WO1994010571A1 - Evaluation de la quantite totale d'une molecule dans un echantillon et procedes bases sur celle-ci - Google Patents

Evaluation de la quantite totale d'une molecule dans un echantillon et procedes bases sur celle-ci Download PDF

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WO1994010571A1
WO1994010571A1 PCT/US1993/010550 US9310550W WO9410571A1 WO 1994010571 A1 WO1994010571 A1 WO 1994010571A1 US 9310550 W US9310550 W US 9310550W WO 9410571 A1 WO9410571 A1 WO 9410571A1
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Prior art keywords
sample
amount
molecule
specific binding
cells
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PCT/US1993/010550
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English (en)
Inventor
Charles W. Rittershaus
Patrick C. Kung
George H. Parsons
Patricia S. Meisner
Alfred E. Fox
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T Cell Sciences, Inc.
T Cell Diagnostics, Inc.
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Priority to JP6511400A priority Critical patent/JPH07509783A/ja
Priority to CA002148219A priority patent/CA2148219C/fr
Priority to AU54571/94A priority patent/AU686577B2/en
Priority to EP93925154A priority patent/EP0666987A4/fr
Publication of WO1994010571A1 publication Critical patent/WO1994010571A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • the present invention is directed to methods for the direct measurement of the total quantity of a molecule in a sample.
  • the molecule may be endogenous to the host cell, or a molecule associated with a pathogenic microorganism invading a cell or a tissue.
  • the method of the present invention is applicable to diagnosis and to monitoring a subject's response to therapy in a variety of disease states.
  • CELL SURFACE ANTIGENS A multitude of molecules, including proteins, glycoproteins, glycolipids, oligosaccharides, and the like, are expressed on cell surface membranes. Also detectable either on the surface, or in the intracellular compartment are molecules produced by intracellular parasites, such as bacteria or viruses. Cell-surface molecules are also present in the cytoplasm, and are often found in soluble or shed form in body fluids. Conventional immunoassays detect or enumerate cells expressing a given molecule on their surface by visualizing or measuring the binding of an antibody to an epitope of that molecule on the cell.
  • Well-known cell surface molecules with clinical significance include human leukocyte antigens such as HLA glycoproteins, B and T cell marker glycoproteins, and B and T cell receptor molecules (Bernard et al . , 1984, Hum . Immunol . 11:1-10; Knapp et al . , 1989, Immunol . Today 10:253:258; Gebel et al . , 1988, ASHI Quarterly 22:11; McMichael, A.J., ed. , Leukocyte antigens such as HLA glycoproteins, B and T cell marker glycoproteins, and B and T cell receptor molecules (Bernard et al . , 1984, Hum . Immunol . 11:1-10; Knapp et al . , 1989, Immunol . Today 10:253:258; Gebel et al . , 1988, ASHI Quarterly 22:11; McMichael, A.J., ed
  • Mycoplasma M. pneumoniae T strains (atypical pneumonia)
  • Candida albica s Candida albicans Trichophyton spp (thrush) (athlete's foot, ringworm, etc.)
  • a leukocyte a mobile cell which is carried through the blood or lymph.
  • a mobile cell such as a leukocyte
  • leukocytes 5 include measles virus, smallpox virus, tubercle bacilli.
  • Some bacteria such as Mycobacterium tubersulosi ⁇ , Salmonella typhi or Brucella abortus do much of their multiplication in macrophages that have ingested them, though they are not obligate 0 intracellular parasites.
  • Table II below gives examples of infections in which microorganisms enter across epithelial surfaces and spread systemically.
  • Table III below lists microorganisms that regularly multiply in macrophages.
  • Organisms examples Viruses Herpes-type viruses, hepatitis virus of mice, measles, distemper, poxviruses,
  • mice 25 lymphocyte choriomeningitis virus, lactic dehydrogenase virus of mice
  • Such organisms multiply inside the cell while avoiding the intracellular defense mechanisms, such as lysosomal degradation. In some cases, the organisms multiply in phagosomes, while in others, they escape from the vacuole and multiply in the cytoplasm. The cells which harbor these organisms appear to give them biochemical and nutritional support.
