Therapeutic and diagnostic methods using total leukocyte surface antigens

The invention relates to the measurement of total leukocyte antigens, or fragments thereof, and the use of such measurements to enumerate cells, especially in whole blood. The term "total" leukocyte antigen used herein refers to the total amount of a leukocyte antigen in a sample, including that present in membrane and intracellular compartments and extracellular soluble compartments. Measurements of a total leukocyte antigen can be used to type cells, detect or diagnose disease or to monitor disease therapy. In a further embodiment, the invention relates to the measurement of both the amount of total leukocyte antigen and the amount of the soluble form of the leukocyte antigen and a comparison of the measured levels.

1. INTRODUCTION 
The present invention is directed to the measurement of total leukocyte 
surface antigens, such as T cell receptors and T cell differentiation 
antigens or fragments thereof, and the application of such measurements in 
the diagnosis and therapy of diseases and disorders. The present invention 
is also directed to the measurement of soluble leukocyte antigens or 
fragments thereof, in conjunction with the measurement of total leukocyte 
surface antigens and the application of such dual measurements in the 
diagnosis and therapy of diseases and disorders. The measurement of such 
molecules, and preferably a plurality of such molecules, can be used in 
monitoring the effect of a therapeutic treatment, detecting and/or 
diagnosing disease. 
2. BACKGROUND OF THE INVENTION 
2.1. LEUKOCYTE SURFACE MOLECULES 
Clusters of differentiation (CD) have been established which define human 
leukocyte differentiation antigens (Bernard and Boumsell, 1984, Hum. 
Immunol. 11:1-10; Knapp et al., 1989, Immunol. Today 10:253:258; Gebel et 
al., 1988, ASHI Quarterly 12:11; Leukocyte Typing III: White Cell 
Differentiation Antigens. Ed., McMichael, A. J. 1987. Oxford University 
Press. Oxford), by the comparison of reactivities of monoclonal antibodies 
directed against the differentiation antigens. The T cell surface 
antigens, their classification into epitope-defined subgroups, and their 
distributions on T cells have been studied by use of monoclonal antibodies 
directed against human T cells (Clark et al., 1983, Immunogenetics 
18:599-615; Hansen et al., 1984, in Leucocyte Typing, Bernard, A., et al., 
eds., Springer-Verlag, New York, pp. 195-212). Some of the T cell clusters 
of differentiation and other cell surface molecules are listed in Table I. 
These cell surface markers serve as markers of cell lineage, the identity 
of the functional T cell subset to which the T cell belongs, and the 
activation state of the T cell. Several of the cell surface molecules have 
been studied in great detail, have been found to be important in 
initiating and regulating immune functions and are critical to 
communication processes between immune cells. 
TABLE I 
______________________________________ 
LEUKOCYTE SURFACE MOLECULES 
Cell Detection 
Surface Monoclonal Refer- 
Marker Expression Antibodies ences 
______________________________________ 
T cell Antigen 
All T cells and 
T40/25, 1, 2, 3, 4, 
Receptor T cell subsets 
.alpha.F1, .beta.F1, 
5, 6 
.delta.TCS1, 
TCR.delta.1,C.gamma.M1 
CD1 Thymocytes & OKT6 
Langerhans NAI/34 
Calls, Lukemia 
Cells 
NK cell NK cells NC-37 7 
receptor specific 
antibodies 
Cell Adhesion 
Molecules 
CD2 All T cells OKT11 8, 9, 10 
Leu5 
B67.1 
CD58 (LFA-3) 
Leukocytes, TS2/9 11 
epithelial 
CD3 Pan T cell OKT3 12 
Leu4 
CD4 Helper/Inducer 
OKT4 12 
Subsets of T Leu3a 
cells 
CD5 T.B subsets UCHT2 11 
T1 
CD7 T Cells 3Al 11 
CD8 Supressor/ .alpha. Chain: 
13, 14 
Cytotoxic OKT8 
Subsets of T Leu2a 
cells .beta. Chain: 
14a 
T8/2T8 
.beta.2 integrins 
LeuCAM leukocyte cell 11 
adhesion 
molecules 
CD11a (LFA-1) 
myeloid, 11, 15 
lymphoid 
CD11b myeloid 11, 15, 
(MAC-1 (CR3)) 16, 17, 
18, 19 
CD11c (CR4) 
myeloid 11, 15 
CD16 (FcR111) 
Natural Killer, 
HUNK2 11 
Macrophages 3G8 
Granulocytes 
CD21 (CR2) B subset B2 11, 20 
HB5 
CD23 (FC.epsilon.R11) 
B subset MHM6 11 
Blast-2 
CD25 TAC IL-2 Anti-TAC 21 
Receptor 7G7/B6 
(Activated T 
Cells) 
CD30 Activated T, B 
Ki-1 11 
Cells HSR4 
Reed-Steinberg 
Cells 
CD35 (CR1) Granulocytes, B 
YZ-1 11, 22 
cells, monocytes 
J3D3 
.beta.3 integrins 
CD41 11, 23 
CD51 11, 23 
Homing Leukocytes, 33-3B3 24 
Receptors brain GRHL1 25, 26 
CD44 
Mel-14 
.beta.1 integrins 
CD49a-f(VLA-1) 
Extra cellular 27, 23 
VLA-2, VLA-3, 
Matrix (ECM) 
VLA-4 
CD56 Natural Killer 
NKH1 11 
(NKH1, NCAM) 
Activated Leu19 
lymphocytes 
CD71 Transferring 11, 28 
Receptor, 
Proliferating 
cells 
______________________________________ 
List of References for Table I 
1 Brenner et al., 1984, J. Exp. Med. 160:541-551. 
2 Henry et al., 1989, Hybridoma, 8:577. 
3 Brenner et al., 1987, J. Immunol., 138:1502. 
4 Wu et al., 1988, J. Immunol., 141:1476. 
5 Band et al., 1987, Science 238:682. 
6 Hochstenbach et al., 1988, J. Exp. Med. 168:761. 
7 Evans, International patent publications #W089/03394, #W088/03395, & 
#W088/03396 published April 20, 1989 
8 Bierer et al., 1989, Annu. Rev. Immunol. 7:579-99. 
9 Verbi et al., 1982, Eur. J. Immunol. 12:81-86. 
10 Perussia et al., 1983, J. Immunol. 133:180. 
11 Knapp et al., 1989, Immunol. Today 10:253. 
12 Kung et al., 1979, Science 206:347-349. 
13 Reinherz et al., 1979, Proc. Natl. Acad Sci. USA 76:4061-4065. 
14 Ledbetter et al., 1981, Monoclonal Antibodies and Tcell Hybridoma, 
Elsevier, North Holland, N.Y. pp 16-22. 
14a Shieu, L., et al., 1988, J. Exp. Med. 168(6):1993-2005. 
15 Kishimoto et al., 1989, Adv. Immunol. 46:149-182. 
16 Altieri & Edgington, 1988, J. Biol. Chem 263:7007-1 
17 Wright et al., 1988, Proc. Natl..Acad. Sci. USA 85:7734-38. 
18 Wright et al., 1989, J. Exp. Med. 169:175-83. 
19 Russell & Wright, 1988, J. Exp. Med. 168:279-92. 
20 Nemerow et al., 1985, J. Virol. 55:347-351. 
21 Uchiyawa et al., 1981, J. Immunol. 126(4):1393-1397. 
22 Klickstein et al., J. Exp. Med. 165:1095-1112. 
23 Hemler, 1990, Annu. Rev. Immunol. 8:365-400. 
24 Berg et al., 1989, Immunol. Rev. 108:1-18. 
25 Lasky et al., 1989, Cell 56:1045-55. 
26 Haynes et al., 1989, Immunol. Today 10:423. 
27. Hynes, 1987, Cell 48:549. 
28. Reinherz et al., 1980, Proc. Natl. Acad. Sci. USA, 77:1588-1592. 
2.2. LEUKOCYTE SURFACE MOLECULE SPECIFIC ANTIBODIES 
Over the last several years, antibodies to determinants of murine and human 
TCRs have been developed. Some of these antibodies appear to recognize all 
members of a V region family, some a subset of V regions within a family, 
and some a particular V region only (TABLE II). These TCR antibodies 
identify minor populations of peripheral blood T cells (1-5%) and 
subdivide T cells in a new way based on TCR V region usage. 
TABLE II 
______________________________________ 
Name Clone Specificity 
Immunogen 
Reactivity 
Ref* 
______________________________________ 
.beta.V5(a) 
1C1 V.beta.5.2 and 
HPB 1-5% 1 
V.beta.5.3 of PBL 
Subfamilies 
.beta.V5(b) 
W112 V.beta.5.3 HPB 0-3% 2 
Subfamily of PBL 
subset of 
.beta.V5(a) 
.beta.V8(a) 
16G8 V.beta.8 family 
JURKAT 1-5% 2 
of PBL 
.beta.V12(a) 
S511 V.beta.12 family 
SEZARY 1-5% 3 
of PBL 
.beta.V6(a) 
OT145 V.beta.6 family 
T-CLL 0-5% 4 
Allotypic of PBL 
V.beta.6.7 
epitope 
.alpha.V2(a) 
F1 V.alpha.2 T-CLL 1-5% 5 
Subfamily of PBL 
.alpha..beta.V(a) 
LC4 V.beta.5.1 SUP-T13 1-5% 6 
Subfamily of PBL 
______________________________________ 
List of References for Table II 
1 Boylston et al., 1986, J. Immunol. 137:741-744. 
2 Tian et al., 1989, FASEB J. 3:A486 Abstr. 
3 Bigler et al., 1983, J. Exp. Med. 158:1000-1005. 
4 Posnett et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7888-7892. 
5 Janson et al., 1989, Canc. Immunol. Immunother. 28:225-232. 
6 Maecker & Levy, 1989, 1989 J. Immunol. 142:1395-1404. 
These reagents have proven valuable in studying the repertoire of TCRs 
expressed under many in vivo and in vitro conditions. For example, in 
vitro stimulation of mononuclear cells with certain bacterial enterotoxins 
(superantigens) leads to the expansion of T cells expressing a limited 
number of TCR V.beta. families (Kappler et al., 1989, Science 
244:811-813). In vivo expansion of T cells bearing particular V.beta. 
regions has been detected in certain animal models of autoimmune diseases. 
Other antibodies recognize common determinants on all .alpha..beta. or 
.gamma..delta. TCRs. These include C.gamma.M1, a monoclonal antibody which 
has been shown to specifically recognize the TCR-.gamma. protein 
(Hochstenbach et al., 1988, J. Exp. Med. 168:761). This monoclonal 
antibody was generated against a constant-region encoded peptide and 
reacts with both C.gamma.1 and C.gamma.2 encoded TCR-.gamma. chains. It 
appears to possess framework reactivity against all TCR-.gamma. 
polypeptides. 
TCR.delta.1 (Band et al., 1987, Science 238:682) and .delta.TCS1 (Wu et 
al., 1988, J. Immunol. 141:1476) are monoclonal antibodies specific for 
the .delta. chain of the human .delta..gamma. TCR. Unlike clone specific 
or idiotypic anti-TCR antibodies, TCR.delta.1 appears to identify all T 
cells which express the .gamma..delta. TCR. .delta.TCS1 identifies a minor 
subset of these .gamma..delta. T cells. 
Additional monoclonal antibodies to the T cell receptor gamma and delta 
chains have also been reported (European Patent Publication #EP289252 
Published Nov. 2, 1988; International Patent Publication #WO88/00209 
published Jan. 14, 1988). 
