Immunoassay for HIV-I antigens using F(AB').sub.2 fragments as probe

The present invention provides a probe for the detection of HIV 1 antigens, comprising anti-HIV 1 F(Ab').sub.2 fragments. The invention further provides an immunoassay with enhanced specificity.

BACKGROUND OF THE INVENTION 
The present invention relates to the detection of the Human 
Immunodeficiency Virus (HIV) in serum, plasma or other body fluids. In 
particular, this invention describes diagnostic assays which employ 
F(ab').sub.2 fragments as a probe for the detection of HIV 1 antigens. 
HIV 1 is believed to be the causative agent in acquired immunodeficiency 
syndrome (AIDS) [Chamberland et al., Ann. Int. Med. (1984) 101:617-623; 
Seligman et al., New Eng. J. Med. (1984) 311:1286-1292]. The virus has 
been isolated from patients with AIDS and AIDS-related complex (ARC) as 
well as from healthy persons at high risk for AIDS (Gallo et al., Science 
(1984) 244:500-502). 
Since Dec. 1986, enzyme immunoassays, for example, the Abbott HIV 1 Antigen 
assay (Abbott Laboratories, North Chicago, Illinois), have been 
commercially available on a research basis for detection of HIV 1 
antigens. These tests are highly sensitive and provide a direct indication 
of the presence of the virus. Consequently, detection of HIV 1 antigens 
may be useful as an aid in the diagnosis and monitoring of HIV 1 infection 
(Pedersen et al., Brit. Med. J. (1987) 295:567-569; DeWolf et al., Brit. 
Med. J. (1987) 295:569-572). 
The sensitivities of the current HIV 1 antigen assays are quite good, 
however, the specificity varies widely. All manufacturers appear to have 
samples which nonspecifically react with components of the assay yielding 
false reactives. For this reason, Abbott provides a confirmatory procedure 
which involves neutralization of HIV 1 antigen in the sample prior to 
assaying. Although this increases specificity to near 100%, the cost 
involved in additional testing warrants efforts to increase the predictive 
value of antigen testing. The invention described herein provides one 
method of significantly increasing specificity while also enhancing assay 
sensitivity and timing. 
SUMMARY OF THE INVENTION 
A probe for the detection of HIV 1 antigens in plasma, serum, tissue 
culture and other biological fluids is provided by the present invention. 
This probe is a chemically modified antibody. The modification involves 
enzymic cleavage of antibodies to HIV 1 to produce F(ab').sub.2 fragments. 
Highly specific diagnostic assays to detect HIV 1 antigens, using a 
F(ab').sub.2 probe, are provided by the invention. When used in 
conjunction with labeled F(ab').sub.2 fragments specific for the 
F(ab').sub.2 probe described above, an assay of the invention is capable 
of detecting HIV 1 antigen with greater specificity and sensitivity than 
previously observed. Immunoassays of the invention comprise: 
(1) coating a solid support with anti-HIV 1 antibody; 
(2) contacting the solid support with a biological sample; 
(3) contacting the solid support with a probe comprising anti-HIV 1 
F(ab').sub.2 fragments from a different animal species than that used in 
step 1; 
(4) contacting the solid support with F(ab').sub.2 fragments specific for 
the probe used in step 3 conjugated to a label; and 
(5) detecting the label as a measure of the presence of HIV 1 antigen in 
the sample. 
In an especially preferred assay of the invention, monoclonal antibodies, 
most preferably those designated 31-42-19 and 31-90-25, are coated on the 
solid support to specifically capture HIV 1 p24 antigens.

DETAILED DESCRIPTION 
The present invention provides an improved means for the detection of HIV 1 
antigens. The use of F(ab').sub.2 fragments as a probe in an immunoassay 
to detect HIV 1 antigens increases assay specificity while at the same 
time enhances assay sensitivity. For example, this probe can be utilized 
advantageously to improve the immunoassay disclosed in U.S. Pat. No. 
