Method for the detection and quantitative determination of antigen in a test sample containing immune complexes of antigen bound to antibodies and to rheumatoid factors

The invention is a simple and rapid method for immune complex dissociation and destruction of rheumatoid factors. It permits the quantitative measurement of the total, i.e., free and immune complex-bound, antigen contained in a test sample. The method involves heating the test sample to a point sufficiently above 65.degree. C. at which the antigen-binding function of antibodies is destroyed. As a consequence, interference by rheumatoid factors is eliminated and the antigen is released from immune complexes, after which it can be measured in an antigen assay that recognizes the heat-denatured antigen.

BACKGROUND OF THE INVENTION 
Antigen (Ag) assays are of increasing importance as diagnostic tools, e.g., 
for the diagnosis of infectious diseases in humans or animals. In 
addition, quantitative measurement of Ag is useful for the follow-up of 
infected individuals. In viral infections, for example, the determination 
of the viral load is important with respect to prognosis, indication for 
antiviral treatment, or assessment of treatment success. Unfortunately, in 
the presence of antibody (Ab) the Ag becomes bound in immune complexes 
(IC). Such complexed Ag is no longer freely available to binding to the 
immune reagents used in antigen assays, i.e., detection by such assays, as 
demonstrated in the case of infection with the human immunodeficiency 
virus, HIV (de Wolf et al., Br. Med. J., 295:569-572, 1987; Pedersen et 
al., Br. Med. J., 295:567-569, 1987; Lange et al., AIDS, 1:15-20, 1987). 
Several groups have developed IC dissociation procedures based on either 
treatment with acids (for an example see Lange et al., AIDS, 1:15-20, 
1987) or bases. While these procedures lead to a significantly higher 
detection rate of antigenemia (Nishanian et al., J. Inf. Dis., 162:21-28, 
1990; Miles et al., N. Engl. J. Med., 328:297-302 1992), convincing data 
showing that they are capable of freeing all IC-bound Ag, thus making 
possible a truly quantitative measurement, have not been presented. 
Another problem that impairs the quality of Ag assays is the presence of 
rheumatoid factors (RF) in a test sample, i.e., of antibodies that have 
specificity for immunoglobulins. Such rheumatoid factors may link the 
capture and tracer antibodies used in an Ag assay, thereby leading to 
overestimation of Ag concentrations or outright false-positive results. 
The usual way to deal with rheumatoid factors is their preabsorption with 
high concentrations of immunoglobulin. However, this does not represent a 
safe remedy, and its effect cannot be readily controlled. 
SUMMARY AND OBJECTS OF THE INVENTION 
It is an object of the invention to propose a simple method permitting the 
detection and quantification of an Ag in a test sample by an Ag assay even 
if a fraction or the whole of said Ag is bound in IC. 
It is a further object of the invention to provide a method for destroying 
rheumatoid factors, thereby eliminating this source of imprecision and 
increasing the specificity and precision of antigen assays. 
With the foregoing and other objects, advantages and features of the 
invention that will become hereinafter apparent, the nature of the 
invention may be more clearly understood by reference to the following 
detailed description of the preferred embodiments of the invention and to 
the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The following are definitions of terms and phrases as used herein: 
The term "antigen (Ag)" is a substance that can induce a detectable immune 
response when introduced into an animal or into man. 
The phrase "antigen tests/assays" refers to any test or assay that detects 
and identifies Ag by a method which requires the specific binding of Ag to 
Ab or to other reagent(s) that exhibit(s) such Ag-specific binding 
properties. 
The phrase "immune complexes (IC)" refers to Ag-antibody (Ab) complexes 
formed when Ab is added to Ag, according to the fundamental law of 
immunology, 
Ag+Ab-&gt;Ag-Ab. 
The phrase "immune complex dissociation" refers to the method or process 
that leads to dissolution of IC, Ag-Ab-&gt;Ag+Ab. 
The phrase "rheumatoid factors (RF)" refers to any type of antibody that 
reacts with any kind of immunoglobulin. 
The phrase "heat denaturation" refers to the heating of a test sample to a 
temperature above 65.degree. C., thereby achieving at least the partial 
loss of Ag-binding function of Ab. 
The phrase "capturing reagent" refers to Ab or other reagent used in an Ag 
assay, which captures the Ag present in a sample and immobilizes it, e.g., 
to the well of a microliter plate, a bead, or a particle. 
The phrase "tracing reagent" refers to an Ab or other reagent used for the 
identification of captured Ag. 
The proposed method involves destroying the Ag-binding function of the 
antibodies present in the sample by heat application, whereby the Ag is 
released from complexes and rendered amenable to quantitative detection by 
the Ag assay's immunological reagents, these reagents being capable of 
recognizing the Ag after such heating. 