.beta.FI (Brenner et al., 1987, J. Immunol. 138:1502-1509) is a murine 
monoclonal antibody specific for a framework, i.e., common or 
nonpolymorphic determinant on the .beta. chain of the .alpha..beta. TCR 
and identifies all T cells expressing the .alpha..beta. TCR. .alpha.F1 
(Henry et al., 1989, Hybridoma 8:577) is a monoclonal antibody specific 
for a framework determinant of the .alpha. chain and identifies all T 
cells expressing the .alpha..beta. TCR. 
Antibodies to CD4 have been widely described (Kung, P. C., et al., 1979, 
Science 206:347-349) and are commercially available. A series of such 
antibodies reacting with non-competing epitopes on the CD4 molecule have 
been described. Such a set has been termed OKT4, OKT4A, OKT4B, OKT4C, 
OKT4D, OKT4E, and OKT4F (Rao, P. E., et al., 1983, Cell. Immunol. 80:310). 
Antibodies directed against the CD4 or CD8 antigens have been shown to 
block cell function. Antibodies against CD4 block most helper T functions, 
mixed lymphocyte reactions and induction of T helper activity (Biddison et 
al., 1984, J. Exp. Med. 159:783). Antibodies against CD8 block the 
cytotoxic activity of CD8 positive cytotoxic T lymphocytes (Swain, S. L., 
1981, Proc. Natl. Acad. Sci. U.S.A. 78:7101-7105). Antibodies against CD4 
have also been described that are capable of activating CD4-positive T 
cells. CD4 is internalized upon treatment of the cells with phorbol esters 
and resulting phosphorylation (Hoxie, J. A., et al., 1986, J. Immunol. 
137:1194-1201). 
Antibodies to CD35 (Wong et al., 1985, J. Immunol. Methods 82:303; Yoon & 
Fearon, 1985, 134:3332; Schrieber, U.S. Pat. No. 4,672,044 issued Jun. 9, 
1987) have also been reported. 
2.3. CLINICAL APPLICATIONS 
These various lymphocyte cell surface markers have enormous clinical 
application potentials for the identification of lymphocyte populations 
and their functional status (Krensky, A. M. and Clayberger, C., 1985, 
Transplant. 39 (4):339-348; Kung, P. C., et al., 1984, Monoclonal 
Antibodies in Clinical Investigations, Clinical Biochemistry-Contemporary 
Theories and Techniques, Vol. 3, Academic Press, pp. 89-115; Kung, P. C., 
et al., 1983, Int. J. Dermatol. 22.(2):67-733; Cosimi et al., 1981, N. 
Engl. J. of Med. 305:308; Knowles et al., 1983, Diagnostic Immunol. 1:142; 
Hoffman, 1984, Amer. Biotechnol. Lab 2:39). 
Existing clinical methods of T cell typing involve the use of monoclonal 
antibodies which define T cell surface markers to detect the presence of 
specific cell surface markers on the T cell surface. Measuring the total 
numbers of T cells expressing a marker on the surface or membrane has been 
useful for the characterization and classification of lymphoid 
malignancies (Greaves, M., et al., 1981, Int. J. Immunopharmac. 3(3): 
283-300). Changes in the relative percentage of T helper and T 
suppressor/cytotoxic cells were found to be associated with immune events 
in renal transplantation due to viral infection (Colvin, R. B., et al., 
1981, Proc. 8th Int. Congr. Nephrol., Athens, pp. 990-996), autoimmune 
diseases (Veys, E. M., et al., 1981, Int. J. Immunopharmac. 3(3):313-319), 
and AIDS (Gupta, S., 1986, Clin. Immunol. Immunopathal. 38:93-100; Ebert, 
E. C., et al., 1985, Clin. Immunol. Immunopathol. 37-283-297). 
The expression of T cell surface markers has also been used for the 
assessment of the immune status of patients. It has been established that 
by measuring the relative number of distinct, functional T cell subsets, 
and/or the relative number of activated T cells in peripheral blood or 
tissues, an assessment of the immunological condition of a patient is 
possible. 
In an HIV-infected individual, the most useful single prognostic indicator 
for progression to overt AIDS is the absolute number of CD4.sup.+ T 
cells/mm.sup.3 of whole blood. Currently, accurate measurement of 
CD4.sup.+ T cells requires the use of a flow cytometer, both costly and 
not widely available. In addition, federal guidelines define eligibility 
for AZT administration to AIDS infected individuals solely on the basis of 
the CD4.sup.+ T cell count (a T cell count which drops below 500 CD4.sup.+ 
T cells/mm.sup.3) regardless of symptomology. The cell count cut off for 
pentamidine treatment is 200 cells/mm.sup.3. However, the costly and 
largely unavailable flow cytometer leaves a large population of clinicians 
and patients without the means of proper immune status evaluation and 
therefore AZT treatment. 
If the CD4.sup.+ T cell count drops below 500 cells/mm.sup.3 (AZT 
treatment) or below 200 cells/mm.sup.3 (pentamidine treatment) (Cowley et 
al, Jun. 25, 1990, Newsweek, pp. 23-27; Mills & Masur, August, 1990, 
Scientific American, pp. 50-57; Fahey et al., 1990, New Engl. J. Med. 
322:166-72; Goedert et al., 1989, New Engl. J. Med. 321:1141-8), then the 
patient is indicated for therapeutic intervention. 
The measurement of CD4 positive or CD8 positive T cells may be used to 
detect or diagnose disease, or monitor disease treatment as described in 
patents and publications U.S. Pat. Nos. 4,709,015, 4,725,543, 4,361,550, 
4,908,203, WO91/10722, WO89/08143, WO91/07985, EP 421,380, WO91/09966, 
WO91/03493, EP 403,935, WO88/04327, U.S. Pat. Nos. 4,695,459, and 
4,649,106. 
2.4. METHODS OF MEASURING LEUKOCYTE SURFACE MOLECULES IN A SAMPLE 
The methods for detecting, staging or diagnosing a disease, or monitoring 
the progress of therapy of a disease discussed above require separation of 
the components of a sample. 
For example, methods of diagnosis or monitoring of a patient with a disease 
may depend on detection of the amount of a soluble leukocyte antigen, and 
comparison of the amount of soluble leukocyte antigen in the sample from a 
patient to the amount in a sample from a normal individual or in the same 
individual at an earlier time. See, e.g., publications WO 87/05912, and WO 
90/04180. However, the assay sample must be isolated from cells and 
cellular debris, which is time consuming. 
Prior to the instant invention, measurement of the amount of leukocytes 
positive for a leukocyte marker was carried out by direct analysis of 
cells. To date, investigators have primarily measured the amount of cell 
surface markers in enriched cell populations derived from whole blood. 
This involved separating whole blood into its serum (or plasma) and cell 
constituents followed by enrichment of the desired cells by procedures 
such as those involving the lysis of red blood cells and subsequent 
isolation of white blood cells on density gradients of polymers, such as 
Ficoll-Hypaque. The enriched cell populations can then be analyzed either 
by direct or indirect immunofluorescence involving flow cytometers or 
fluorescent microscopes, or alternatively lysed with appropriate buffers 
followed by analysis of either the total lysate or membrane and cytosolic 
components individually. Limitations of these procedures include (1) the 
requirement for fresh samples, (2) the need to use enriched cell 
populations rather than whole blood, (3) the requirements of expensive 
equipment, (4) the time involved in preparing the samples and (5) the need 
for fairly large sample sizes or cell numbers for analysis, since cells 
are lost during the sample preparation steps and because flow cytometric 
analyses require that a statistically significant number of cells be 
analyzed for reliable measurements to be obtained. In diseases where the 
cells of interest are steadily declining, (for example, the decrease in 
the number of CD4 cells during HIV infection and progression to AIDS) 
larger sample volumes must be used in order to obtain a significant number 
of cells to analyze in the enriched cell population. 
3. SUMMARY OF THE INVENTION 
The present invention overcomes the deficiencies of the prior methods of 
detecting leukocyte antigens and of estimating the number of cells 
positive for a particular leukocyte marker by providing a reliable, 
quantitative, easy-to-use method to measure total amount of a leukocyte 
marker in a sample containing cells in the biological fluid in which the 
cells are obtained. Alternately, the sample can contain just cells. 
Surprisingly, it has been discovered that treatment of cells in a sample 
with concentrated detergent followed by dilution of the sample solubilizes 
the total amount of leukocyte markers in the sample. Thus, the method for 
determining the total amount of a leukocyte marker in a sample comprises 
adding a concentrated non-ionic detergent solution to the sample to form a 
detergent-treated sample, and allowing the detergent to lyse the cells, 
and/or disrupt membranes. Having released any intracellular and/or 
membrane bound marker in this manner, the total amount (i.e., inclusive of 
marker so released as well as any soluble marker that may be present 
originally in the sample) of leukocyte marker in the sample can be 
detected. After the cells have been lysed, the sample is diluted to reduce 
the detergent concentration. Detection of the amount of leukocyte marker 
in the sample is by immunological detection means, e.g., immunoassay. 
Remarkably, measurements of total leukocyte markers can be used to 
determine the approximate number of leukocytes positive for the leukocyte 
marker, in a sample i.e., measurement of total leukocyte marker can be 
used to enumerate cells expressing that antigen with an appropriate 
correlation. Even more remarkable is the linearity of the correlation 
between the measurements obtained according to the method of the invention 
and cell counts achieved by conventional means. Hence, the instant 
invention represents a highly advantageous substitute for the more 
cumbersome methods of the prior art. 
Measurements of total leukocyte markers are useful in monitoring the 
effectiveness of a treatment of a subject, in predicting therapeutic 
outcome or disease prognosis, and in evaluating and monitoring the immune 
status of patients. Measurements of total leukocyte markers can be 
accomplished by sandwich enzyme immunoassays where the samples are treated 
so that the total amount of a leukocyte marker present in membrane, 
intracytoplasmic and soluble compartments can be measured. 
In particular embodiments, the invention is directed to the measurement of 
amounts of total CD4 antigen. Total CD4 antigen measurements are 
particularly useful in diseases where the absolute number of CD4.sup.+ 
cells is the best indicator of disease prognosis or treatment outcome. 
Such diseases include, but are not limited to, AIDS. 
In another embodiment, the invention directed to the measurement of the 
total amount of CD8 antigen. Total CD8 antigen measurements are especially 
useful in diseases associated with modulation of the CD8.sup.+ subset of 
leukocytes. These include but are not limited to infectious disease, 
autoimmune disease, and transplantation rejection illness. 
In yet another embodiment, the invention is directed to the measurement of 
the total amount of T cell antigen receptor (TCR) present in a sample. 
Total TCR measurements can include the measurement of total TCR on all 
cells expressing any .alpha..beta. or .gamma..delta. TCR or can include 
the measurement of total TCR on particular subsets of cells including, but 
not limited to, subsets expressing specific V.alpha., V.beta., V.gamma. 
and/or V.delta. peptides. 
In a further embodiment, the invention is directed to the measurement of 
both the amount of total leukocyte marker and the amount of the same 
soluble leukocyte marker and a comparison of the measured levels. The 
change in the total levels and soluble levels relative to one another 
during disease progression or disease treatment can be superior to the 
measurement of either total or soluble levels alone. Such measurements are 
useful for the detection, diagnosis or monitoring of treatment of a 
disease or disorder. 
In another embodiment, the invention provides for the measurement of the 
total amount of two or more leukocyte markers, and comparison of the 
amounts of the markers. The relative amounts of the markers, or time 
dependent variation in the relative amounts of the markers during disease 
progression, can be used to detect, diagnose, or monitor treatment or 
progress of a disease or disorder. Significantly, the measurement of total 
leukocyte markers can be used to estimate the number of cells positive for 
each marker. The ratios of total leukocyte marker or approximate numbers 
of cells positive for each leukocyte marker can be compared with the same 
ratios in a normal individual, and this comparison used to detect, 
diagnose, or stage a disease or monitor treatment of a disease or 
disorder. 
