizing a statistical 
package provided by Du Pont. Qualitative controls for each assay included 
serum samples containing viral lysate equivalent to 15 ng/l p24 protein. 
The reproducible lower limit of antigen detection was less than 31 ng/l 
yet greater than 15 ng/l. Quantitative values less than 31 ng/l were 
considered to be 30 ng/l for statistical analysis. 
p24 antigen concentrations for the antigen solution were determined for 
each assay using the same p24 assay protocol. Controls consisted of 
aliquots of sera with varying titers of p24 binding capacity which were 
included with each assay. The nonspecific binding of serum for p24 was 
established among subjects at no risk for HIV infection. 
The binding capacity (p24 BC) of a sample in ng/ml was calculated from the 
following formula, where A is the p24 antigen concentration in ng/ml of 
the antigen solution, B is the final p24 antigen concentration in ng/ml of 
the test mixture, V is the volume of the test mixture (mcl), and Q is the 
volume of the undiluted serum sample (mcl): 
EQU p24 BC=[A-B].times.[V/Q]. 
As discussed above, one particularly important advantage of the anti-p24 
antibody assay of the present invention is the ease with which it can be 
integrated with a p24 antigen assay. Thus, for the protocol described 
above, both an anti-p24 antibody assay and a p24 antigen assay can be 
performed on a patient's serum by simplying taking one or a few extra 
samples of serum, mixing them with the antigen solution, incubating them 
at 37.degree. C., and then running a standard p24 antigen assay for these 
extra samples along with the samples one would normally run to determine 
the patient's level of p24 antigen. Plainly, these few added steps are 
easy to teach to clinical laboratory personnel already familiar with the 
p24 antigen assay. 
Without intending to limit it in any manner, the present invention will be 
more fully described by the following examples. 
EXAMPLES 
I. Summary of Results 
Two hundred fourteen untreated subjects were followed (range 12-42 months) 
to study the natural history of HIV infection: 110 were classified as CDC 
II/III, 11 as CDC IV A, 15 as CDC IV D, 31 as CDC IV C, and 47 were 
HIV-seronegative controls. p24 binding capacity and quantitative levels of 
HIV p24 antigen in serum were determined at regular intervals. For people 
in all diagnostic groups, a p24 BC below 31 ng/ml was more closely 
associated with progressive disease than was p24 antigen positivity: 87% 
of CDC IV C, 89% of CDC IV A, 69% of CDC IV D, and 21% of CDC II/III 
subjects had p24 BC less than 31 ng/ml at entry, while 65% of CDC IV C, 
64% of CDC IV A, 60% of CDC IV D, and 13% of CDC II/III subjects were p24 
antigenemic. Prospective analysis of 47 CDC II/III men followed for 3 
years who showed actuarial progression rates to ARC at 4%, 13%, and 23% 
and to AIDS at 5%, 8%, and 8% at 1, 2, and 3 years, indicated that levels 
of p24 BC below 31 ng/ml were more strongly associated with progression 
than was p24 antigenemia (X.sup.2 32 20.6 versus 4.6). Overall, it was 
concluded that quantitative measures of p24 binding capacity and p24 
antigen were powerful indicators of progressive disease. 
II. Methods 
A. Patient Population 
Two hundred and fourteen (n=214) subjects were recruited between Jan. 1984 
and Mar. 1987. These included 110 asymptomatic HIV-seropositive and 47 
seronegative people, 11 patients with AIDS-related complex (ARC, Centers 
for Disease Control ("CDC") classification CDC IV A), 15 with Kaposi's 
sarcoma (KS, CDC IV D), and 31 with the opportunistic infection, 
Pneumocytis carinii pneumonia (AIDS/OI, CDC IV Cl). Study participants 
were seen at six month intervals or more frequently when clinical symptoms 
were reported. All subjects gave informed consent for HIV virus testing in 
accordance with the guidelines of the U.S. Public Health Service. Initial 
serological evaluation was performed by one of several ELISAs (Abbott, Du 
Pont, or Cellular Products, Inc.) with confirmation by Western blotting of 
serum (Biotech Research Laboratories, E. I. Du Pont, Bethesda, Md.). 
B. Assays 
p24 BC values were determined using the assay protocol described above. p24 
antigen concentrations were determined using the Du Pont assay described 
above. 