4,748,110, issued May 31, 1988. 
Alternately, any antigen binding fragment, such as Fab monomers, produced 
by cleavage of anti-HIV 1 antibodies with the enzyme papain, can be 
employed as a probe in the inventive assays. 
In addition to using anti-HIV 1 F(ab').sub.2 fragments as a probe, 
F(ab').sub.2 fragments specific for the probe can be labeled and used to 
measure the amount of HIV 1 antigen present in the sample. Any label 
capable of producing a detectable signal can be used in the assays of the 
invention. Representative labels include enzymes, radioisotopes, 
fluorescent and chemiluminescent labels. Further, hapten/labeled 
anti-hapten systems such as a biotin/labeled anti-biotin system can be 
utilized in the inventive assays. 
Both polyclonal and monoclonal antibodies are useful as reagents to capture 
HIV 1 antigen on the solid support used in an assay of the invention. In 
an especially preferred embodiment of the invention, mouse monoclonal 
antibodies to HIV 1 p24 designated 31-42-19 and 31-90-25, disclosed in an 
U.S. application, entitled "Mouse Monoclonal Antibodies to HIV 1 p24 and 
Their Use in Diagnostic Tests," filed by S. Mehta et al. concurrently with 
this application and deposited at the ATCC, Rockville, Maryland under 
accession numbers HB 9726 and HB 9725, respectively, are utilized to 
specifically capture HIV 1 p24 antigen. In addition, both IgG and IgM 
antibodies may be used in the assays of the invention. 
Biological samples which are easily tested by the method of the present 
invention include human and animal body fluids such as whole blood, serum, 
plasma, cerebrospinal fluid and Iymphocyte or cell culture supernatants. 
Solid supports which can be used in immunoassays of the invention include 
wells of reaction trays, test tubes, polystyrene beads, strips membranes, 
microparticles and other solid matrices known to those skilled in the art. 
In addition, reagents for the assays of the invention are ideally suited 
for preparation of a kit. Such a kit may comprise carrier means being 
compartmentalized to receive in close confinement, one or more container 
means such as vials, bottles, test tubes and the like. Each of the 
container means comprises one of the separate elements to be used in the 
assay. 
Current immunoassays for detecting HIV 1 have limited specificity in that 
they routinely detect as reactive, or positive, samples which have no HIV 
1 antigens. These false positives are caused, in large part, by 
nonspecific reactions of the antibodies used with interfering substances 
(for example, rheumatoid factor). These reactions are often mediated by 
the Fc portions of the antibody molecules. By utilizing F(ab').sub.2 
fragments, one can eliminate most of these nonspecific interactions. The 
enhanced specificity afforded by the F(ab').sub.2 fragments greatly 
increases the utility of the assays of the present invention in the 
diagnosis and monitoring of patients infected with HIV 1. The following 
examples illustrate other advantages of the invention. 
EXAMPLE 1 
Preparation of F(ab').sub.2 Fragments 
Rabbit anti-HIV 1 antibodies were subjected to cleavage with pepsin 
according to standard procedures (Fanger et al., J. Immunol. (1970) 
105:1484-1492; Parham, J. Immunol. (1983) 131:2895-2902). The F(ab').sub.2 
fragments were isolated from the digestion mixture by gel filtration 
chromatography. Analysis by sodium dodecyl sulfate polyacrylamide gel 
electrophoresis (Laemmli, Nature (1970) 227:680-685) confirmed the 
presence of intact F(ab').sub.2 fragments. This product retains the 
specificity for recognizing HIV 1 proteins, while eliminating the 
nonspecific reactions frequently mediated by the Fc portion of the whole 
molecule antibody. 