Simultaneous measurement of the total amount of a marker is an improvement 
over separately measuring the cell bound, cell lysate or soluble marker 
for the following reasons. Firstly, the measurement can include the total 
amount of markers present in all three compartments, not just the amounts 
present in one or two compartments. Secondly, the measurement of total 
markers is easier than other procedures that involve greater sample 
preparation, complex equipment and more steps. Thirdly, small quantities 
of sample, e.g., 100 .mu.l, and as little as 5-10 .mu.l of whole blood, 
can be directly analyzed in a simple immunoassay format without prior 
enrichment of the samples. The small volume of whole blood necessary has 
major benefits in pediatric applications with infants and small children. 
This represents a significant cost reduction per sample analyzed, and the 
elimination of an expensive equipment requirement, thereby making the 
analysis widely available to many laboratories or clinics. Fourthly, the 
measurement of total marker is an improvement, since it involves minimum 
sample preparation and does not create aerosols that are hazardous in the 
case of infectious samples from patients. Most important, for infectious 
samples such as those containing HIV, the solubilization procedure, i.e., 
treatment with concentrated detergent, inactivates the virus, thereby 
making subsequent analysis safer. Fifthly, the total marker assay does not 
require fresh samples. Each patient sample can be treated and stored 
frozen. This is especially useful for a series of samples obtained from 
the same patient over a period of time as in a longitudinal study. Each 
sample can quickly be treated and frozen so that all samples can be thawed 
and analyzed simultaneously. This is a definite improvement over flow 
cytometric analysis where the cells need to be fresh and intact. It also 
eliminates variance obtained in assay results that arise from interassay 
variability, since all of the assays may be performed at one time. 
3.1. DEFINITIONS 
As used herein, the following terms will have the meanings indicated: 
______________________________________ 
Total Leukocyte = the total amount of a 
Marker leukocyte antigen or 
fragment thereof 
(including 
membrane-associated, 
intra- and extracellular) 
present in a sample. 
WBCC = White blood cell count 
AZT = azido-deoxthymidine 
HTLV III/LAV/HIV = 
Human T cell Leukemia 
Virus Type 
I/Lymphadenopathy 
Associated Virus/Human 
Immunodeficiency Virus 
OPD = O-phenylenediamine 
mAb = monoclonal antibody 
Spontaneous release = 
release by normal or 
pathologic physiological 
processes of the cell 
AIDS = Acquired immunodeficiency 
disease syndrome 
TCAR = T cell antigen receptor 
______________________________________

5. DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to the measurement of total leukocyte 
surface antigens, total T cell differentiation antigens, or related total 
markers or fragments thereof, and the use of such measurements in the 
diagnosis and therapy of diseases and disorders. 
The measurement of total markers according to the invention is valuable in 
cell enumeration, cell typing, monitoring the effect of a therapeutic 
treatment on a subject, detecting and/or diagnosing a disease in a 
subject, in predicting therapeutic outcome or disease prognosis and in 
evaluating and monitoring immune status of patients. A plurality of total 
markers can be measured, such as total CD4 antigen and total CD8 antigen. 
In a preferred embodiment in relation to AIDS, assays are configured such 
that total CD4 and total CD8 antigens can be measured simultaneously. 
As used herein, the term "total" shall mean the total amount of the marker 
present in the sample. For example, in a particular sample such as a 
sample that comprises whole blood, the total marker includes the amount of 
marker present in the cell membrane, intracytoplasmic and soluble serum 
compartments. The soluble compartment can include both spontaneously 
released soluble marker as well as soluble recombinant markers that may 
have been administered as a therapeutic treatment. In another sample, such 
as in a sample that comprises cells in tissue culture, the total marker 
includes the amount of marker present in the membrane, intracytoplasmic 
and cell culture media compartments of the sample. In yet another 
embodiment in which the sample comprises a tissue, e.g., a biopsy 
specimen, the total marker includes the amount of marker present in the 
membrane, intracytoplasmic and interstitial compartments. 
As used herein, the term "compartment" modified by membrane, 
intracytoplasmic, and soluble elements of a sample refers to the total 
amount of all membrane, the total amount of intracytoplasmic contents, and 
the total amount of soluble material included in the sample. 
As used herein, the term "leukocyte marker" refers to an antigen or 
polypeptide found on the cell surface of a leukocyte. Such markers include 
but are not limited to cell surface antigens, CD (clusters of 
differentiation) antigens, receptors, or other cell surface polypeptides 
or proteins. The term leukocyte marker is further intended to include an 
immunologicaly active fragment, e.g., an epitope, of the foregoing 
molecules. The leukocyte markers for detection according to the present 
invention, and the particular cells known to express these markers, are 
summarized in Table I in Section 2.1, supra. In specific embodiments, the 
leukocyte marker can be CD4, CD8 or T cell antigen receptor (TCAR). 
As used herein, the term "leukocyte" refers to the usual meaning of that 
terms, i.e., immune associated cells derived from hematopoietic stem 
cells, such as lymphocytes (B cells and T cells) and myeloid lineage cells 
(neutrophils, macrophages, eosinophils, megakaryocytes, erythrocytes, mast 
cells). 
As used herein, a "sample" refers to a collection of cells in the milieux 
in which they were obtained, i.e., a biological fluid, or to membrane 
and/or intracytoplasmic components of the cells. Total leukocyte antigens 
may be measured in samples derived from a biological fluid, e.g., whole 
blood, plasma, serum, blood cells, saliva, urine, synovial fluid, pleural 
effusions, tumor and tissue infiltrates, amniotic fluid, spinal fluid or 
cranial fluid. In another embodiment, the biological fluid may be cell 
culture fluid. The sample can comprise tissue, including interstitial 
fluid. Preferably when the sample is a tissue sample, the tissue is 
treated to disrupt the connective tissue matrix, e.g., by trypsin 
digestion or homogenization. In another embodiment, the sample comprises 
cells derived from the foregoing sources. 
5.1. DETECTION AND MEASUREMENT OF TOTAL LEUKOCYTE MARKER 
The total amount of a leukocyte marker exists in three compartments: the 
membrane, intracytoplasmic and released/soluble compartments ("total 
leukocyte marker"). The invention includes immunoassays that 
simultaneously measure the total amount of a leukocyte marker present in 
all three compartments or in the membrane and/or intracytoplasmis 
compartment, depending upon how the sample is prepared. To determine the 
total amount of a leukocyte marker, an "original" sample, such as whole 
blood or blood cells, is first treated to solubilize the cellular 
components (step 1). The preferred method of solubilizing the cells in the 
sample without interfering with immunospecific binding is to treat the 
cells with a concentrated non-ionic detergent or detergents to lyse the 
cells efficiently, thus forming a "detergent-treated" sample. After the 
cells have been lysed the detergent-treated sample is diluted prior to 
assay. 
Non-ionic detergents for use in present invention include but are not 
limited to TRITON.RTM. X-100, NONIDET.RTM. P-40 (NP-40), Tween-20 
(polyoxyethylenesorbitan), CHAPS 
(3-[(3-cholamidopropyl)-dimethylammoniol]-1-propane-sulfonate), to mention 
a few. In a preferred embodiment, cells are solubilized with TRITON.RTM. 
X-100, NONIDET.RTM. P-40, Tween-20 and/or CHAPS. In a more preferred 
embodiment, cells are solubilized with concentrated detergent to give a 
final detergent concentration of about 2% to about 4% in the 
detergent-treated sample. 
According to the invention, the total volume of non-ionic detergent added 
to the sample should not dilute out any leukocyte marker in the sample. 
Preferably, the volume of detergent does not exceed about 25% of the 
sample volume; more preferably, it does not exceed about 20%. The 
non-ionic detergent or detergents can be added to the original sample 
neat, or they can be prepared in a concentrated solution. In addition to 
the non-ionic detergent or detergents, the concentrated detergent solution 
can comprise distilled water or buffer. The concentration of non-ionic 
detergent or detergents in the solution will preferably be 5 times, and 
more preferably 6 times the final concentration of the non-ionic detergent 
or detergents after addition to the sample, i.e., in the detergent-treated 
sample. 
In an even more preferred embodiment, more than one non-ionic detergent is 
used to lyse the cells. For example, a high concentration of Tween-20 and 
TRITON.RTM. X-100, or TRITON.RTM. X-100 and NONIDET.RTM. P-40, or NP-40 
and Tween-20 can be used. The concentration of each detergent ranges from 
about 1% to about 2% after addition to the sample; the total concentration 
of detergent in the detergent-treated sample ranges from about 2% to about 
4%. More preferably the total detergent concentration ranges from about 2% 
to about 3%; and even more preferably from about 2% to about 2.5%. In a 
specific embodiment, the final detergent concentration in the sample is 
1.5% TRITON.RTM. X-100 and 1% "NONIDET.RTM." P-40. This is concentration 
preferred if both CD4 and CD8 are to be assayed. 
In another embodiment, the detergent concentration is a concentration that 
inactivates virus, especially HIV. It is a particular advantage of the 
invention that the lytic concentration of non-ionic detergent, such as the 
preferred ranges set forth above, is also a virus-inactivating 
concentration. 
Other methods of solubilizing cells, e.g., repeated freeze-thaw cycles, 
sonication, hypotony, or the addition of lower concentrations of 
detergents, are not as effective. Ionic detergents such as SDS are not 
effective, since SDS interferes with the subsequent antibody-antigen 
binding. 
After solubilization for at least about one minute, preferably for about 
one to about five minutes, the sample is diluted with buffer (step 2) 
prior to analysis in an immunoassay. The dilution can be 2-fold; 
preferably it is 5-fold or greater. The buffer is chosen to be compatable 
with the immunoassay for detecting the leukocyte antigen. Buffers 
preferred for various immunoassays are well known in the art. In a 
specific embodiment, sample buffer is 1% bovine serum albumin, 0.25% 
"NONIDET.RTM." p-40 and 0.01% thimerosal in phosphate buffered saline 
(PBS). 
The present method is not limited by the amount of sample available. In one 
embodiment, about 100 .mu.l of a detergent-treated sample of whole blood 
may be assayed for total leukocyte antigen. In a more preferred 
embodiment, about 2.5 to about 25 .mu.l of a detergent-treated sample of 
whole blood may be used. Similar amounts of a sample of culture 
suspension, pleural effusion, or other biological fluid can be used. The 
actual amount of sample assayed can be varied by adjusting the dilution 
factor. Determination of specific parameters can be accomplished by a 
simple dilution series assay, and is well within the level of ordinary 
skill in the art. 
Furthermore, a solubilized sample may be stored frozen so that samples 
taken at different times may be assayed in a single experiment. In one 
embodiment, the sample is stored at about -20.degree. C. In a preferred 
embodiment, the sample is stored at about -70.degree. C. 
Any method of detecting and measuring leukocyte antigens may be used in the 
practice of this invention. Such methods include but are not limited to 
competitive and non-competitive assay systems using techniques such as 
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" 
immunoassays, immunoradiometric assays, fluorescent immunoassays, and 
protein A immunoassays, to name but a few. U.S. Pat. No. 4,845,026, issued 
Jul. 4, 1989, entitled "Assay Systems for Detecting Cell-Free T Cell 
Antigen Receptor Related Molecules and Clinical Utilities of the Assays" 
and U.S. Pat. No. 5,006,459, issued Apr. 9, 1991, entitled "Therapeutic 
and Diagnostic Methods Using Soluble T Cell Surface Molecules" teach 
preferred methods of immunoassay. Their teachings are incorporated herein 
by reference. 