Levels of anti-p24 were also measured on selected samples by an 
experimental enzyme immunoassay utilizing a recombinant gag gene product 
expressed in E. coli (E. I. Du Pont, Glascow, Del.). Serial dilutions of 
serum samples (1:20 to 1:43740 in three-fold steps) were incubated in 200 
microliter volumes with recombinant HIV p24 fixed in wells of a 
polystyrene microtiter plate for 1 hour at 37.degree. C. Following washing 
with PBST, the wells were incubated with alkaline phosphatase conjugated 
anti-human globulin for 30 minutes at 37.degree. C. Following further 
washes, para-nitrophenylphosphate (pNPP) substrate was added to the wells 
and incubated for 30 minutes at 37.degree. C. The reaction was stopped by 
the addition of 50 microliters of 3N NaOH and the absorbance of the 
solution was read at 405 nm by a spectrophotometer. Positive results were 
defined as a sample absorbance twice the mean of negative control values. 
The reciprocal of the maximum serum dilution to be scored positive was 
taken as the measure of the antibody in the sample. 
Complete blood counts (Coulter Electronics, Hialeah, Fla.) with white cell 
counts (WBC) and lymphocyte subset profiles were obtained at 6 month 
intervals for the asymptomatic seropositive men and seronegative controls. 
Study subjects in advancing stages of infection underwent complete blood 
counts and lymphocyte subset profile analysis at varying intervals, 
depending upon clinical status. Lymphocyte subsets were quantitated 
utilizing monoclonal antibodies Leu 4, Leu 3a, and Leu 2a (Becton 
Dickinson, Sunnyvale, Calif.) which label the lymphocyte receptors CD3, 
CD4, and CD8 respectively, and by FACS (fluorescein-activated cell 
sorting). 
C. Statistical Analysis 
Analysis of data was initially performed with the patients grouped by 
admitting diagnosis. The variables associated with outcome were 
established by discriminant analysis. The data were also analyzed with 
samples grouped by diagnosis, outcome, and visit number. Group differences 
were compared by one-way ANOVA with Fisher's least significance test. 
Grouped data were also analyzed by nonparametric methods, particularly the 
Mann-Whitney two sample test and the Kruskal-Wallis test of multiple group 
samples. Relationships among variables were established by correlation 
analysis and chi squares. Prospective data were analyzed by the 
product-limit method and the differences among the actuarial progression 
rates were evaluated by ANOVA. See Cox, et al., Analysis of Survival Data, 
Chapman and Hall, London, 1984. Statistical significance implies a p 
value of less than or equal to 0.05 unless stated otherwise. 
III. Results 
A. Analysis of entry data with patients grouped by diagnosis 
Table 1 shows selected entry laboratory values by diagnostic category. All 
HIV-infected groups showed significant reduction in total numbers of white 
blood cells (WBC) and CD4+ lymphocytes (helper cells) from levels in 
seronegative controls. Patients with ARC or AIDS/OI had significantly 
decreased numbers of WBC and CD4+ cells compared to subjects classified as 
CDC II/III, while KS patients had levels intermediate between asymptomatic 
men and ARC/AIDS patients. Although there was overlap of the 95% 
confidence level ranges, both asymptomatic subjects and patients with 
Kaposi's sarcoma had somewhat higher numbers of CD8+ lymphocytes than 
other patients (n.s., p=0.08) or controls. 
While HIV p24 antigen was detected at entry in subjects from all groups, 
the frequency of positive samples and the quantitative level of antigen 
(range 30-832 ng/l) was greater in symptomatic patients than in 
asymptomatic subjects (X.sup.2 =61.6, p less than 0.001). Of 31 patients 
with a history of opportunistic infection, 20 (65%) were p24 antigenemic 
at entry (range 31-832 ng/l). Sixty-four percent (7/11) of ARC patients 
were antigenemic at entry (range 30-623 ng/l); and for the 15 patients 
presenting with KS, 60% were p24 positive at entry (range 30-204 ng/l). 
Asymptomatic subjects were less frequently p24 positive at entry (14/110, 
13%, range 30-240). If persistent antigenemia was defined as two or more 
p24-positive samples collected at least 4 weeks apart, 12/15 AIDS 
patients, 3/5 ARC patients, and 8/47 asymptomatic subjects were found to 
be persistently antigenemic. 
The level of p24 BC of serum varied among the diagnostic groups (range 
0-114 ng/ml): the background p24 BC among seronegative controls (n=47) at 
no risk for occult HIV infection was 9.+-.1 ng/ml. p24 BC levels were 
highest among asymptomatic subjects (mean 50.+-.2, range 6-114 ng/ml). 