The following examples are presented in order to more fully illustrate the 
preferred embodiments of the invention. They should in no way be 
construed, however, as limiting the broad scope of the invention. 
EXAMPLE 1 
Quantitative and Sensitive Detection of Immune-complexed and Free HIV 
Antigen After Boiling of Serum 
The following Example is described in Schupbach J. & Boni J; J. VIROL 
METHODS 1993; 43:247-256, which is hereby incorporated by reference in its 
entirety. 
MATERIALS AND METHODS 
Artificial HIV IC were formed by overnight incubation of HIV 
antibody-positive human serum with HIV culture supernatant. For IC 
disruption these samples or, alternatively, sera or plasma from 
HIV-positive patients or controls were diluted 1:3 with distilled water or 
a 0.5% solution of Triton X-100. The diluted samples were boiled in 1.5 ml 
Eppendorf tubes on a dry heat block (Techne DRI-BLOCK.RTM. DB-2A), or in a 
water bath, and assayed in duplicate by commercial HIV Ag detection kits 
from Du Pont de Nemours or Coulter. For comparison, Ag was also assayed 
without previous denaturation, or after acid disruption of immune 
complexes, using the Coulter Immune Complex Disruption (ICD) kit. Ag tests 
were performed according to the manufacturers' procedures, with the 
exception that the first incubation was for 2 hrs at room temperature on a 
microplate shaker. 
RESULTS 
The efficacy of various methods for immune complex dissociation was 
assessed in model systems using artificial complexes of HIV antigen and 
HIV-specific antibodies formed under controlled conditions. Pooled 
HIV-positive serum with a high liter of antibodies to p24, as assayed by 
Western blot, was serially diluted in antibody-negative serum and IC were 
formed by the addition of a constant amount of HIV Ag from supernatant of 
a HIV-positive cell culture to each serum dilution, resulting in a final 
concentration of added Ag of approximately 100 pg/ml. The samples were 
subjected to heat denaturation, acid treatment, or simple 3-fold dilution 
(to compensate for the dilution required in both IC disruption procedures) 
and assayed by the Coulter HIV-1 p24 Ag assay. FIG. 1A shows that heat 
denaturation yielded basically constant Ag concentrations in all samples, 
independent of the antibody/Ag ratio. In contrast, Ag concentrations 
obtained from both untreated or acid-treated samples were highly dependent 
on the antibody/Ag ratio and ranged from highly-positive to completely 
negative. The untreated HIV-positive serum had such a high concentration 
of p24-specific antibodies that a dilution of 1:2000 was sufficient to 
complex virtually all added Ag. Acid treatment yielded a quantitative 
recovery of Ag down to a dilution of 1:200. However, 90% of the added Ag 
remained undetectable at 1:20 and none was detectable at 1:2. 
FIG. 1B shows the effect of heat denaturation time on Ag detection. 
Denaturation at 100.degree. C. for 2, 3, or 5 min was increasingly 
efficient in the release of complexed exogenous Ag. Denaturation for 2 min 
had an incomplete effect similar to that achieved by acid disrupture. 
Additional experiments showed unchanged good effect of denaturation for 6 
or 7 min, but a slight decrease after 10 min (not shown). Temperatures of 
less than 100.degree. C. were insufficient; 5 min/80.degree. C. had no 
effect at all and 5 min/90.degree. C. released only 5 to 10% of the input 
Ag (not shown). 
FIG. 2A shows the effect on the recognition of the same amount of exogenous 
Ag (in the order of 200 to 300 pg/ml) of adding to it each of 6 human sera 
and incubating overnight. Three of these sera (P1 to P3) were from 
HIV-positive patients and three (N4 to N6) from HIV-negative controls. No 
endogenous Ag was detected with or without previous heat denaturation in 
any of these samples (not shown). When mixed with the exogenous Ag results 
of Ag testing of the undenatured samples ranged from highly positive 
(absorbance 2,304) to completely negative. Western blot analysis for 
p24-specific antibodies revealed that serum P1 had only low reactivity, 
while P2 and P3 had medium and strong reactivity, respectively (not 
shown). Heat denaturation of the mixtures led to the detection of very 
similar amounts of Ag (mean absorbance 0.487, standard error 0.058) in 
all, independent of the presence and titer of antibodies to p24. Thus, 
although the absorbantes were significantly reduced, the variation was not 
different from that observed in those undenatured sera (P1,N4-N6) that 
permitted the detection of exogenous Ag (mean 2.076, standard error 
0.102). Experiments with six additional antibody-negative plasma resulted 
in a mean absorbance of heat-denatured Ag that corresponded to 19%.+-.3.2% 
of that of the undenatured sample (not shown). FIG. 2B shows that heat 
denaturation per se did not impair the recognition of Ag. Ag tests of Ag 
standards serially diluted from 100 to 3 pg/ml in serum-free buffer 
yielded the same absorbance values as undenatured or acid-denatured 
standards. When, however, the dilutions of Ag were made in HIV-positive or 
negative human serum, the resulting concentrations of heat-denatured Ag 
were reduced to about one-third (34%.+-.3%) of those without serum; 
otherwise, the concentration curves had the same characteristics, i.e., 
they showed the same slope and did not vary by more than a factor of two 
from each other (FIGS. 2C, 2D). 