In a preferred embodiment, a sandwich enzyme immunoassay can be used. One 
description of such an embodiment follows: An antibody (capture antibody, 
Ab1) directed against the leukocyte marker is adsorbed onto a solid 
substratum. The leukocyte marker present in the sample binds to the 
antibody, and unreacted sample components are removed by washing. An 
enzyme-conjugated antibody (detection antibody, Ab2) directed against a 
second epitope of the leukocyte marker binds to the antigen captured by 
mAb1 and completes the sandwich. After removal of unbound Ab2 by washing, 
a substrate solution is added to the wells. A colored product is formed in 
proportion to the amount of antigens present in the sample. The reaction 
is terminated by addition of stop solution and absorbance is measured with 
a spectrophotometer. A standard curve can be prepared from known 
concentrations of the leukocyte marker, from which unknown sample values 
can be determined. 
In a preferred embodiment for the measurement of total CD8 antigen levels, 
anti-CD8 mAbs 4C9 and 5F4 can be used as the capture and detection 
antibodies, respectively, in a sandwich enzyme immunoassay; in a more 
preferred embodiment, a CELLFREE.RTM. CD8 assay (T Cell Sciences, Inc., 
Cambridge, Mass.) can be used (described in U.S. Pat. No. 5,006,459; see 
Section 8, infra). In a preferred embodiment for the measurement of total 
CD4 antigen levels, anti-CD4 mAbs 8F4 and R2B7 can be used as the capture 
and detection reagents, respectively, in a sandwich enzyme immunoassay; in 
a more preferred embodiment, a CELLFREE.RTM. CD4 assay (T Cell Sciences, 
Inc., Cambridge, Mass.) can be used (described in International Patent 
Publication WO 90/04180; see Sections 6 and 7, infra). In a preferred 
embodiment for the measurement of total V.delta.1 antigen levels, 
anti-TCR.delta. mAbs TCR.delta.1 and .delta.Tcs1 can be used in a sandwich 
enzyme immunoassay (see Section 9, infra). In a preferred embodiment for 
the measurement of total .beta. TCR antigen levels, anti-.beta.TCR mAbs 
.beta.F1 and W4 can be used in a sandwich enzyme immunoassay (see Section 
10, infra). The foregoing antibodies have been deposited with the ATCC, as 
described in Section 10, infra. 
Various procedures known in the art may be used for the production of 
antibodies to leukocyte marker. Such antibodies include but are not 
limited to polyclonal, monoclonal, chimetic, single chain, Fab fragments 
and an Fab expression library. For the production of antibodies, various 
host animals, including but not limited to rabbits, mice, rats, etc., may 
be immunized by injection with a leukocyte marker. In one embodiment, 
leukocyte marker may be conjugated to an immunogenic carrier. In another 
embodiment, leukocyte marker epitope, e.g., a hapten, is conjugated to a 
carrier. As used herein, an "epitope" is a fragment of an antigen capable 
of specific immunoactivity, e.g., antibody binding. Various adjuvants may 
be used to increase the immunological response, depending on the host 
species, including but not limited to Freund's (complete and incomplete), 
mineral gels such as aluminum hydroxide, surface active substances such as 
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, 
keyhole limpet hemocyanin, dinitrophenol, and potentially useful human 
adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium 
parvum. 
Monoclonal antibodies to leukocyte marker may be prepared by using any 
technique which provides for the production of antibody molecules by 
continuous cell lines in culture. These include but are not limited to the 
hybridoma technique originally described by Kohler and Milstein, (1975, 
Nature 256:495-497), the more recent human B-cell hybridoma technique 
(Kosbor et al., 1983, Immunology Today 4:72) and the EBV-hybridoma 
technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, 
Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the 
invention, monoclonal antibodies specific for leukocyte marker may be 
produced in germ-free animals utilizing recent technology 
(PCT/US90/02545). According to the invention, human antibodies may be used 
and can be obtained by using human hybridomas (Cote et al., 1983, Proc. 
Nat'l. Acad. Sci., U.S.A. 80:2026-2030) or by transforming human B cells 
with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and 
Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the 
invention, techniques developed for the production of "chimeric 
antibodies" (Morrison et al., 1984, Proc. Nat'l. Acad. Sci. U.S.A. 
81:6851- 6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 
1985, Nature 314:452-454) by splicing the genes from a mouse antibody 
molecule specific for leukocyte marker together with genes from a human 
antibody molecule of appropriate biological activity can be used; such 
antibodies are within the scope of this invention. 
According to the invention, techniques described for the production of 
single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to 
produce a leukocyte marker-specific single chain antibody. An additional 
embodiment of the invention utilizes the techniques described for the 
construction of Fab expression libraries (Huse et al., 1989, Science 
246:1275-1281) to allow rapid and easy identification of monoclonal Fab 
fragments with the desired specificity for leukocyte marker. 
Antibody fragments which contain sites specific for leukocyte marker may be 
generated by known techniques. For example, such fragments include but are 
not limited to: the F(ab').sub.2 fragments which can be produced by pepsin 
digestion of the antibody molecule and the Fab fragments which can be 
generated by reducing the disulfide bridges of the F(ab').sub.2 fragments. 
The above-described method can also be used to assay any total leukocyte 
surface marker, e.g., the markers described in Sections 2.1 supra. For 
example, and not by way of limitation, total CD4, total CD8, or total TCR 
may be measured in the practice of this invention. In another embodiment, 
a plurality of two or more total leukocyte surface molecules or markers 
can be measured. In yet a further embodiments, the relative amount of one 
marker can be compared to another. 
5.2. ENUMERATION OF LEUKOCYTES POSITIVE FOR A MARKER 
The measurement of a total leukocyte marker according to the invention 
provides a method to enumerate the number of leukocytes that are positive 
for the leukocyte marker in a sample. In another embodiment, the activity 
of a leukocyte marker in an assay can be compared to the activity of a 
standard that comprises a known number of cells positive for the leukocyte 
marker. 
This aspect of the invention is based on the discovery that the total 
leukocyte marker level in a sample, which is proportioned to the activity 
of the leukocyte mark in an assay for the leukocyte marker, correlates 
directly with the number of cells positive for that marker. As shown in an 
Example, infra (Section 7), the correlation between the number of CD4+ 
cells determined according to the present invention and the number of 
cells determined by fluorescence-activated cell sorting (FACS) has a 
correlation coeffecient of 0.946, which indicates that enumeration of a 
leukocyte marker-positive cell by the total leukocyte marker method of the 
invention can be as reliable as enumeration by flow cytometry. 
Furthermore, as pointed out in the Summary of the Invention, supra, the 
present methods do not require complicated sample preparation, careful 
storage conditions, or expensive analytical equipment. 
In another embodiment, the amount of leukocyte marker in the membrane 
and/or intracytoplasmic compartments of a sample, i.e., not including 
soluble leukocyte marker, correlates with the number of cells in the 
sample. As shown in an Example, Section 8 infra, the correlation between 
the number of cells positive for a leukocyte marker and the amount of 
leukocyte marker in the membrane and intracytoplasmic compartments is 
good. 
According to the present invention, the number of cells positive for a 
leukocyte marker in a sample can be determined by (1) determining the 
total amount of leukocyte marker in a sample, and (2) calculating the 
total number of cells positive for the leukocyte marker from the total 
amount of leukocyte marker. In one embodiment, the calculation can be made 
by extrapolating from a standard curve of total leukocyte marker versus 
total number of cells positive for the leukocyte marker. In another 
embodiment, a formula can be derived from a standard curve, and the total 
amount of leukocyte marker plugged into the formula. In yet another 
embodiment, only the activity of the assay for a total leukocyte marker is 
used to calculate the total number of cells positive for the marker. In 
this embodiment, the total amount of leukocyte marker is reflected in the 
relative activity in the detection assay. The method of calculating a 
total number of cells, e.g., extrapolation or a formula, remains the same. 
The present invention provides simple, straightforward methods to calculate 
a standard. In one embodiment, the total amount of leukocyte marker 
relates to the total number of cells positive for the marker. In a more 
preferred embodiment, a linear regression analysis of the total leukocyte 
marker to total cell number positive for the marker is performed. The 
linear regression analysis can yield a straight line curve, an estimate of 
error, and a formula for calculating total cell number from total 
leukocyte marker amount. 
In another embodiment, the activity of the leukocyte marker in the 
detergent-treated sample can be detected by immunological detection means 
and compared with an assay of a known amount of leukocyte marker in a 
standard sample, and the number of cells positive for the leukocyte marker 
determined from the comparison. In a more preferred embodiment, the 
activity in an assay of the leukocyte marker in the detergent-treated 
sample can be compared with the activity in an assay of the leukocyte 
marker in a standard containing a known number of cells positive for the 
marker, and the number of cells positive for the leukocyte marker in the 
sample determined from the comparison. 
In another embodiment, the correlation of total leukocyte marker with cell 
number positive for that marker can be corrected for an excess amount of 
leukocyte marker in the soluble or total compartment. The amount of 
soluble leukocyte marker can be assayed by the same immunological 
detection means, e.g., using the same immunoassay. The amount of soluble 
marker can be subtracted from the amount of total marker. The corrected 
cell-associated leukocyte marker value can correlate more accurately with 
the cell count. In a specific embodiment, the total number of CD8+ cells 
correlates with the total amount of CD8 minus soluble CD8 (see Section 8, 
infra). 
In preparing a standard, total cell number can be independently determined 
by any means known in the art, e.g., FACS, immunofluorescence microscopy, 
and complement lysis assays, to mention a few. In a preferred embodiment, 
the independent total cell number is obtained by FACS. 
A total CD4 assay may be used to estimate the member of CD4.sup.+ T cells 
in a patient sample. The estimate of the number of CD4.sup.+ T cells in a 
sample can be used for diagnosis or monitoring of a therapeutic treatment, 
e.g., for AIDS patients. 
In a preferred embodiment, CD8.sup.+ cells can be enumerated by measuring 
the total amount of CD8 antigen in the sample and subtracting the amount 
of soluble CD8 antigen in the same sample. Such an analysis yields 
information on both the immune system activation and the CD8.sup.+ cell 
count. 
In yet a further embodiment the total number of T cells positive for 
.alpha..beta. or .gamma..delta. TCAR can be determined. Similarly, the 
total number of T cells positive for subsets of V.alpha., V.beta., 
V.gamma. or V.delta. can be determined. Thus, whole blood samples can now 
be analyzed quickly, safely and reliably for the number of specific 
subsets of T cells. 
5.3. KITS 
The reagents necessary for the practice of the present invention can be 
conveniently provided in a kit. The essential elements of a kit of the 
invention are a concentrated non-ionic detergent solution and a means for 
immunologically detecting the presence of an antigen. 
The kit can comprise any immunological detection means known in the art, 
e.g, those discussed in Section 5.1., supra. Preferably the immunological 
detection means are a first capture antibody specific for a leukocyte 
marker, and a second detectable antibody specific for the leukocyte 
marker. The first antibody can be provided on a solid support, such as a 
glass or plastic bead, membrane, a plastic stick, a microwell, a glass or 
plastic test tube, or other solid supports known in the art for 
immunoassays. Alternatively, the first antibody can be provided for later 
immobilization. The first and second antibodies can be provided as a 
lyophilized preparation for reconstitution, or as a concentrated solution. 
The second antibody can be detectable by labeling means. In a preferred 
embodiment, the labeling means is an enzyme. 
The detergent solution is provided in a container having sufficient volume 
to hold the amount of solution necessary for the number of assays 
contemplated by the kit. Preferably the detergent solution comprises two 
or more non-ionic detergents. In a preferred embodiment, the detergent 
solution comprises 9% TRITON.RTM. X-100 and 6% NONIDET.RTM. " p-40 in 
distilled water. That particular detergent concentration is optimum with 
respect to an assay for both CD4 and CD8. 