Patients with KS showed lower mean levels at entry (33.+-.4, p 0.05) than 
asymptomatic subjects. Patients with ARC or AIDS/OI had still lower levels 
of p24 BC (mean 14.+-.3, 13.+-.3 respectively, p=0.01). When the 
quantitative level of p24 BC was dichotomized above or below 31 ng/ml (a 
break point which discriminated between asymptomatic and symptomatic 
subjects, X.sup.2 =39.2, p less than 0.001), 19/89 (21%) asymptomatic 
subjects showed less than 31 ng/ml of p24 BC at entry. Among symptomatic 
patients, 9/13 (69%) patients with KS, 8/9 (89%) of patients with ARC, and 
13/15 (87%) of patients with AIDS/OI had p24 BC less than 31 ng/ml at 
entry. 
B. Prospective analysis of laboratory data from all diagnostic groups 
For all patient groups, p24 antigenemia greater than or equal to 31 ng/l 
was strongly associated with progression (X.sup.2 =11.2, p less than 
0.001). While the difference in level of association was small, an entry 
level p24 BC less than 31 ng/ml was somewhat better associated with 
progressive disease than p24 antigen positivity at entry (X.sup.2 =41.3 
and 6.8, respectively). When levels of CD4+ lymphocytes were dichotomized 
below or above 400 cells/mm.sup.3, both entry p24 antigenemia and levels 
of p24 BC less than 31 ng/ml were more closely associated with symptoms 
than numbers of CD4+ cells (X.sup.2 =61.6, 39.2, and 17.9). 
Among the HIV-seronegative high-risk control group, 2 subjects were noted 
with outlying values for p24 BC (greater than 3 standard deviations above 
mean for low-HIV-risk seronegative individuals, 9.+-.1). One man, a 31 
year old homosexual with a history of high risk sexual activity with a 
rising p24 BC (initially 5 ng/ml followed 6 months later by 18 ng/ml), has 
been culture and serum antigen negative on several occasions but has HIV 
DNA in uncultured lymphocytes by enzymatic amplification and oligomer 
cleavage detection (polymerase chain reaction (PCR)). See Kwok et al., 
"Identification of human immunodeficiency virus sequences by using in 
vitro enzymatic amplification and oligomer cleavage detection", J. Virol., 
1987, Vol. 61, pages 1690-94. The second man, a 43 year old homosexual 
reporting occasional high risk sexual activity, has also shown an elevated 
level of p24 BC (25 ng/ml). This subject has had several weakly positive 
HIV serologies by indirect immunofluorescence followed by negative 
serologies by the same technique. Although cultures and seru antigen 
levels have been consistently negative, occult infection remains to be 
excluded. 
C. Prospective analysis of asymptomatic subjects 
A cohort of asymptomatic seropositive homosexual men (n=47) who were seen 
on at least 3 occasions during the 3 year study period and who were not 
treated with antiviral agents were studied to evaluate the use of several 
laboratory markers to establish prognosis. The actuarial progression rate 
to ARC was 4%, 13%, and 23% at 1, 2, and 3 years respectively: the 
actuarial progression rate to AIDS was 5%, 8% and 8% at 1, 2, and 3 years 
respectively (FIG. 1). Groups for analysis were established by outcome. 
At entry, there were no significant differences in the mean numbers of WBC, 
CD4+ or CD8+ lymphocytes among the outcome groups. Subjects who progressed 
to AIDS had lower entry levels of CD4+ cells (not significant), with 
marked variation in the number of cells among these subjects. There was a 
significant difference in the mean entry level of p24 antigen and p24 BC 
between those progressing to AIDS and the stable subjects or those 
progressing to ARC (FIGS. 4a and 4b, p=0.01). Levels of p24 BC below 31 
ng/ml at entry were strongly associated with progression (X.sup.2 =20.6, p 
less than 0.001); levels of p24 BC between 31-47 ng/ml were also 
associated with progression (X.sup.2 =7.8, p less than 0.01) (Table 2). 
p24 antigenemia at any quantitative level was significantly associated 
with progression (X.sup.2 =4.6, p less than 0.05), although not as 
strongly as p24 BC. If p24 antigenemia was defined as p24 greater than or 
equal to 31 ng/l, the association of p24 antigenemia with progression 
became stronger (X.sup.2 =14.3, p less than 0.001). Taken independently, 
p24 antigen positivity with quantitative levels less than 31 ng/l did not 
correlate with progression. The combination of p24 antigenemia and p24 BC 
less than 31 ng/ml at entry was a powerful predictor of progression 
(X.sup.2 =25.2). Using dichotomized entry laboratory values, numbers of 
CD4+ lymphocytes above and below 400 cells/mm.sup.3 failed to predict 
progression (X.sup.2 =1.42). Levels of anti-p24 antibody as measured by 
the recombinant gag assay did not correlate with outcome for the group. 