The efficacy of the various methods for immune complex dissociation was 
then assessed with samples from blood donors and HIV-infected patients, 
respectively. FIG. 3 shows Du Pont Ag test results with sera negative or 
positive for antibodies to HIV-1, as determined by enzyme immunoassay and 
Western blot. Similar results were found when some of the testing was done 
with Coulter kits (data not shown). FIG. 3A shows the results of 38 sera 
negative by Western blot tested undenatured, heat-denatured, or 
acid-denatured. Both undenatured and heat-denatured sera had a mean 
absorbance of 0.005 and a standard deviation (SD) of 0.004. Mean and SD 
were, with 0.009 and 0.012, resp., slightly but significantly different 
for acid-treated samples (p.ltoreq.0.006, two-sided paired t-test). The 
cut-off (mean+3 SD) was set at 0.017 for both the untreated and 
heat-treated samples, while it was 0.045 for acidified samples. These 
values corresponded to 1.5 to 2 pg/ml for untreated, 4 to 5 pg/ml for 
heat-denatured, and 5 to 6 pg/ml for acid-denatured samples. One sample 
was consistently borderline to low-positive in all procedures; antigen 
neutralization could not be performed due to the lack of serum. FIGS. 3B 
to F show Ag detection in HIV-positive sera that were ranged into five 
groups of similar antibody reactivity to p24 by visual examination of 
Western blot strips, but were otherwise unselected. Clinical information 
on these patients is not available. Each of FIGS. 3A-F shows the results 
of 10 sera. All sera exhibiting very high (FIG. 3B) or high (FIG. 3C) 
anti-p24 titers were Ag-negative when tested undenatured or after 
acidification; heat denaturation, however, yielded 4 samples above cut-off 
in FIG. 3B and 3 in FIG. 3C. Neutralization assays confirmed the presence 
of Ag in all of the three sera whose absorbance/cut-off ratio was equal to 
or greater than 5.0 and in one serum that was just borderline. 
Neutralization was not successful in the three others. The effect of 
acidification increased with decreasing titer of p24 antibodies (FIG. 3D 
to F), but undenatured sera became also positive more frequently. Eight 
out of ten undenatured samples were above cut-off in the absence of 
anti-p24 antibodies (FIG. 3F). Statistics by t test (two-sided, paired) 
indicated superiority of heat denaturation versus no treatment 
(p.ltoreq.0.004) and versus acid denaturation (p.ltoreq.0.002) in the 30 
samples with the highest titers of anti-p24 antibodies (FIGS. B,C,D), 
while there were no significant differences for the 20 low-titered sera of 
FIGS. 3E and F. 
Independent of whether the samples were tested denatured or undenatured, 
the mean absorbances were lowest in the patients with the highest antibody 
reaction to p24. The concentration of total Ag was reversely correlated 
with the titer of anti-p24 (FIGS. 3B to F). 
The above results show that boiling of diluted serum or plasma samples 
releases IC-bound Ag. Heat denaturation permits a quantitative measurement 
of both IC-bound and free Ag present in serum (FIGS. 1A, 2A). The Ag 
release depends on time and temperature: 5 to 7 min/100.degree. C. were 
sufficient (FIG. 1B). The use of heat-denatured standards permits 
quantification of the Ag despite the fact that absorbances are reduced in 
the presence of human serum (FIGS. 2B to D). The study of clinical samples 
showed that heat denaturation resulted in an increased presence and 
concentration of Ag among HIV antibody positives; this effect was most 
pronounced in individuals with high-titered antibody to p24 (FIG. 3). 
Acidification of serum is currently the method of choice for the disrupture 
of IC. The data show, however, that acidification is not capable of 
detecting antigen present in concentrations as high as 100 pg/ml when 
antibodies to p24 are present at very high concentration (FIGS. 1A, 3). 
Others have found a two- to four-fold increase in the rate of Ag 
positivity after acidification. With the small selection of 50 
HIV-positive samples investigated here, the effect of acidification was 
not significant (17 versus 13 positives; p=0.384; t-test). This 
discrepancy may be due to two factors: Firstly, the present samples were 
selected on the basis of anti-p24 antibody titers; this selection does not 
necessarily represent the clinical sample cohorts tested by others. 