Preferably the kit comprises a leukocyte marker standard, either in 
solution or lyophilized for reconstitution. More preferably, the kit 
comprises a standard comprising a known number of cells positive for the 
leukocyte marker of interest. Thus, the activity in an assay for the 
leukocyte marker in an unknown sample can be directly compared with a 
known sample, and the number of cells in the unknown sample that are 
positive for the leukocyte marker enumerated thereby. 
The kit can further comprise a reagent reactive with label, when the second 
antibody is detectable by attachment of a label to the antibody. For 
example, when the label is an enzyme, the kit can provide the enzyme 
substrate. The kit can also provide a dilution buffer, in final 
concentration, high concentration for dilution, or dry for reconstitution. 
In yet a further embodiment, a kit provides immunological detection means 
for more than one leukocyte antigen. In a preferred embodiment, the kit 
comprises immunological detection means for both CD4 and CD8. 
5.4. METHODS OF DIAGNOSIS AND MONITORING OF A DISEASE 
5.4.1. METHODS OF DIAGNOSIS 
In another embodiment of the present invention, measurement of a total 
leukocyte marker can be used to detect, diagnose or stage a disease or 
disorder in a subject. The measured amount of the total leukocyte marker 
is compared to a baseline level, i.e., the amount of total-leukocyte 
marker in normal individuals. This baseline level can be the amount which 
is established to be normally present in the body fluid of subjects prior 
to the onset of disease or the amount present during remission of disease. 
Disease or disorders which may be detected, diagnosed or staged in a 
subject according to the present invention include but are not limited to 
those listed in Table III. 
TABLE III 
______________________________________ 
DISEASES AND DISORDERS WHICH MAY BE 
DETECTED AND/OR DIAGNOSED AND/OR 
MONITORED IN A SUBJECT ACCORDING TO 
THE PRESENT INVENTION 
______________________________________ 
I. Infectious Diseases Induced by virus 
Herpesvirus 
Cytomegalovirus 
Epstein-Barr Virus 
HTLV-I 
HTLV-III / LAV / HIV (AIDS) 
II. Cancer 
B or T cell leukemia 
HTLV-I- associated adult T cell leukemia 
B or T cell lymphoma 
Burkitt's lymphoma 
Hairy cell leukemia 
Sezary syndrome 
Hodgkin's disease 
Chronic lymphocytic leukemia 
Non-Hodgkin's lymphoma 
B-cell acute lymphoblastic leukemia 
Solid tumors 
III. Autoimmune Diseases 
Rheumatoid arthritis 
Diabetes 
Multiple sclerosis 
Systemic lupus erythematosis 
IV. Organ Allograft Rejection 
______________________________________ 
The methods for detecting diseases or disorders based on the number of 
cells in a sample from the patient that are positive for a leukocyte 
marker are discussed in Section 2.3 supra and in the references cited 
therein. Those methods can be practiced by the present methods of the 
invention more cheaply and easily than heretofore thought possible. 
In one embodiment, the amount of total CD4 antigen can be used to enumerate 
CD4 positive cells which in turn can be used to diagnose AIDS. In the 
past, the diagnosis of AIDS was determined when a patient who was 
HIV-positive demonstrated CD4 positive cell counts of &lt;500 cells/mm.sup.3 
in the presence of one or more opportunistic infections. This definition 
of AIDS is expected to change to HIV-positive in the presence of &lt;200 
cells/mm.sup.3 whether or not opportunistic infections are present (see 
Section 2.3., supra). 
Responses to viral infections can also be monitored by measuring total CD8 
levels in a patient. For example, patients infected with herpes virus or 
an AIDS virus can present modified levels of total CD8. In other 
embodiments, total levels can be measured in transplant patients, and used 
as a diagnostic indication of allograft rejection. Detection of increased 
levels of total CD8 can be associated with rheumatoid arthritis and 
infectious diseases such as EBV-induced mononucleosis. Detection of 
elevated levels of a CD8 antigen can indicate the involvement of 
significant numbers of suppressor/cytotoxic T cells with a specific 
pathological event, distinct from immune activation. 
In yet another embodiment, the measurement of total CD4 in a cell culture 
can be relied on as an indication of the CD4.sup.+ phenotype of the 
lymphocytes present. For example, CD4.sup.+ leukemias or lymphomas can be 
classified using total CD4 antigen measurements. In a further embodiment, 
the measurement of total CD4 antigen can be used to detect CD4.sup.+ cells 
and to enumerate them. Similar embodiments of the invention include the 
measurement of total CD8 antigen to classify CD8.sup.+ leukemias or 
lymphomas, to detect CD8.sup.+ cells and to enumerate them. 
5.4.2. METHODS OF MONITORING 
The present invention provides a method for monitoring the effect of a 
therapeutic treatment on a subject who has undergone the therapeutic 
treatment. This method comprises measuring at suitable time intervals the 
amount of a total leukocyte marker. Any change or absence of change in the 
amount of the total leukocyte marker can be identified and correlated with 
the effect of the treatment on the subject. 
In one embodiment, total CD4 can be measured (and CD4.sup.+ cells 
enumerated) and used in the prediction of therapeutic outcome of AIDS 
patients following administration of therapeutic compounds such as AZT, 
interferon or CD4. 
In another embodiment, total CD8 antigen can be measured and correlated 
with disease progression or treatment outcome. 
Measurement of total T cell antigen receptor can be especially useful in 
monitoring the effectiveness of treatment with agents such as T cell 
receptor specific antibodies. In specific embodiments, the total TCR 
antigen in a specific subset of T cells expressing specific variable 
regions can be measured and correlated with treatment outcome. 
The therapeutic treatments which may be evaluated according to the present 
invention include but are not limited to radiotherapy, drug 
administration, vaccine administration, immunosuppressive or 
immunoenhansive regimens, etc. The immunosuppressant regimens include, but 
are not limited to administration of drugs such as Cyclosporin A, 
chlorambucil, cyclophosphamide, or azathioprine, and anti-T cell antibody 
such as anti-T3 monoclonal antibody, anti-T cell antigen receptor 
antibody, and anti-thymocyte globulin, etc. The immunoenhansive regimens 
include, but are not limited to administration of interleukin-1, 
interleukin-2, interleukin-4 and other T cell growth factors. 
5.4.3. METHODS OF DIAGNOSIS OR MONITORING OF THERAPY BASED ON DETECTION OF 
A PLURALITY OF LEUKOCYTE MARKERS 
The present invention also provides for the detecting or diagnosis of 
disease, or the monitoring of treatment by measuring a plurality (at least 
two) of total leukocyte markers. For example, a plurality of T cell 
markers either in total form, for example but not limited to CD4 and CD8, 
and TCAR to mention but a few, can be measured to diagnose, detect, or 
monitor treatment of diseases or disorders. Such diseases or disorders 
include those indicated in Table III. Total marker levels can represent a 
measure of immune system function, paralleling disease course or treatment 
efficacy. In a preferred embodiment, the prognostic indicator is the 
observed change over time in different marker levels relative to one 
another, rather than the absolute levels of the markers present at any one 
time. Since CD4, CD8 and TCAR are indicators of the immune system 
function, they should provide a much improved measure of the relative 
health of the immune system during various stages of disease or disorders. 
In a particular embodiment, diseases and disorders caused by HIV (the 
causative agent of AIDS) infection may be monitored by measurements of a 
plurality of leukocyte surface markers. AIDS therapies include the 
treatment of AIDS patients with drugs such as AZT (azido-deoxythymidine), 
.gamma. or .beta. interferons, and with soluble CD4, or its fragments and 
derivatives. In another embodiment, the efficacy of potential AIDS 
vaccines, such as gp120 peptides can be tested by monitoring a plurality 
of markers. Practitioners in AIDS therapy very much need a procedure that 
can be used to monitor the efficacy of these treatments or vaccines. To 
date, the levels of the HIV antigen p24 have not proved sensitive enough. 
Total CD4 relative to total CD8 can be detected in HIV-infected patients 
with different manifestations of disease, providing a sensitive 
immunoassay to monitor AIDS therapies and vaccines. The measurement of 
total CD4 and total CD8 is an inexpensive and easy immunoassay format and 
is a valuable clinical tool for predicting disease prognosis and treatment 
outcome in AIDS patients. Detection of total CD4 and CD8 according to the 
method of the invention antigen provides a particularly useful way to 
follow HIV infection and AIDS therapy since the relative level of CD4 
positive T cells decrease dramatically relative to the total number of CD8 
positive cells in the progress of AIDS etiology. 
In a preferred aspect, the approach that can be taken is to determine the 
levels of total CD4 and total CD8 levels in longitudinal time studies and 
to compare these values with a baseline level. The baseline level can be 
either the level of the marker present in normal, disease-free individuals 
or the level present in a patient prior to treatment, during remission of 
disease, or during periods of stability. These levels can then be 
correlated with the disease course or treatment outcome. 
The present invention also provides for the detection or diagnosis of 
disease or the monitoring of treatment by measuring the amounts of total 
leukocyte marker and of soluble leukocyte marker in a sample and comparing 
the two measurements. The change in the levels of the leukocyte markers 
relative to one another can be an improved prognostic indicator. In one 
embodiment, the level of soluble CD8 (a measure of immune system 
activation, see International Patent Publications WO 87/05912 published 
Oct. 8, 1987 and WO 90/04180 published Apr. 19, 1990) is compared with the 
level of CD8 antigen obtained by subtracting the amount of soluble CD8 
antigen from the amount of total CD8 antigen (e.g., the difference yields 
cell membrane bound plus cytoplasmic CD8, which is equivalent to 
cell-associated total CD8). Such a comparison gives information on the 
relative level of immune system activation and on changes in the number of 
cells in the CD-positive cell subset, both useful in the monitoring of 
disease progression or treatment. In a preferred embodiment, both the 
soluble and total CD8 antigen levels can be determined using one 
immunoassay configuaration, but with different sample treatment before 
assay. 
The instant invention will be further clarified by the following Examples, 
which are provided as purely exemplary of the invention and are not 
intended as limiting of the invention. 
6. EXAMPLE: A TOTAL CD4 ANTIGEN ASSAY 
The present example demonstrates the development of an assay that 
correlates total CD4 values with the number of CD4.sup.+ T cells in a 
sample. 
6.1. MATERIALS AND METHODS 6.1.1. SAMPLE PREATION 
Samples. Patient samples included 26 sero-positive HIV-infected individuals 
and 13 healthy adult volunteers. Blood was obtained by venipuncture into a 
blood collection tube. 
Sample Treatment. Prior to assay, whole blood samples were treated as 
follows. 
Step 1: 
100 .mu.l of anticoagulated blood was removed from the blood collection 
tube and mixed with 20 .mu.l of concentrated 6.times. detergent (6% 
TRITON.RTM. X-100, 6% NONIDET.RTM. "p-40 in distilled water) in a 
12.times.75 mm glass tube. This mixture was then incubated for one minute 
at room temperature. After one minute the treated sample was either used 
in a CD4 immunoassay or stored at -70.degree. C. until assayed. Samples 
stored at -70.degree. C. were allowed to thaw at room temperature before 
use. 
Step 2: 
Prior to assaying the treated samples in a CD4 immunoassay, the samples 
were diluted as indicated in the results section with sample buffer (1% 
bovine serum albumin, phosphate buffered saline, 0.25% NONIDET.RTM." p-40 
and 0.01% thimerosal) to dilute the concentration of detergent. Total 
sample volumes added to the immunoassay were maintained at 50 .mu.l. 