Product-moment analysis of the relationship between the levels of the 
variables and progression are shown in FIG. 2. Subjects antigen-negative 
at entry were less likely to develop progressive disease than subjects 
antigen-positive at entry (FIG. 2a, p less than 0.001). The difference in 
the rates of progression based upon the stratification of subjects by the 
quantitative level of p24 antigen in serum was nonsignificant. The 
difference between the progression rates for subjects with p24 BC greater 
than 47 ng/ml and subjects with p24 BC 31-47 ng/ml was significant at the 
p=0.03 level (FIG. 2b), while the difference in progression rates for 
subjects with levels of p24 BC at and above 47 ng/ml or less than 31 ng/ml 
was significant at the p=0.0001 level. Progression rates for the levels of 
CD4+ cells were different (FIG. 2c), although entry levels (greater than 
or less than 400 cells/mm.sup.3) did not significantly correlate with 
outcome. When the progression rates were compared (FIG. 2d), p24 BC 
identified the progressors most effectively, significantly more 
effectively than numbers of CD4+ cells at entry (p=0.05). 
The actuarial rate of the appearance of index laboratory values, i.e., p24 
positivity, p24 BC less than 31 ng/ml, or CD4+ cells under 400 
cells/mm.sup.3, was markedly different between progressors (FIG. 3a) and 
nonprogressors (FIG. 3b). For progressors, there was no significant 
difference in the rates of appearance of index values. For nonprogressors, 
p24 antigen-positivity was seen more frequently than abnormal levels of 
p24 BC (p less than 0.001), suggesting that p24 BC greater than 31 ng/ml 
at entry was a more reliable indicator of a stable course than the absence 
of p24 antigen. 
Mean levels of p24 antigen and p24 BC during the course of observation are 
shown in FIGS. 4a and 4b. Detectable antigen fluctuated sharply among 
progressors, while levels of p24 BC showed gradual change. Among subjects 
who remained stable, low or falling levels of p24 antigen correlated with 
increasing numbers of CD4+ lymphocytes, while increasing levels of p24 
antigen correlated with decreasing numbers of CD4+ cells (FIG. 4c). The 
presence of p24 antigen in serum was also associated with increased 
numbers of CD8+ lymphocytes (r=0.702, p=0.02). Levels of p24 and p24 BC in 
all groups showed an overall decline as patients progressed (FIG. 5). 
IV. Discussion 
As shown by the above results, p24 antigen level is a meaningful parameter 
with which to monitor HIV-infected patients. In this study, the percent of 
subjects with detectable p24 antigen in serum ranged from a low of 13% in 
asymptomatic men to a high of 65% in patients with AIDS/OI (Table 1). Not 
all HIV-infected subjects were antigenemic during observation; 21 of 57 
(37%) symptomatic patients (CDC IV+) were p24 negative, while 96 of 110 
(87%) HIV-seropositive asymptomatic subjects were p24 antigen negative. 
Prospective analysis of the data revealed that the quantity and the 
persistence of detectable antigen were related to outcome. Only three of 
thirty-two asymptomatic subjects who remained stable over a three-year 
period had p24 antigen-positive samples with a quantitative p24 level of 
greater than or equal to 31 ng/l in the first year, while ten of fifteen 
who were initially asymptomatic but progressed in 3 years had p24 antigen 
levels greater than or equal to 31 ng/l during the first year (X.sup.2 
=18.4, p less than 0.001). If persistent antigenemia was defined as two or 
more p24-positive samples drawn at intervals of 4 weeks or more, then 
persistent antigenemia was also strongly associated with progression 
(X.sup.2 =20.6, p less than 0.001). While persistent antigenemia was an 
indicator of poor prognosis regardless of the clinical symptoms of the 
patient, the absence of p24 antigenemia did not necessarily mean that the 
patient was going to become or remain stable. Five of 11 asymptomatic men 
progressing to ARC showed no detectable antigen on at least 2 visits 
before clinical advancement. In 2 of 4 men progressing to AIDS from the 
asymptomatic stage, levels of p24 antigen were negative in the 6-month 
period preceding diagnosis. As the levels of p24 antigen varied widely in 
any individual over time, it was essential that several values were 
obtained before an overall assessment was made. 