EXAMPLE 2 
F(ab').sub.2 Fragments Used as Probe in Enzyme Immunoassay (EIA) for 
Detection of HIV 1 Antigens 
A representative assay of the invention is described below: 
(1) A human polyclonal anti-HIV 1 IgG coated 1/4 inch polystyrene bead was 
contacted with 200 .mu.l of plasma, serum or other biological fluid. After 
an overnight incubation (16-20 hours) at room temperature, the bead was 
washed three times with 5 ml water to remove unbound sample. 
(2) The washed bead was contacted with 200 .mu.l of rabbit F(ab').sub.2 
anti-HIV 1 and incubated for 1 hour at 40.degree. C. in a waterbath. The 
bead was then washed as described in step 1 to remove unbound reagent. 
(3) The washed bead was contacted with 200 .mu.l of goat F(ab').sub.2 
anti-rabbit F(ab').sub.2 conjugated to horseradish peroxidase (HRPO) 
[Pel-Freeze Biologicals, Rogers, Arkansas], and incubated for 2 hours at 
40.degree. C. in a waterbath. The bead was washed again as described in 
step 1. 
(4) The washed bead was contacted with 300 .mu.l of 
o-phenylenediamine-hydrogen peroxide solution, at room temperature for 
approximately 30 minutes, which formed a yellow-orange colored product in 
the presence of horseradish peroxidase. The reaction was quenched with 1N 
H.sub.2 SO.sub.4 ; then, absorbance was read at 492 nm. 
Alternately, steps 2 and 3 may be combined into one step to facilitate the 
procedure. Species of antibodies other than human can be used to capture 
the HIV 1 antigens, and species of F(ab').sub.2 fragments other than 
rabbit also can be used as the probe. Better specificity is achieved when 
the capture and probe antibodies are from different animal species. 
F(ab').sub.2 fragments also can be used to coat the beads, in addition to 
its use as the probe and the conjugate. 
Additionally, shorter incubation times are possible by incubating the 
reaction mixtures at 40.degree. C. in a waterbath or in a dynamic 
incubator. For example, in the above-described assay, the incubation times 
of sample, anti-HIV 1 F(ab').sub.2 fragments, and conjugate can be reduced 
to 1.5 hours, 0.5 hours and 2 hours, respectively, when incubations take 
place at 40.degree. C. in a waterbath. 
EXAMPLE 3 
The specificity of the assay described in Example 2 was determined using 
eight nonspecific samples. These samples are repeatably reactive but 
nonconfirming in the Abbott HIV 1 Antigens assay. This assay uses whole 
molecule antibodies as a probe. For both assays, an absorbance value which 
was 0.05 O.D. units greater than that of the negative control was 
considered the cutoff value. Samples showing higher absorbance values than 
the cutoff value were considered reactive for HIV 1 antigens. Therefore, 
samples having a sample to cutoff absorbance value (S/C) greater than or 
equal to 1.0 were considered reactive. All reactive samples were tested by 
the Abbott HIV 1 neutralization test, commercially available from Abbott 
Laboratories for research use. A sample which was neutralized by the 
procedure was considered to be confirmed positive for HIV 1 antigens. 
The data provided in Table 1 shows that all eight specimens were negative 
using two different production lots of rabbit F(ab').sub.2 fragments as 
the probe. 
TABLE 1 
______________________________________ 
Comparative Profile of Nonspecific Samples. 
Sample to Cutoff Value 
Sample Abbott 
Number HIV 1 Ags Assay 
F(ab').sub.2 Lot #1 
F(ab').sub.2 Lot #2 
______________________________________ 
1 1.328 0.502 0.420 
2 1.670 0.742 0.735 
3 1.233 0.459 0.378 
4 3.226 0.469 0.420 
5 1.044 0.404 0.389 
6 4.934 0.426 0.452 
7 6.641 0.480 0.504 
8 1.452 0.448 0.410 
______________________________________ 
EXAMPLE 4 
To analyze the assay sensitivity, defined as picograms of HIV 1 p24 per ml 
that gave an absorbance value equal to the cutoff value, a serial dilution 
panel of purified HIV 1 p24 antigen was assayed, and the sensitivity 
determined by linear regression. The signal to noise ratio (S/N), defined 
as the ratio of the positive control absorbance value mean to the negative 
control absorbance value mean, also was compared. 