However, in none of FIGS. 3A to F, which together represent the whole 
spectrum of HIV infection, did acid treatment result in a doubling of 
positivity (except in FIG. 3D where positivity doubled from one to two). 
Of more importance may be the fact that a low cut-off (mean+3 SD) was used 
for all testing independent of whether it was done with undenatured, 
acid-denatured, or heat-denatured samples. Others have used the 
manufacturer's cut-off (Nishanian et al., J. Inf. Dis., 162:21-28, 1990), 
not specified it (Portera et al., J. Med. Virol., 30:30-35, 1990), or have 
even used the manufacturer's cut-off for undenatured samples and a 
modified cut-off, e.g., mean+2 SD, for interpretation of acid-denatured 
samples (Miles et al., N. Engl. J. Med., 328:297-302, 1992). Such 
interpretation is much in favor of the method that uses the mean+2 SD 
cutoff, since the cut-off of, e.g., the Du Pont Ag test is calculated by 
adding 0.080, i.e., 12 to 20 SD, to the mean of three negative controls. 
The use of such a high cut-off in a population in which Ag positives are 
frequent is not reasonable. Thus, a much lower cut-off was chosen (mean+3 
SD), which will result in one to two false-positives in a test population 
of 1000 Ag-negatives. These can be easily identified with the 
neutralization test which should be used consequently. Using this cut-off, 
the detection limit of the Du Pont Ag test with undenatured sera was 1.5 
to 2 pg/ml, i.e., considerably lower than the 15.+-.3 pg indicated for the 
same test but using undiluted serum (Nishanian et al., J. Inf. Dis., 
162:21-28, 1990). The use of the mean+3 SD cut-off instead of the mean +20 
SD (corresponding to 8-16 pg/ml) increased the number of positive 
undenatured samples from 7 to 13 (FIG. 3). Had a cut-off corresponding to 
10-20 pg/ml been chosen, acidification would indeed have resulted in at 
least a doubling of positives in FIGS. 3E and F. In FIG. 3F, e.g., only 2 
to 4 of the undenatured, but 7 to 9 of the acid-denatured samples would 
have been positive and in FIG. 3E the number would have increased from 1 
to 6. In this respect, our findings are in complete accordance with those 
of others. 
The mean absorbance/cut-off ratio as well as the mean concentration of p24 
present in patients with high-titered antibodies to p24 were lower than in 
those with low-titered antibodies, independent of whether and how the 
samples were denatured (FIG. 3). Since heat denaturation is capable of 
releasing all IC-bound Ag, this finding represents a true difference in 
the amount of total Ag present at these different stages. This is in 
agreement with the results of other tests for virus that are not 
influenced by the titer of anti-HIV antibodies, such as the detection of 
virion genome by RNA-PCR. Testing with this method has indicated a lower 
average presence and concentration of virus at early stage of HIV 
infection (Zhang et al., AIDS, 5:675-681, 1991). It has been well 
established that the early stages of HIV infection are correlated with 
high-titered antibody to p24 (Schupbach et al., N. Engl. J. Med., 
312:265-270, 1985; Lange et al., Br. Med. J., 293:1489-1492, 1986; Weber 
et al., Lancet, i:119-122, 1987). It is assumed, therefore, that the sera 
shown in FIGS. 3B to D represent early stages while those in FIGS. 3E and 
F may represent late-stage infection. A different sample population has 
been tested by both heat-denatured Ag test and RNA-PCR and a good 
correlation of the two methods has been found. 
Taken together, heat denaturation of sera (i) improves the early detection 
of antigenemia and (ii) permits the quantitative assessment of total 
antigen. The first will be of benefit in the selection of patients for 
early antiviral treatment intervention and the second represents an easily 
measurable endpoint parameter in the assessment of treatment success, 
particularly in patients in whom antigen was not previously detectable. In 
addition, heat denaturation is superior to all other described methods of 
IC disruption with respect to cost, ease of handling, and assay time. A 
further advantage is that HIV or other infectious agents are instantly 
rendered uninfectious. Finally, heat destruction of antibody binding 
function also destroys the IgG binding functions of rheumatoid factors and 
thus a possible cause of false-positive results. 
Although the present investigations were restricted to the analysis of HIV 
Ag, they have a more general bearing: They show a simple way of solving 
the familiar problem of incomplete Ag detection in the presence of 
high-titered antibody. It is suggested that heat-denatured Ag be employed 
for the production of antibodies that will be used as capturing or tracing 
antibodies in Ag detection tests. Heat denaturation of samples before 
antigen testing will then become a standard procedure for antigen testing. 
While the invention has been described and illustrated herein by references 
to various specific material, procedures and examples, it is understood 
that the invention is not restricted to the particular material 
combinations of material, and procedures selected for that purpose. 
Numerous variations of such details can be implied as will be appreciated 
by those skilled in the art.