6.1.2. CD4 IMMUNOASSAY 
Total CD4 antigen was measured in a CD4 specific immunoassay (see 
International patent publication WO 87/05912 published Oct. 8, 1987) 
involving a one-step, three hour format using microtiter plates that had 
been precoated with capture antibody overnight. Briefly, each well of a 96 
well microtiter well plate was coated with 100 .mu.l of murine anti-CD4 
coating antibody in phosphate-buffered saline (PBS) overnight at 4.degree. 
C. Any remaining protein-binding sites were blocked with 300 .mu.l per 
well of blocking buffer (0.5% casein, 0.008% NONIDET.RTM." P-40, 0.005% 
EDTA in PBS) for 2 hours at 37.degree. C. The wells were washed three 
times with 350 .mu.l per well of wash buffer (PBS, ph 7.4, with 0.05% 
Tween 20). After aspirating the final wash buffer from the wells, 50 .mu.l 
of horseradish peroxidase (HRP) conjugated murine monoclonal anti-human 
CD4 antibody (in PBS with 15% FCS and 0.15% NONIDET.RTM." p-40) and 50 
.mu.l of sample or standard were added to each well of the microtiter 
plate in duplicate. The combined volume of sample and HRP conjugated 
antibody was 100 .mu.l. Samples and antibodies were incubated for 3 hours 
at room temperature. After washing the plate as described above, 100 .mu.l 
of OPD substrate (OPD tablets, BioDesign Intl, Catalogue #A45104T, 
dissolve 1 tablet in 4 ml citrate buffer-peroxide, BioDesign Intl, 
Kennebunkport, Me. Catalog number A45105B) was added to all the wells and 
incubated for 30 minutes at room temperature. At the end of this last 
incubation, 50 .mu.l of 2 N H.sub.2 SO.sub.4 was added to each well to 
stop the reaction and absorbance of each well was read at 490 nm. Results 
were plotted as values obtained for each sample at O.D. 490 against total 
CD4.sup.+ cells/mm.sup.3 or sample volume. Correlation coefficients were 
calculated using linear regression analysis. 
Whole blood samples from normal and HIV-infected patients were assayed for 
absolute CD4.sup.+ T-cells/mm.sup.3 using the formula: 
EQU Abs. CD4.sup.+ T cells/mm.sup.3 =WBC.times.% Lymphocyte.times.% CD4.sup.+ T 
cells. 
WBC (White blood cell count) was determined using a hemacytometer. % 
Lymphocyte was determined by a differential count, and %CD4.sup.+ T cells 
were determined using the Ortho cytoflurograph II and Leu-3a (anti-CD4) 
fluorescein-conjugated antibody (Becton Dickinson, Mountain View, Calif.) 
6.2. EXPERIMENTAL RESULTS 
Experiment 1: Measurement of total CD4 antigen in whole blood. This study 
was conducted to determine whether detergent treatment of whole blood 
would yield reliable measurements of total CD4 antigen in a CD4 
immunoassay. Samples of whole blood were treated with a 6.times. detergent 
solution using a ratio of 20 .mu.l detergent to 100 .mu.l anticoagulated 
whole blood. Serial dilutions were made of these samples to produce a 
series of samples containing decreasing amounts of total CD4 antigen. 
These samples were then analyzed in the CD4 assay to determine which 
dilutions produced values that were linear with sample dilution. As the 
assay configuration became saturated with excess CD4, the optical density 
values representing total CD4 values became nonlinear. 
The results of this experiment can be seen in FIG. 1. Total CD4 
preparations made from three healthy volunteers were treated with 
concentrated non-ionic detergent, and diluted in serial 1:2 increments 
(from 1/2 to 1/32). Sample volumes were maintained at 50 .mu.l by dilution 
into sample buffer. Linear regression analysis demonstrated that samples 
containing less than 20 .mu.l and preferably 5-10 .mu.l of detergent 
treated whole blood were optimal for total CD4 measurement as they fell 
within the linear range. Samples containing greater than 20 .mu.l of whole 
blood saturated the assay and total CD4 could not be accurately 
determined. 
Experiment 2: Specificity of the total CD4 antigen measurement. The ability 
of an anti-CD4 antibody to block the total CD4 signal detected in whole 
blood samples of three normal controls is presented in Table IV. The assay 
was run as described above, except that before adding the HRP-conjugated 
murine anti-CD4 monoclonal antibody to the wells, 5 .mu.l of the 
unconjugated murine anti-CD4 monoclonal antibody used in the coating 
procedure was added to the wells, followed by the HRP-conjugated antibody. 
Samples were diluted with sample diluent to a final volume of 50 .mu.l. 
The unconjugated antibody was able to block total CD4 detection by 50%, 
51% and 51% in samples #1, 2, and 3, respectively, at the level of 
competing antibody used. This demonstrates the specificity of the assay to 
detect total CD4. Other proteins released during detergent lysis of the 
whole blood samples (such as hemoglobin) did not interfere with the assay. 
TABLE IV 
TABLE IV 
______________________________________ 
SPECIFICITY OF TOTAL CD4 DETECTION IN 
DETERGENT TREATED WHOLE BLOOD 
O.D. 495 
sample # 
10 .mu.l* 10 .mu.l + anti-CD4** 
% inhibition 
______________________________________ 
1 0.433 0.216 50% 
2 0.928 0.472 51% 
3 0.833 0.422 51% 
______________________________________ 
*10 .mu.l of detergent treated whole blood prepared from normal 
uncoagulated whole blood 
**5 .mu.l of the antiCD4 coating antibody from the CD4 assay was also 
added to compete for binding. 
Experiment 3: Enumeration of cell number using total CD4 values. This study 
was done to determine whether the measurement of total CD4 levels in 
treated whole blood samples would correlate with absolute CD4 positive 
cell numbers. Data in FIG. 2 demonstrate a statistically significant 
correlation between total CD4 measured in whole blood samples and the 
total number of CD4.sup.+ cells/mm.sup.3 of blood (r=0.904). Samples 
measured in this assay contained 2.5 .mu.l of detergent treated whole 
blood diluted to a final volume of 50 .mu.l from a total of five normal 
controls. The normal range of CD4.sup.+ cells/mm.sup.3 is 800-1000 
cells/mm.sup.3. The normal values in this assay appear lower than usual 
due to the fact that the WBC count used to determine the number of CD4 
cells/mm.sup.3 was performed on the uncoagulated blood samples three days 
after the CD4 assay was run. The relationship between total CD4 detected 
in the assay and the total CD4.sup.+ cells/mm.sup.3 for each individual 
was determined by linear regression analysis and a statistically 
significant r value of 0.904 was obtained. This indicates that the 
measurement of total CD4 antigen can be used to directly enumerate CD4 
positive cells. 
Experiment 4: Enumeration of CD4.sup.+ cells in HIV-infected and normal 
individuals using measurements of total CD4 antigen. After the properties 
of the total CD4 method were established using healthy donors, further 
study was conducted to establish a relationship between the total CD4 as 
measured in the ELISA to the total number of CD4.sup.+ T cells in HIV 
infected individuals. 
The total CD4 antigen values of HIV-infected individuals and normals were 
determined using the total CD4 method on two separate occasions. The 
result of these experiments are presented in FIGS. 3 and 4. Ten .mu.l of 
whole blood samples diluted to a total sample volume of 50 .mu.l from 
either the disease group or control group were used to determine the total 
CD4 antigen values. The total CD4.sup.+ cells/mm.sup.3 cells was 
determined on fresh uncoagulated blood samples on the day of the CD4 
assay. 
Both FIGS. 3 and 4 demonstrate a statistically significant correlation 
between total CD4 antigen measured in the assay and the total CD4.sup.+ 
cells/mm.sup.3 for both the HIV-infected group (single squares) and the 
control group (double squares) (r=0.860 and 0.867 for FIGS. 3 and 4, 
respectively). Thus, the measurement of total CD4 antigens from detergent 
treated whole blood accurately reflects the absolute number of CD4.sup.+ 
T-cells in whole blood for both normal and HIV infected individuals. 
6.3. DISCUSSION 
As described herein, we have developed a method that measures the level of 
total CD4 antigen in a patient sample. Whole blood samples were detergent 
treated and assayed using a CD4 immunoassay in a one-step, three hour 
format. It was demonstrated that total CD4 antigen could be detected in 
whole blood samples of normal individuals by a simple detergent lysis step 
and CD4 immunoassay. Optimal detection could be measured using 10 .mu.l or 
less of detergent treated whole blood samples. Accurate and reproducible 
measurements can be detained from 2.5 .mu.l to 5 .mu.l of whole blood. The 
detection of total CD4 antigen was specific as a murine anti-CD4 
monoclonal antibody was capable of blocking the total CD4 antigen signal 
when added to the assay format. The release of other proteins during 
detergent treatment of whole blood did not interfere with the specificity 
of the assay. It was further demonstrated that the total CD4 antigen 
values obtained in the ELISA correlated with the total CD4.sup.+ T 
cells/mm.sup.3 in the whole blood of normal donors (r=0.9). 
A comparison between the total CD4 antigen obtained in the CD4 ELISA and 
the total number of CD4.sup.+ T Cells/mm.sup.3 of whole blood yielded a 
statistically significant correlation in both HIV-infected individuals and 
normals. This comparison was determined on two separate occasions using 
two different groups of HIV-infected individuals and normals. Both 
comparisons yielded statistically significant correlations between total 
CD4 antigen values and total CD4.sup.+ T cells/mm.sup.3 (r=0.86 and 0.87). 
Since the measurement of total CD4 antigen accurately reflects the number 
of CD4 positive cells, the total CD4 method can be used to enumerate CD4 
positive cells and to determine the numbers of CD4 positive cells in 
disease diagnosis or monitoring of treatment, etc. 
7. EXAMPLE: ENUMERATION OF CD4 POSITIVE LYMPHOCYTES IN PERIPHERAL BLOOD BY 
THE TOTAL CD4 METHOD 
This Example outlines further development and characterization of the total 
CD4 method for enumeration of CD4 positive cells. The method consists of 3 
steps: treatment of samples with concentrated detergent, dilution of the 
concentrated detergent lysate and CD4 immunoassay. 
7.1. MATERIALS AND METHODS 
Samples: Ninety-five samples of whole blood were collected in EDTA 
containing vacutainer tubes and mixed thoroughly. Samples were analyzed 
for white blood cell count and differential and CD4.sup.+ lymphocytes by 
flow cytometry. 
Sample Treatment: Samples were treated as described in section 6, except 
for the following: 
Step 1: 
1 volume of concentrated detergent solution was added to 5 volumes of fresh 
EDTA whole blood, followed by gentle mixing. Usually 100 .mu.l of whole 
blood was treated with 20 .mu.l of detergent solution. A comparison of 
detergents of various concentrations indicated that the best results were 
obtained using a combination of detergents such as TWEEN-20 with 
TRITON.RTM." X-100 or TRITON.RTM." X-100 with NP40. Any of the three 
detergents alone was not as effective. The detergent treated sample (100 
.mu.l sample plus 20 .mu.l concentrated detergent) yielded to a final 
combined detergent concentration of 2.5%. The optimal detergent solution 
for measuring both total CD4 and total CD 8 using the same concentrated 
detergent solution was 9% TRITON.RTM." X100 and 6% NONIDET.RTM." (15% 
total in the concentrated detergent solution) which corresponded to 1.5% 
TRITON.RTM." X100 and 1% NONIDET.RTM." (2.5% total) in the treated sample. 
In other experiments, the final concentration of the detergents in the 
treated sample was similarly effective over a range of at least 1-2% 
TRITON.RTM." p-40 X100 combined with 1-1.5% NONIDET.RTM.". 
Other methods of lysis, solubolization or disruption of cells including 
hypotonic treatment with distilled water, sonication, or addition of 
detergent at lower final concentrations (e.g. one detergent at 1% 
concentration in the treated sample) were tested and found not effective. 