As the above results also show, p24 BC is a further meaningful parameter 
with which to monitor HIV-infected patients. Cross-sectional analysis of 
the data showed that the levels of p24 BC generally provided mirror images 
of the levels of p24 antigenemia (FIG. 5). Prospective analysis 
illustrated the usefulness of the determination of p24 BC. Among 
asymptomatic subjects who progressed to ARC/AIDS, a p24 BC less than 31 
ng/ml (abnormal for the asymptomatic group) was found more frequently at 
entry (n.s.) and significantly more frequently by 18 months of follow-up 
than p24 antigenemia (FIG. 3a, p less than 0.001). Among nonprogressors 
from the same group, an abnormal p24 BC was seen less frequently than p24 
antigenemia both at entry and throughout the 3 year period (FIG. 3b, p 
less than 0.001). For asymptomatic subjects developing ARC, 5 of 11 
subjects were p24 antigen negative during the year prior to progression, 
while all 11 of these men had intermediate to low levels of p24 BC (FIGS. 
4a and 4b). 
It should be noted that the anti-p24 antibody assay of the present 
invention does not directly measure antibody to p24, but instead measures 
the ability of a sample to complex known amounts of antigen. Levels of 
anti-p24 as measured by the recombinant p24 ELISA used in this study did 
not correlate with levels of p24 BC. This may, in part, be due to the 
small number of samples for which both assay techniques were used. Also, 
although not wishing to be bound by any particularly theory or theories of 
operation, it is believed that the in vitro immune complex formation which 
occurs in the p24 BC assay may favor measurement of high affinity 
antibodies, while the recombinant ELISA may detect antibodies of lower 
affinity. Because of these theoretical considerations, the term "p24 
binding capacity" has been used herein to describe the observed data 
rather than the term "antibody level". In addition, it is considered 
possible that high affinity antibodies may disappear prior to the loss of 
low affinity antibody. 
Although not wishing to be bound by any particular theory or theories of 
operation, it is believed that a reduction in p24 BC levels probably 
reflects binding of p24 antibody into antigen-antibody complexes, the 
antigen being the result of chronic viral proliferation. See Ujhelyi et 
al., "A simple method for detecting HIV antibody hidden in circulating 
immune complexes", AIDS, 1987, Vol. 1, pages 161-165. An excess of 
antibody to antigen, particularly during the asymptomatic stage of 
infection, could produce a situation in which antigen is undetectable 
while levels of p24 BC might show measurable decline during low-grade 
viral proliferation. It is believed that this probably accounts for the 
earlier decline in p24 BC levels than the appearance of p24 antigen in 
asymptomatic men. Yet in some groups of patients with advanced disease, 
the levels of p24 BC seem disproportionately low compared to levels of 
detectable antigen. Stacked bar graphs of the detectable p24 antigen and 
p24 BC by diagnostic groups show that patients with advanced disease had 
somewhat less p24 BC than would be expected by comparison to other 
diagnostic groups (FIG. 5). The general debilitation of the immune system 
may be responsible for this finding. 
In sum, the foregoing data demonstrates that: 1) the presence of p24 
antigenemia, particularly with a quantitative level of greater than 31 
ng/l or with persistence of antigenemia, is an useful indicator of 
progressive disease, and 2) quantitative measurement of p24 BC is more 
strongly associated with prognosis than is antigenemia. The results 
regarding p24 BC are in contrast with those of Moss et al., 
"Seropositivity for HIV and the development of AIDS or AIDS related 
condition: three year follow up of the San Francisco General Hospital 
cohort", Br. Med. J., 1988, Vol. 296, pages 745-750. Moss et al., however, 
used the technique of visual inspection of Western blots for estimation of 
anti-p24. This technique is imprecise and does not always correlate with 
measurement of p24 BC (data not shown). It is believed that these factors 
probably account for the difference between the above data and the Moss et 
al. report. 
The identification of a useful anti-retroviral agent, zidovudine, has 
encouraged both the medical community and the HIV-infected patient 
population to invest time, energy, and resources toward further treatment 
refinements. See Yarchoan et al., "Development of antiretroviral therapy 
for the acquired immunodeficiency syndrome and related disorders: a 
progress report", New England J. Med., 1987, Vol. 316, page 258. 
