Method for determining total analyte concentration in a sample having both free and bound analyte

A method for determining total analyte concentration in a sample having both free and bound analyte is described. This method involves (a) disassociating immune complexes, (b) assaying for the concentration of free analyte, (c) assaying at least once for the concentration of free analyte in the sample wherein said sample contains a known quantity of analyte which has been added to the sample, and (d) determining total analyte concentration from the assayed concentrations obtained in steps (b) and (c).

FILED OF THE INVENTION 
This invention relates to a method for determining the quantity of analyte 
in a sample and, more particularly, to method for determining total 
analyte concentration in a sample containing both free and bound analyte. 
BACKGROUND OF THE INVENTION 
Assays and, in particular, immunoassays are widely used as diagnostic tools 
in bacterial, viral and parasitic diseases as well as infectious diseases 
such as AIDS which constitute major health problems around the world. 
Immunoassays are generally utilized for detecting and/or quantifying the 
amount of analyte in serum or other biological fluids and are based 
principally on the binding of specific binding substance, such as an 
antibody, to a particular analyte which might be present in a specimen. 
Unfortunately, detection of analyte is often hindered by the presence of 
endogenous binding substances because analyte is masked by the formation 
of endogenous binding substance-analyte complexes. For example, analytes 
such as Human Immunodeficiency Virus (HIV)-1 p24 core protein are present 
in serum, plasma, etc. bound in equilibrium to human antibody that has 
been produced as a consequence of viral infection. Masking by antibody 
(due to formation of antigen-antibody complexes) interferes with detection 
of analyte in a bound state by conventional methods such as enzyme-linked 
immunosorbent assay (ELISA) and radioimmunoassay (RIA). Consequently, only 
free analyte can be detected. However, determination of the amount of free 
analyte will be affected by a variety of factors such as the concentration 
of analyte, and the concentration and affinity of the antibody. 
The inability of current methods to detect total concentration of analyte 
creates a serious problem where measurement of total analyte is important. 
For example, measurement of HIV antigen levels in sera or plasma of 
HIV-infected individuals is important in determining the existence of 
antigen or infectious virus before seroconversion and for prognosis and is 
a useful tool for monitoring antiviral drug therapy. Diagnosis of HIV 
infection in children during the first year of life is hampered by the 
inability of standard serologic assays to diagnose HIV due to persisting 
maternally derived antibody. Thus, to understand the role of HIV in the 
process of disease development, control and prognosis, reliable 
measurement of serum antigen and viral antigen production in HIV-infected 
individuals is very important. 
Efforts to increase sensitivity and specificity of assay systems to improve 
detection and/or quantification of analyte have been undertaken as 
illustrated by the following: 
U.S. Pat. No. 4,604,348, issued to Neurath on Aug. 5, 1986, describes a 
process for detecting antibodies or antigens in a sample where the 
antigens or antibodies are present in the form of an immune complex. This 
process involves irreversibly attaching the antigen or antibody to a 
protein sorbing solid support in the presence of a dissociating buffer. 
Once antigen or antibody derived from immune complex has been adsorbed to 
the solid support, the existence thereof can be determined by RIA or 
ELISA. 
Similarly, Neurath et al., Proc. Natl. Acad. Sci. USA, 79: 4415-4419 (July 
1982), describe a solid-phase method for separating antigens from 
antibodies. Specifically, immune complexes are precipitated from serum by 
polyethylene glycol, dissociated with NaSCN, and adsorbed onto 
nitrocellulose or polystyrene supports. Detection is then effected using 
RIA. 
European Patent Application Publication No. 0 155 104 published Sep. 18, 
1985 describes a free analyte assay in which analyte is present partly 
bound to natural protein binders and in which free analyte and an analyte 
derivative compete for reaction with a specific binder. A differential 
blocking agent is used to reduce binding of the analyte derivative but 
without reducing binding to analyte to natural protein binders. 
U.S. Pat. No. 4,703,001, issued to Vodian et al. on Oct. 27, 1987, 
describes an immunoassay for detecting serum analytes using pH dependent 
chaotropic acids so that the analyte is substantially free from its 
antibody and/or other serum proteins are largely denatured. 