Step 2: 
After 5 minutes, 100 .mu.l of treated sample was diluted with 400 .mu.l of 
dilution buffer to yield a 1:5 dilution. Fifty .mu.l of this diluted 
sample (about 10 .mu.l of whole blood) was used for each assay well. The 
assay configuration allowed whole blood volumes of 5-25 .mu.l to be 
measured, since the resulting values fell within the range of the standard 
curve. Blood volumes above or below these amounts yielded values that fell 
outside the range of the standard curve. The importance of step 2 is the 
dilution of the detergent concentration in the treated sample to prevent 
"stripping" in the following assay and loss of signal. Samples 
corresponding to 50 .mu.l of whole blood performed poorly in the CD4 
immunoassay because the resulting values were outside the assay range, and 
because the detergent concentration was disruptive to the antibody 
sandwich formation or stability. 
Assay. Samples were stored at -70.degree. C. prior to assay. The samples 
were assayed in the one-step sandwich immunoassay as described in section 
6. Total CD4 values were determined from a standard curve. Standards were 
soluble, recombinant CD4 antigen prepared by removing the transmembrane 
region of the CD4 gene, followed by expression of the truncated gene in 
mammalian cells and recovery of recombinant CD4 antigen in the supernatant 
of the cell culture (see International Patent Publications WO88/01304 and 
WO89/02922). 
7.2. EXPERIMENTAL RESULTS 
Experiment 5: A standard curve for the total CD4 antigen method was 
generated using culture supernatant containing recombinant CD4 receptor 
(see FIG. 5). For the assay configuration used, the standard range was 
determined to be 0 to 200 units/.mu.l with a correlation coefficient of 
0.999. 
Experiment 6: Specificity of the assay for the CD4 antigen. Blood samples 
were drawn from 4 normal volunteers and treated as follows: A sample from 
each was analyzed for white blood cell count and differential (the 
percentage of WBCs that were lymphocytes; see the formula in Section 
6.1.2). The rest of the sample was divided; one-half was depleted of 
CD4.sup.+ cells by absorption with anti-CD4 coated Dynabeads (Dynal, Inc. 
Great Neck, N.Y.) while the other half was untreated. The depleted and 
untreated samples were again divided and one fraction analyzed by flow 
cytometry while the other was treated using the total CD4 method. CD4 
positive cell number was calculated from the white blood cell count, 
lymphocyte differential, and percentage of CD4 cells by flow cytometry 
using the formula described in Section 6.1.2. The comparison of CD4 
positive cell number to total CD4 antigen is given in Table V. 
TABLE V 
______________________________________ 
Specificity of the Total CD4 Antigen Assay for CD4 Antigen 
Untreated Fractions 
Depleted Fractions 
Flow Total CD4 Flow Total CD4 
Cytometry Antigen Cytometry 
Antigen 
# cells/.mu.l 
units/.mu.l 
cells/.mu.l 
units/.mu.l 
______________________________________ 
1 71 320 0 7 
2 934 445 0 11.5 
3 1109 345 40 15 
4 912 395 45 15 
______________________________________ 
Both the flow cytometry and the total CD4 methods indicated the removal of 
CD4 positive cells from all depleted fractions indicating that the assays 
were specific for the CD4 receptor. 
Experiment 7: Assay dilution and linearity. This study was done to further 
verify assay sensitivity. Whole blood samples from normal volunteers were 
collected in EDTA. A and a portion of each was depleted of CD4 positive 
cells to serve as the sample diluent in order to keep the sample matrix as 
normal as possible. Samples were prepared neat and at 1/2, 1/4 and 1/8 
dilutions with concentrated detergent; and run in the CD4 immunoassay. 
Table VI shows the results of this experiment on 6 different samples. 
Values are presented as raw data as run in the assay. The zero value 
represents the assay value obtained for the depleted portion used as 
sample diluent. This value is included in the calculations of expected 
values. 
TABLE VI 
______________________________________ 
Dilution and Linearity of Assay 
Units Units Percent 
Sample Dilution Detected Expected 
Recovery 
______________________________________ 
585 Neat 64 64 100 
1:2 35 35.5 99 
1:4 24 21 114 
1:8 14 14 100 
0 7 
586 Neat 89 89 100 
1:2 58 51 114 
1:4 34 31 110 
1:8 22 21 105 
0 11.5 
593 Neat 79 79 100 
1:2 38 41 91 
1:4 26 22 119 
1:8 22 24 92 
0 16 
598 Neat 63 63 100 
1:2 40 39 103 
1:4 30 27 111 
1:8 26 21 124 
0 15 
599 Neat 69 69 100 
1:2 36 42 86 
1:4 28 28 100 
1:8 22 22 100 
0 15 
600 Neat 65 65 100 
1:2 36 40 90 
1:4 27 27 100 
1:8 21 21 100 
0 15 
______________________________________ 
These results indicate that normal whole blood samples dilute linearly in 
the assay. The assay background is &lt;20 units. Neat normal samples fall in 
the middle of the assay range, so dilution of even 1:2 should be rarely 
necessary. 
Experiment 8: Cell enumeration using the total CD4 method. This study 
confirmed that it was possible to enumerate CD4 positive cells in whole 
blood by the total CD4 method. Both flow cytometry and total CD4 antigen 
assays were run by Maryland Medical Laboratory on normal and abnormal 
blood specimens that were less than 24 hours old. Flow cytometry was 
performed on a Coulter Epics Flow Cytometer; the CD4 immunoassay was read 
using a Molecular Devices VMAX ELISA reader. Samples with low CD4 positive 
cell counts were selected from HIV positive specimens. Cells were dually 
stained with anti-CD4 and anti-CD2 antibodies in order to make sure that 
the cells counted by Flow were CD4 positive T cells. A total of 95 samples 
(46 normal and 49 abnormal) were assayed for white blood cell number, 
lymphocyte differential, and percent CD2.sup.+ CD4.sup.+ cells by flow 
cytometry and for the amount of CD4 by the total CD4 antigen method. The 
number of CD4.sup. + T lymphocytes was calculated from the cell count, 
differential and flow data and compared to the data obtained from the 
total CD4 method by linear regression. The distribution of the two types 
of samples was: 
______________________________________ 
46 Normals 
CD4+ T cell Number 500-1600 cells/.mu.l 
total CD4 antigen 222-600 units/ml 
49 Abnormals 
CD4+ T cell Number 1-690 cells/.mu.l 
total CD4 antigen 48-295 units/ml 
______________________________________ 
The results of the linear regression analysis are shown in FIG. 6, where 
units/ml from tile total CD4 antigen method are compared to cells/.mu.l 
from the flow analysis. The slope of the line (0.26) and Y-intercept 
(89.7) define the linear regression curve equation Y=0.26.times.+89.7, 
where the total CD4 antigen units/ml is calculated to be 0.26 times the 
total number of cells/.mu.l plus 89.7. The correlation coefficient for the 
comparison is 0.946, which indicates that enumeration of CD4 positive 
cells via the total CD4 antigen method is as reliable as the same 
enumeration by flow cytometry. 
7.3. DISCUSSION 
The quantitation of CD4 receptor protein in treated samples of whole blood 
has been shown to be equivalent to flow cytometry for the enumeration of 
CD4 positive T lymphocytes (r=0.946). For the configuration of the assay 
used, sensitivity is 100 units/ml or 50 cells/.mu.l, which allows the 
total CD4 antigen method to be used over a wide range (50-1600 
cells/.mu.l) of samples. In the 95 samples tested, the possible presence 
of CD4 positive monocytes did not interfere with the assay results. Since 
only 10 .mu.l of whole blood is needed per test, the total CD4 method will 
be useful for detecting CD4.sup.+ T cells in samples that are difficult to 
obtain. The total CD4 method can be run in laboratories without access to 
flow cytometry. The method is simple and can be competed in less than 4 
hours. Samples can be treated quickly and assayed immediately or treated 
and stored frozen for batch testing. Such batch testing of longitudinal 
patient samples following a course of treatment is not subject to the 
errors obtained with flow cytometry measurements where samples are run 
separately on different days. The detergent treatment step of the total 
CD4 method also provides the added benefit of inactivating enveloped 
viruses which increases operator safety. 
8. EXAMPLE: ENUMERATION OF CD8 POSITIVE CELLS BY THE TOTAL CD8 METHOD 
8.1. MATERIALS AND METHODS 
Samples: Patient samples included 49 sero-positive HIV-infected individuals 
and 46 healthy adult volunteers with CD8.sup.+ cell counts ranging from 28 
to 2983 cells/.mu.l. 
Sample Treatment: Whole blood samples were prepared as described in Section 
7, supra. Two hundred .mu.l of concentrated detergent solution (9% 
TRITON.RTM." X100 and 6% NONIDET.RTM." P-4 were added to 1 ml of whole 
blood. Following gentle mixing, the sample was diluted 1:5 (50 .mu.l 
treated sample plus 200 .mu.l diluent) with diluent before assay. Treated 
samples were stored at -70.degree. C. prior to assay. 
Assay. Total CD8 was measured in detergent treated whole blood in a 
CELLFREE.RTM. CD8 immunoassay (T cell Sciences, Inc.) using a one-step, 
three hour format. Briefly, each well of a 96 well microtiter plate was 
coated with 100 .mu.l of the murine anti-CD8 coating antibody in 
phosphate-buffered saline overnight at 4.degree. C. The coating buffer was 
removed and 300 .mu.l of blocking buffer (0.5% casein, 0.008% 
NONIDET.RTM." P-40, 0.005% EDTA in PBS) were added per well and incubated 
for 2 hours at 37.degree. C. After washing the wells three times with 400 
.mu.l of wash buffer (PBS, pH 7.4, with 0.05% Tween 20), 50 .mu.l of 
horseradish peroxidase (HRP) conjugated murine monoclonal anti-human CD8 
antibody (10% FCS, 0.025% thimerosal, 0.01% gentamicin, 0.05% tween in 
Tris Buffered Saline, TBS) were added to all wells except those used as 
blanks. Fifty .mu.l of CD8 standard or treated whole blood was added to 
200 .mu.l of diluent (1% bovine serum albumin, phosphate buffered saline, 
0.25% NONIDET.RTM." P-40 and 0.01% thimerosal) and 50 .mu.l of this were 
added to the appropriate microtiter wells. The plate was covered with 
plate sealer and incubated for three hours at 20.degree. C. with shaking 
at 150 rpms. After washing the plate as described above, 100 .mu.l of OPD 
substrate were added to all the wells, and the plate was incubated at 
20.degree. C. for 30 minutes. Fifty .mu.l of 2N H.sub.2 SO.sub.4 were 
added to all wells to stop the reaction and absorbance was read at 490 nm. 
A standard curve (correlation &gt;0.999; range 0-2300 units/.mu.l; assay 
background &lt;100 units) was constructed by plotting O.D. against the 
concentration of the standards. The concentration of total CD8 in the 
whole blood samples was then determined from the standard curve. 
Correlation coefficients were calculated using linear regression analysis. 
Whole blood samples from normal and HIV-infected patients were assayed for 
absolute CD8.sup.+ T-cells/mm.sup.3 using the formula: 
EQU Abs. CD8.sup.+ T-cells/mm.sup.3 =WBC.times.% lymphocyte.times.% CD8.sup.+ T 
cells. 
WBC (white blood cell count) was determined using a hemacytometer. % 
Lymphocyte was determined by a differential count, and %CD8.sup.+ T-cells 
were determined using the Ortho cytoflurograph II and Leu-2a (anti-CD8, ) 
fluorescein conjugated antibody (Becton Dickinson, Mountain View, Calif.) 