  • An enzyme immunoassay for chlamydia trachomatis is commercially available (CHLAMYDIAZYME® Diagnostic Kit from Abbott Laboratories, North Chicago, IL) .
  • This method utilizes a solid phase enzyme immunoassay which detects chlamydial antigen from urogenital, endocervical, conjunctival or nasopharyngeal swabs.
  • the method is based on adsorption of antigen from the sample onto beads coated with an anti-chlamydial antibody which are then reacted with a rabbit antibody specific for a chlamydial antigen followed by an enzyme-conjugated anti-rabbit immunoglobulin antibody.
  • the method does not employ detergent treatment of a sample, and is therefore limited to that amount of chlamydial antigen in the sample which exists in a form readily available for binding to the solid phase antibody.
  • sample treating fluids which may contain a detergent, for example, saponin (Dorn, U.S. Patent 3,932,222; 1/13/76).
  • a detergent for example, saponin
  • Tumor antigens that serve as markers for the presence of a preneoplastic or a neoplastic cell.
  • Tumor antigens are classified into two broad categories: tumor-specific antigens (TSA) and tumor- associated antigens (TAA) .
  • TSA tumor-specific antigens
  • TAA tumor-associated antigens
  • Simple, rapid and sensitive assays for analysis of a sample of cells, tissue or body fluids for the total amount of a tumor antigen are highly desirable for the detection and diagnosis of cancer, as well as for monitoring cancer therapy and research efforts relating to this area.
  • the ability to detect low levels of a TSA or TAA early in the process of tumor development, when only few cells may express the antigen, is especially desirable.
  • RBC blood group antigens
  • a well-known representative of this group is the human ABO blood group which comprises antigens referred to as the ABHLe blood groups substances.
  • the antigenicity of the ABH and Le (Lewis) blood group substances resides in oligosaccharide chains of varying length. Some chains are only a few sugar residues long, while others contain up to 60 residues. The chains can exist in three forms: free, attached to proteins, or attached to lipids.
  • Free ABHLe oligosaccharides have been isolated from milk colostrum and urine.
  • Protein bound ABHLe antigens are glycoproteins secreted by goblet cells, mucus glands of the gastrointestinal tract, genitourinary tract, and respiratory tract. In addition such glycoproteins are present in amniotic fluid, milk, sweat and tears.
  • the oligosaccharide side chains are attached through N-acetyl-D- galactosamine (GalNAc) to a serine or threonine residue of the protein backbone.
  • GelNAc N-acetyl-D- galactosamine
  • Lipid-bound ABHLe antigens are glycolipids incorporated into the plasma membrane of RBC and cells of other tissues including liver, spleen, kidney, pancreas and stomach.
  • the oligosaccharide is attached by a glucose (Glc) to a sphingolipid (consisting of N-acetyl-sphingosine or ceramide and fatty acids) .
  • Glc glucose
  • sphingolipid consisting of N-acetyl-sphingosine or ceramide and fatty acids
  • GalNAc glycolipids may or may not contain this sugar.
  • ABHLe ABHLe system
  • Rh system a human blood group system of clinical importance
  • examples of other known human blood group systems include Duffy, Kidd, Lutheran, Kell, MN, Gerbich, P, I, Sda, and Pr.
  • Alterations in the expression of blood group antigens on cells other than RBC are associated with pre-malignant and tumorous conditions, in particular in cells of the gastrointestinal mucosa (Hakomori, S. et al . , J. Natl . Cane . Inst . 71 : 231 (1983); Keshvara, L. et al . , Glycocon jugate J. 9 : 16 (1992)).
  • the enzyme known as A-transferase ⁇ (l-3)N-acetylgalactosaminyl transferase converts the H-glycolipid precursor to the mature form of A-glycolipid.
  • CRl number has also been found to correlate inversely with serum levels of immune complexes, with serum levels of C3d, and with the amounts of erythrocyte-bound C3dg, perhaps reflecting uptake of complement-activating immune complexes and deposition on the erythrocyte as an "innocent bystander" (Ross et al . , 1985, J. Immunol . 135:2005- 2014; Holme et al . , 1986, Clin . Exp. Immunol . 63:41- 48; Walport et al. , 1985, Clin . Exp. Immunol . 59:547).