Nishanian et al., J. Infect. Dis., 162: 21-28 (July 1990), von Sydow et 
al., Br. Med. J., 296: 238-240 (January 1988), and Kageyama et al., J. 
Virol. Meth., 22: 125-131 (1988), describe methods to enhance detection of 
HIV-1 antigen (present in immune complexes) in serum or plasma samples by 
pretreating samples at an acidic pH to dissociate immune complexes and 
denatured antibodies with little or no compromise of antigen 
immunoreactivity. 
While these methods facilitate detection of undetectable or poorly 
detectable analytes, they do not provide for determination of total 
analyte concentration. 
SUMMARY OF THE INVENTION 
This invention relates to a method for determining total analyte 
concentration in a sample which comprises: 
(a) assaying for the concentration of free analyte in the sample; 
(b) assaying for the concentration of free analyte in the sample wherein 
said sample contains a known quantity of analyte which has been added to 
the sample, said determination being made at least once; and 
(c) determining total analyte concentration from the assayed concentrations 
obtained in steps (a) and (b). 
In another embodiment this invention concerns, a method for determining 
total HIV antigen concentration in a sample which comprises: 
(a) assaying for the concentration of free HIV antigen in the sample; 
(b) assaying for the concentration of free HIV antigen in the sample 
wherein said sample contains a known quantity of analyte which has been 
added to the sample, said determination being made at least once; and 
(c) determining total HIV antigen concentration from the assayed 
concentrations obtained in steps (a) and (b).

DETAILED DESCRIPTION OF THE INVENTION 
The method of this invention can be used to determine total analyte 
concentration in a sample which comprises: 
(a) assaying for the concentration of free analyte in the sample; 
(b) assaying for the concentration of free analyte in the sample wherein 
said sample contains a known quantity of analyte which has been added to 
the sample, said determination being made at least once; and 
(c) determining total analyte concentration from the assayed concentrations 
obtained in steps (a) and (b). 
Any conventional assay used to detect and/or quantify free analyte in a 
biological sample such as plasma, serum, urine, etc. can be used in the 
method of the invention to provide a quick and precise determination of 
total analyte concentration in a biological sample containing analyte in 
both a free and bound form. If analyte does not exist in a free form, it 
can be rendered free using conventional techniques such as those described 
above. Examples of analytes which can be evaluated using the method of the 
invention include viral proteins, bacterial proteins, hormones, drugs, 
etc. In particular there can be mentioned blood-borne infectious agents 
such as HIV, hepatitis, Epstein-Barr virus, cytomegalovirus, HTLV-1, 
HTLV-II, etc. 
An example of a commercially available assay which can be used is the Du 
Pont HIV-1 p24 Core Profile ELISA used in conjunction with the Du Pont 
HIV-1 p24 Acid Disruption Difference Immune Complex Disruption Kit which 
is designed to dissociate antigen/antibody complexes in serum and plasma 
using a combination of low pH and heat. 
An important aspect of the method of the invention to determine total 
analyte concentration involves assaying for free analyte at least twice, 
once in the absence of a spike and at least once in the presence of a 
known quantity of analyte ("spike") which has been added to the sample. As 
is shown below, use of an analyte "spike" enables determination of the 
total concentration of analyte whether free or bound. 
Generally, free analyte concentration in a sample is determined "as is" 
(without use of a spike) in an assay. The same assay is then run at least 
a second time using a spike. In other words, the sample in the second 
assay not only contains analyte "as is" but, in addition, also contains a 
known quantity of analyte ("spike") in addition to whatever analyte was 
present in the sample. At least one spike must be used in addition to 
determining analyte concentration in the absence of a spike. It is also 
possible to use three or more spikes if desired. 
Total analyte concentration in the original sample can then be determined 
from the measurements obtained by calculating the following: 
Total Analyte Concentration=Measured Unspiked Sample.times.Value of 
Spike/(Measured Spiked Sample-Measured Unspiked Sample). 
If two spikes were used (e.g. one at a high level and one at a low level), 
then the calculation for total analyte concentration would be: Measured 
Unspiked Sample.times.(Value of High Spike-Low Spike)/(Measured High 
Spiked Sample-Measured Low Spiked Sample). 