8.2. EXPERIMENTAL RESULTS 
Experiment 1. The specificity and linearity of the assay for CD8 were 
determined using similar depletion and dilution studies as described in 
Section 7. Total CD8 antigen values were obtained from whole blood of 43 
normal and 43 HIV-infected, sero-positive individuals and compared to the 
total number of CD8.sup.+ T-cells/mm.sup.3 of whole blood. Table VII 
summarizes the population statistics for the normal and abnormal samples 
by flow cytometry and the total CD8 method. The normal samples have a 
linear correlation of r=0.772 and the HIV.sup.+ samples have a linear 
correlation of r=0.718. 
TABLE VII 
______________________________________ 
AVERAGE RANGE FOR 45 NORMAL PATIENT SAMPLES 
26 patients were male 
19 patients were female 
ages ranged from 23-69 years 
ages ranged from 18-65 years 
______________________________________ 
Total 
CD8 
Samples 
WBC* % Lymph % CD8* #CD8* units/ml 
______________________________________ 
average 
6.48 31 23 483 949 
2 STD 3.04 10 14 181 343 
High 10.2 41 42.3 898 2300+ 
Low 4.0 22 8.2 173 423 
______________________________________ 
AVERAGE RANGE FOR 49 HIV.sup.+ PATIENT SAMPLES 
45 patients were male 
4 patients were female 
ages ranged from 24-65 years 
ages ranged from 21-43 years 
______________________________________ 
Total 
CD8 
Samples 
WBC* % Lymph % CD8* #CD8* units/ml 
______________________________________ 
average 
4.66 36 55 871 1368 
2 STD 5.76 26 28 1050 1244 
High 19.9 61 79.5 2983 2300+ 
Low 1.0 0.2 12.7 28 364 
______________________________________ 
FIG. 7 demonstrates a statistically significant correlation between total 
CD8 antigen measured in the assay and the total number of CD8.sup.+ cells 
(r=0.75). In this study of 86 individuals, it was found that when total 
CD4 values were normal, so were total CD8 values. As CD4 values declined 
in HIV.sup.+ patients, the total CD8 values varied widely. Samples with 
low CD4 (&lt;180 units/ml) and low CD8 values generally had low white blood 
cell counts as well. The HIV.sup.+ patients had some of the highest CD8 
counts, perhaps to replace their diminished CD4.sup.+ T cells. 
Experiment 2. Although it would be possible to enumerate CD8 positive cells 
using the total CD8 method of experiment 1 (r=0.75), this quantitation was 
not as accurate as the enumeration of CD4 positive cells by the total CD4 
method (r=0.946). Since activation of CD8 positive cells results in the 
release of large amounts of soluble CD8 antigen, it is likely that the 
levels of sCD8 were affecting the correlation of total CD8 antigen levels 
with CD8 positive cell counts. To test this hypothesis, total CD8 antigen 
and soluble CD8 antigen were measured in 30 normal and 11 cancer (mainly 
melanoma and lymphoma) whole blood samples. Total CD8 antigen measurements 
were made as described above with the detergent treatment, dilution and 
immunoassay, and soluble CD8 levels were determined directly by 
immunoassay of plasma preparations. The same CD8 immunoassay was used for 
both the total CD8 antigen and soluble CD8 antigen. Since one volume of 
plasma (sample size) has twice the amount of plasma as an equivalent sized 
sample of whole blood, the soluble CD8 amounts in plasma were divided by 
two. The amount of cell associated CD8 (membrane bound plus cytoplasmic) 
was determined by subtracting the soluble levels of CD8 (divided by two) 
from the amount of total CD8 antigen. The results of this analysis are 
shown in FIG. 8. Linear regression analysis of this data gave an improved 
correlation coefficient of 0.859 as compared to 0.75 for total CD8 antigen 
alone. Cell enumeration could be more accurately quantitated from the 
equation: 
EQU Y (cells/mm3)=1.1.times.(units/ml total CD8 antigen)+7.4. 
8.3. DISCUSSION 
As described herein, we have developed a total CD8 method that measures the 
level of total CD8 antigen in a patient sample. Whole blood samples were 
detergent treated, diluted, and assayed using a CD8 immunoassay in a 
one-step, three hour format. Total CD8 antigen values obtained in the 
total CD8 method correlated with total CD8.sup.+ T-cell number (r=0.75) in 
whole blood. A significant improvement in the ability to enumerate 
CD8.sup.+ cells in whole blood was found when the level of soluble CD8 
antigen (released from activated cells) was subtracted from the level of 
total CD8 antigen (r=0.859). Monitoring the relationship of soluble and 
total antigen provided improved information on cell count and immune 
activation. Thus, the total CD8 assay alone, or in combination with other 
soluble or total marker assays, can be used to monitor the immune profile 
of patients. 
9. EXAMPLE: ENUMERATION OF TCAR POSITIVE CELLS BY TOTAL TCR METHODS 
Total TCAR .beta. Experiments. An assay to detect total T cell antigen 
receptor (TCAR) .beta. chain or total V.beta.5 specific TCAR chain was 
performed using detergent treated whole blood samples in a total TCR 
method. Briefly, wells in a 96 well plate were coated with 5 .mu.g/ml of 
coating antibodies which consisted of either a negative control antibody 
(.delta.TCS1 which is specific for the V.delta.1 region of the 
.gamma..delta. T cell receptor), or W76 which recognizes the constant 
region of the .beta. chain (C.beta.) or a V.beta.5 specific monoclonal 
antibody such as W112 (Tian et al., 1989, FASEB J. 3:A486 Abstr). A 
horseradish peroxidase conjugated (HRP)-.beta.F1 antibody, which 
recognizes a different epitope of the .beta. chain constant region 
(C.beta.) than W76, was used as the detection antibody. Whole blood 
lysates were prepared as described in Section 7, supra. The assay format 
was similar to that described in U.S. Pat. No. 4,845,026, issued Jul. 4, 
1989 and entitled "Assay Systems for Detecting Cell-Free T Cell Antigen 
Receptor Related Moleucles and Clinical Utilities of the Assays." In 
addition, a one-step, three hour format as described in Section 7, supra, 
was performed with similar results. Twenty-five .mu.l of whole blood 
lysate was diluted in 75 .mu.l of sample diluent for a starting dilution 
of 1:4. O.D. values from the negative control wells were subtracted from 
all values obtained (negative control wells using .delta.TCS1 as the 
coating antibody averaged 0.10 and blank wells which had no antibody 
averaged 0.095). 
The results of the total TCAR assay can be seen in FIG. 9. TCAR .beta. 
chain was optimally detected at dilutions of 1:4 and 1:8. V.beta.5 
specific TCAR was detected at low levels (1:16 dilution) as well. Lower 
levels of V.beta.5 were expected, since the V.beta.5 positive subset of 
cells represents only a small portion of all .beta. TCAR positive T cells. 
The assay demonstrated good specificity as the background O.D. was no 
higher when the control antibody .delta.TCS1 was used as the coating 
antibody as compared to O.D.s obtained from blank wells which contain no 
coating antibody (0.100 and 0.095 respectively). 
These data demonstrate that a total TCAR method can be used to detect the 
total amount of TCR .beta. chain in a whole blood sample by using two 
monoclonal antibodies which recognize different regions of the constant 
region of the .beta. chain. The total amount of .beta. chain is an 
indication of the total amount of .beta. positive T cells in the sample. 
Furthermore, specific subsets such as the V.beta.5 family, which represent 
only 1-5% of the total TCAR .beta. chains in normal blood, can be 
detected. 
Total TCAR V.delta.1 Experiments. The treatment of samples was similar to 
that of section 7 with a concentrated detergent solution of 9% 
TRITON.RTM." X-100 and 6% NONIDET.RTM." P-40 in 1.times. PBS that was 
added to whole blood at a ratio of 200 .mu.l concentrated detergent 
solution to 1 ml whole blood. Samples were then diluted 1:2 prior to 
immunoassay. The immunoassay procedure was similar to the CD4 or CD8 
immunoassay except with antibody TCR.delta.1 as capture antibody and 
.delta.TCS1 as detection antibody. The TCAR .delta. positive cell line 
PEER was used as a positive control. A standard curve was generated with 
the units arbitraily assigned based upon a PEER cell sample preparation 
stored at -70.degree. C. and assigned a value of 20,000 units. The 
specificity of the assay for TCAR .delta. chain was determined using an 
antibody-affigel stripping procedure. Affigel 10 was coupled to either 
BSA, anti-CD8 antibody 4C9 (an isotype control antibody), .delta.TCS1, 
TCR.delta.1, V.delta.2 or V.gamma.2 at 1 mg/ml concentration. A 50/50 
slurry was made of each sample. Three hundred .mu.l of a detergent treated 
PEER cell sample was added to 50 .mu.l of each antibody-affigel slurry. 
The sample/gels were incubated for 1 hour at 4.degree. C. The samples were 
then centrifuged for 5 minutes at 10 K. The supernatant fluid was removed 
and added to 50 .mu.l more of the antibody-affigel slurries. The 
samples/gels were incubated for another hour at 4.degree. C. This process 
was repeated for a total of three sample/affigel incubations. After the 
last centrifuge spin, the supernatants were removed and run in the assay 
as samples. The supernatants were not diluted 1:2 prior to assay, because 
the PEER sample was diluted 1:25 in sample diluent buffer before the 
antibody-affigel stripping. Samples that were not stripped with 
antibody-affigel were kept at 4.degree. C. while the other samples were 
being stripped. The only two antibodies to completely strip the TCR 
.delta. reactivity from the samples were the TCAR .delta. chain specific 
antibodies .delta.TCR1 and TCS.delta.1, indicating that the assay was 
specific for TCR .delta. chain. When 8 samples were run in the total TCAR 
.delta. chain method, total TCAR .delta. ranged from 55 to 108 units (FIG. 
10). These all fell at the lower end of the standard curve. 
The success of these total TCAR methods for measuring total .beta. or total 
.delta. TCR antigen amounts can be extended to total V.alpha., total 
.gamma..delta., total .alpha..beta. or to total V.gamma., and related 
subset assay procedures as well. Detection of the total amount of the 
above .beta. and .delta. antigens is useful in cell typing and in 
correlating specific V region expression to disease state or treatment 
outcome. 
10. DEPOSIT OF HYBRIDOMAS 
The following hybridoma cell lines, producing the indicated monoclonal 
antibody, have been deposited with the American Type Culture Collection, 
Rockville, Md. and have been assigned the listed accession numbers: 
______________________________________ 
Accession 
Hybridoma Number 
Monoclonal Antibody 
-- 
______________________________________ 
Cell line 4C9 4C9 (anti-CD8) HB 9340 
Cell line 5F4/7B12 
5F4/7B12 (anti-CD8) 
HB 9342 
Cell line 8F4 8F4 (anti-CD4) HB 9843 
Cell line R2B7 R2B7 (anti-CD4) HB 9842 
Cell line 8A3.31 
8A3.31 (.beta.F1) 
HB 9283 
Cell line .delta.TCS1 
.delta.TCAR3 = .delta.TCS1 
HB 9578 
Cell line W112 W112 (V.beta.5) HB 9927 
Cell line W4F.5B 
W4F.5B (anti-C.beta.) 
HB 9282 
Cell line 5A6.E9 
TCR.delta.1 HB 9772 
______________________________________ 
The present invention is not to be limited in scope by the cell lines 
deposited since the deposited embodiments are intended as single 
illustrations of one aspect of the invention and any cell lines which are 
functionally equivalent are within the scope of this invention. Indeed, 
various modifications of the invention in addition to those shown and 
described herein will become apparent to those skilled in the art from the 
foregoing description and accompanying drawings. Such modifications are 
intended to fall within the scope of the appended claims. 
Various references are cited herein, the disclosures of which are 
incorporated by reference herein in their entireties.