The experiment, results of which are shown in Table 2, compared the 
performance of the F(ab').sub.2 probe assay, using either human polyclonal 
anti-HIV 1 IgG (the same as that in the Abbott HIV 1 Antigens assay) or 
mouse monoclonal anti-HIV 1 IgG (the monoclonal antibodies designated 
31-42-19 and 31-90-25) on the solid support. Assay sensitivities were 
measured using a standard procedure for all three assays, with incubation 
times of 16-20 hours, 4 hours and 2 hours, respectively, for sample, 
anti-HIV 1 probe and conjugate. In both assays where F(ab').sub.2 
fragments were used, the signal to noise ratio increased, and the 
detectability of HIV 1 antigen improved significantly. In particular, when 
the F(ab').sub.2 probe was used in conjunction with the monoclonal 
antibodies, the sensitivity was less than 1 pg/ml. 
TABLE 2 
______________________________________ 
Mean 
Positive Sensitivity 
Control S/N (pg p24/ml) 
______________________________________ 
Abbott HIV 1 1.342 30.5 5.1 
Antigens Assay 
F(ab').sub.2 Probe 
2.444 37.6 2.2 
(Human IgG Capture) 
F(ab').sub.2 Probe 
2.896 36.6 0.6 
(Monoclonal IgG Capture) 
______________________________________ 
Sensitivity was also compared on a panel of HIV 1 antigen positive 
specimens consisting of neat and diluted samples from six different HIV 1 
antigen positive donors. The same beads, coated with human anti-HIV 1 IgG 
were used for both assays. The results, illustrated in Table 3, show that 
the F(ab').sub.2 probe assay, was capable of detecting 23 out of 29 
samples as positive, as compared to 16 out of 29 for the Abbott HIV 1 
antigens assay. 
TABLE 3 
______________________________________ 
Sample to Cutoff Value 
Abbott HIV 1 
Donor Member Antigen Assay 
F(ab').sub.2 Assay 
______________________________________ 
1 A 1.56 2.95 
B 1.26 2.02 
C 1.08 1.48 
D 0.75 1.02 
E 0.62 0.74 
2 A 1.89 2.26 
B 1.15 1.65 
C 0.92 1.24 
D 0.66 0.92 
E 0.60 0.57 
3 A 2.90 6.73 
B 1.89 3.86 
C 1.37 2.24 
D 0.89 1.30 
E 0.65 0.87 
4 A 4.19 5.57 
B 3.76 5.73 
C 3.26 4.31 
D 2.46 3.50 
E 1.96 2.49 
F 1.73 1.73 
G 0.89 1.02 
H 0.50 0.68 
5 A 1.02 1.82 
B 0.88 1.29 
C 0.50 0.35 
6 A 1.53 2.18 
B 0.98 1.65 
C 0.79 1.20 
Total Positive 16 23 
______________________________________ 
Because of the enhanced signals and sensitivity obtained with F(ab').sub.2 
fragments, it is possible to significantly shorten the time required to 
assay specimens. Several short configurations have been employed, 
including one assay of 3.5 hours, with incubation times of 1 hour each, at 
40.degree. C., for antigen capture on solid support by monoclonal anti-HIV 
1, incubation of solid support with rabbit F(ab').sub.2 anti-HIV 1 and 
incubation of solid support with goat F(ab').sub.2 anti-rabbit 
F(ab').sub.2, followed by color development with o-phenylenediamine. 
Despite eliminating over 20 hours of incubation time, the calculated 
sensitivity for this procedure was 2.7 pg p24/ml. 
While specific examples have been given to illustrate the invention, it is 
to be understood that those skilled in the art will recognize variations 
without departing from the spirit and scope of the invention.