If more than two spikes are used, then these equations can be modified as 
needed depending upon the number of spikes used. 
The following example illustrates the practice of the invention but should 
not be construed as a limitation thereon. 
EXAMPLE 1 
Part A: Preparation of Antigen and Antibody Positive Reference Samples 
Each of twenty-two antigen and antibody positive reference samples having 
HIV-1 p24 antigen at a concentration of 194 pg/ml were prepared by mixing 
44 .mu.l of an HIV-1 antibody positive, antigen negative serum/plasma with 
375 .mu.l of normal human serum/plasma and 45 .mu.l of HIV-1 viral lysate 
containing 2000 pg/ml p24 antigen. 
Part B: Sample Evaluation 
Each of the twenty-two reference samples was assayed in the absence and 
presence of at least one spike. In this evaluation two different spike 
levels, approximately 200 and 400 pg/ml were used. The Du Pont HIV-1 p24 
Core Profile ELISA antigen assay in conjunction with the Du Pont HIV-1 p24 
Acid Disruption Difference (ADD) kit described above were used to assay 
the samples. The ADD kit was used to disrupt antigen/antibody complexes in 
serum and plasma using a combination of low pH and heat. Samples were 
acidified with 1.5M glycine reagent, pH 1.85 and were incubated at 
37.degree. C. for one hour. After one hour, the samples were neutralized 
with 1.5M TRIS, pH 11 and were assayed in the Du Pont HIV-1 p24 Core 
Profile ELISA. 
The Du Pont HIV-1 p24 Core Profile ELISA antigen assay utilizes an anti-HIV 
p24 mouse monoclonal antibody which is immobilized to microtiter plate 
wells. The immobilized monoclonal antibody then captures HIV-1 p24 antigen 
released upon lysis of virus in the samples. The captured antigen is 
complexed with biotinylated polyclonal antibodies to HIV-1 p24 core 
antigen and probed with a streptavidin-HRP (horseradish peroxidase) 
conjugate. The complex is then detected by incubation with 
orthophenylenediamine--HCl (OPD) which produces a yellow color that is 
directly proportional to the amount of HIV-1 p24 core antigen captured. 
The absorbance of each well is determined using a microplate reader and 
calibrated against the absorbance of an HIV-1 p24 core antigen standard 
curve. 
1. Reagents 
Glycine Reagent 
This is used to acidify sample to about pH 2.0. The Glycine Reagent is 1.5M 
glycine, pH 1.85 
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% Comp 
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Glycine 11.3% 
HCl 9.6% 
Distilled Water 79.1% 
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Tris Reagent 
This is 1.5M Tris, pH 11 which is used to neutralize samples after 
disruption. 
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% Comp 
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Tris Base 17.0% 
Distilled Water 83.0% 
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2. Sample Preparation 
The following were added to the wells of a blank microtiter plate: 
a) 10 .mu.l of 5% Triton X-100, 
b) 90 .mu.l of sample, 
c) 10 .mu.l of either negative serum (unspiked), negative serum spiked with 
approximately 2000 pg/ml HIV-1 p24 antigen (low spike) or negative serum 
spiked with approximately 4000 pg/ml HIV-1 p24 antigen (high spike). 
It should be noted that the actual values of the spike levels were assayed 
by substituting 90 .mu.l of negative serum for the 90 .mu.l of sample. 
3. Assay 
90 .mu.l of 1.5M glycine, pH 1.85, and 90 .mu.l of sample or standard were 
incubated for one hour at 37.degree. C. After one hour, 90 .mu.l of 1.5M 
Tris pH 11 was added to each well and incubated for ten minutes at room 
temperature. This sample mixture (150 .mu.l ) was then transferred to an 
HIV-1 p24 Core Profile ELISA plate and incubated for 2 hours at 37.degree. 
C. The microtiter plate wells were then washed and 100 .mu.l biotinylated 
detector antibody was added to each well and incubated for one hour at 
37.degree. C. The wells were then washed and streptavidin-HRP conjugate at 
a 1:25 dilution was added, incubated for 15 minutes at 37.degree. C. and 
the wells were then washed again. OPD substrate was added for thirty 
minutes at room temperature, the reaction was stopped with kit stop 
solution (4N sulfuric acid) and the absorbance at 490-650 nm was 
determined. 