Pretreatment patient stratification is critical for successful comparison 
of therapeutic regimens; regimens that might treat not only the end-stage 
of HIV infection, AIDS, but also the earlier stages of asymptomatic 
infection. Measures of virus activity will play an increasingly important 
role in the initial stratification and follow-up of patients undergoing 
therapy. The foregoing data shows that the p24 BC assay and the p24 
antigen/p24 BC assay system of the present invention provide effective and 
convenient methods for making this stratification. 
TABLE 1 
__________________________________________________________________________ 
CROSS-SECTIONAL LABORATORY PROFILES AT ENTRY 
BY DIAGNOSTIC GROUP 
p24.sup.2 
p24 BC.sup.3 
Diagnosis.sup.1 
WBC # CD4+ 
# CD8+ 
# +/# 
ng/l 
# &lt; 31/# 
ng/ml 
__________________________________________________________________________ 
Control 
6160 803 467 0/50 47/47 9 
(486) 
(131) 
(89) 100% (1) 
CDC II/III 
5002*+ 
493* 642 14/110 
0 19/89 50* 
(114) 
(29) (32) 13% (0-37) 
21% (2) 
CDC IV A 
3700*+ 
162*+ 
337+ 7/11 
33+ 8/9 14+ 
(424) 
(69) (121) 
64% (0-397) 
89% (3) 
CDC IV D 
4263*+ 
414* 577 9/15 
30+ 9/13 33*+ 
(339) 
(84) (88) 60% (0-68) 
69% (4) 
CDC IV C1 
3380*+ 
170*+ 
342 20/31 
31+ 13/15 13+ 
(816) 
(64) (152) 
65% (0-191) 
87% (3) 
__________________________________________________________________________ 
.sup.1 Diagnosis by Centers for Disease Control classification. For WBC, 
CD4+ cells, and CD8+ cells, the values reported are mean values with 
(standard error of the mean). 
.sup.2 For p24 antigen, the values reported are # positive patients/total 
# of patients with %, and median values in ng/l with (10-90% quartile 
range). 
.sup.3 For p24 BC, the values reported are number of patients with entry 
p24 BC less than 31 ng/ml over the total number of patients with entry 
samples with %, and the mean levels in ng/ml with (S.E.M.) 
*Difference from Control, p = 0.01. 
+ Difference from CDC II/III, p = 0.05. 
Note: 
Not all tests were performed on all subjects at each visit so that the 
totals do not equal 251 in all cases. 
TABLE 2 
__________________________________________________________________________ 
INDEX LABORATORY VALUES BY YEAR FOR THE 
ASYMPTOMATIC SEROPOSITIVE GROUP AND PROGRESSORS 
CDC II/III CDC IV A CDC IV C 
Year 
1 2 3 1 2 3 1 2 3 
__________________________________________________________________________ 
Actuarial 4% 13% 23% 5% 8% 
progression 
rate 
# progressed/ 2/11 
4/11 
5/11 
2/4 
2/4 
# total 
Index value 
p24+ 8/43 
3/24 
1/13 
1/2 4/5 8/9 2/2 
1/1 
N.A. 
p24 .gtoreq. 31 
3/43 
0/24 
0/13 
2/2 2/5 5/9 1/2 
1/1 
N.A. 
p24 BC &gt; 48 
30/43 
20/23 
10/13 
0/2 1/5 0/9 0/2 
0/1 
N.A. 
p24 BC 31-47 
4/43 
2/23 
0/13 
2/2 3/5 0/9 1/2 
0/1 
N.A. 
p24 BC &lt; 31 
2/43 
1/23 
0/13 
2/2 4/5 4/9 1/2 
1/1 
N.A. 
CD4+ &gt; 400 
20/28 
15/23 
3/8 0/2 0/5 0/3 0/2 
0/1 
N.A. 
CD4+ &lt; 400 
8/28 
8/23 
5/8 2/2 5/5 3/3 2/2 
1/1 
N.A. 
__________________________________________________________________________ 
*Index value denotes the first appearance of a laboratory value as define 
with number positive over the total number of available values for the 
group. p24 (ng/l; p24 BC (ng/ml); CD4+ (cells/mm.sup.3). Not all patients 
and/or samples were available for each time point, so totals do not equal 
47 in all cases.