4. Determinations 
Values for all samples were determined by interpolation from an HIV-1 p24 
core antigen standard curve. The actual values of the spikes were 
determined to be 228 pg/ml (low spike) and 448 pg/ml (high spike) for 
samples 1-11, and 234 pg/ml (low spike) and 438 pg/ml (high spike) for 
samples 12-22. As was noted above the actual values of the spike levels 
were assayed by substituting 90 .mu.l of negative serum for the 90 .mu.l 
of sample. 
Total analyte concentration was determined using each of the following 
combinations: 
a) unspiked sample and low spike; 
b) unspiked sample and high spike; and 
c) unspiked sample and both low and high spikes. 
Determination of Total Antigen Concentration for a single spike was made as 
follows: 
Measured Unspiked Sample.times.Value of Spike/(Measured Spiked 
Sample-Measured Unspiked Sample). 
Determination of Total Antigen Concentration for both spikes combined was 
made as follows: 
Measured Unspiked Sample.times.(Value of High Spike--Low Spike)/(Measured 
High Spiked Sample-Measured Low Spiked Sample) 
Total Antigen Concentration Determinations for Sample 1 using data provided 
in Table 1 and actual spike values provided above are set forth below. The 
same determinations were made for the remaining twenty-one reference 
samples. 
(a) unspiked sample and low spike 
##EQU1## 
(b) unspiked sample and high spike 
##EQU2## 
(c) unspiked sample and both spikes 
##EQU3## 
The results of these calculations are summarized for all 22 samples in 
Table 2 below. 
5. Results 
Results are presented in Tables 1 and 2 below. The data presented in Table 
1 is the measured HIV-1 p24 antigen level for each sample run in the assay 
in the absence and presence of both spikes, a low spike and a high spike. 
The data presented in Table 2 is the total antigen concentration determined 
from the data presented in Table 1 and actual values of each spike which 
were determined as discussed above. A comparison is also set forth between 
what was actually measured versus total antigen concentration determined. 
These data show that the presence of an endogenous binding substance, such 
as an antibody, yields highly inaccurate values for many of the samples 
when measuring antigen directly in an assay. 
The method of the invention overcomes this problem by providing a means to 
more accurately and precisely determine total analyte concentration in a 
sample containing analyte in a free and bound form. 
TABLE 1 
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Measurement of HIV-1 p24 Antigen 
In Unspiked and Spiked Samples 
HIV-1 p24 Antigen, pg/ml 
Measured Measured Low 
Measured High 
Sample Unspiked Sample 
Spiked Sample 
Spiked Sample 
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1 50 111 162 
2 35 81 126 
3 79 173 250 
4 32 85 127 
5 57 120 189 
6 49 98 146 
7 46 123 204 
8 175 372 568 
9 124 288 443 
10 175 387 568 
11 215 429 660 
12 192 391 588 
13 202 415 618 
14 164 330 465 
15 146 331 479 
16 183 403 564 
17 137 286 426 
18 185 396 576 
19 205 400 657 
20 199 430 639 
21 197 392 600 
22 173 347 540 
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TABLE 2 
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Comparison of Measured Unspiked and Determination 
of HIV-1 p24 Antigen in Samples Containing 194 pg/ml 
Using the Method of the Invention 
Measured Total HIV-1 p24 Antigen Determined 
Using the Method of the Invention 
Both Spikes 
Sample Unspiked Low Spiked High Spiked 
Combined 
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1 50 187 200 216 
2 35 173 172 171 
3 79 192 207 226 
4 32 138 151 168 
5 57 206 193 182 
6 49 228 226 225 
7 46 136 130 125 
8 175 203 199 196 
9 124 172 174 176 
10 175 188 199 213 
11 215 229 216 205 
12 192 226 212 199 
13 202 222 213 203 
14 164 231 239 249 
15 146 185 192 201 
16 183 195 210 232 
17 137 215 208 200 
18 185 205 207 210 
19 205 246 199 167 
20 199 202 198 194 
21 197 236 214 193 
22 173 233 206 183 
Avg. 137 202 198 197 
S.D. 65 30 24 27 
C.V. (%) 
48 15 12 14 
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