  • a patient with SLE lacking CRl on erythrocytes was found to have an auto-antibody to CRl (Wilson et al . ,
  • Abnormalities of complement receptor expression in SLE are not limited to erythrocyte CRl. Relative deficiencies of total cellular CRl of neutrophils and plasma membrane CRl of B lymphocytes of the SLE patients have been shown to occur (Wilson et al . ,
  • Cytokines are a large, diverse group of bioactive proteins and peptides generally having relatively low molecular weights which regulate a large number of cellular activities. For a recent reviews, see: Arai,
  • cytokines regulate immunoglobulin production by B lymphocytes and the biosynthetic activities of various cell types.
  • cytokine research is based on measurement of the biological activity of cytokines in various bioassays. More recently, cytokine levels in fluids such as blood have been measured in immunoassays, such as ELISA, for example, utilizing a pair of appropriate antibodies directed against the cytokine.
  • immunoassays such as ELISA
  • This approach suffers from several limitations when used to detect cell-derived cytokines in serum or plasma because of their vanishingly low concentrations and extremely short half lives in vivo .
  • the detectability of cytokines in serum or plasma by ELISA with monoclonal antibodies is affected by several factors, such as: (1) locally produced cytokine in vivo is quickly absorbed by target cell receptors and may never reach the sampled compartment, e .g.
  • cytokine proteins are usually unstable in plasma or serum during sample storage. These factors often render a cytokine undetectable even in a sample obtained from the particular site or tissue in which it is produced. In general, immunoassays of molecules present in serum are not useful for detecting proteins that are do not survive storage of the plasma or serum. Finally, most cytokines are known to be present in the extracellular and intracellular compartment. However, some cytokines such as TNF- ⁇ are also expressed on the membrane of producing cells (Kriegler et al . , 1988, Cell 53 (1) :45-53) . Presently, there are no available rapid and simple methods for simultaneously measuring the total amount of cytokine on the cell surface, the intracellular compartment and in soluble form in the fluid surrounding the cells.
  • Bioassays of cytokines in vitro typical measure the ability of a cytokine to stimulate or sustain growth or proliferation a cell line which is dependent on that cytokine for growth. Such assays suffer from the drawback of requiring at least many hours, and more typically, several days.
  • the cell lines used in these bioassays are responsive not only to the cytokine being assayed but to other factors which may "contaminate" the sample. Thus, to attribute a bioactivity to the suspected cytokine, it is often necessary to use a monoclonal antibody to neutralize the cytokine's activity.
  • An additional important drawback of these bioassays is the influence that various inhibitory factors present in the sample, known or not yet discovered, may have on the measured bioactivity.
  • the ability to easily detect in a sample of cells, tissue or a body fluid the total amount of a molecule, including a molecule associated with an intracellular pathogenic microorganism, would, in addition to serving as a tremendous research tool, also have great clinical potential for detection, diagnosis, staging and monitoring therapy in a number of disease states, such as infectious diseases, cancer and immunological disorders.
  • the present invention provides methods for measurement of the total amount of a molecule (termed hereinafter "target molecule”) in a sample in the presence of a non-ionic detergent, and the use of such measurements in the detection, diagnosis, staging, and determining the severity of diseases or disorders, monitoring of therapy, various research endeavors, or enumeration of cells expressing the target molecule.
  • Measurements of the total amount of a target molecule expressed by a cell, which is uniformly expressed, for example a platelet antigen can be used to determine the approximate number of cells, e .g. , platelets, bearing the antigen in a sample such as a body fluid sample.
  • thrombocytopenia and other disorders involving aberrant levels of platelets can be diagnosed.
  • the method of the present invention can serve in early diagnosis, prognosis, or in monitoring antimicrobial therapies.
  • blood group antigens such as the ABH antigens
  • such measurements may be used in blood typing as well as in early detection of pre-malignant changes in mucosal cells.
  • the measurements of the total amount of a target molecule is accomplished by treating a sample containing cells, such as a body fluid, in a manner that make available for binding to a binding partner the total amount of the target molecule present in the cell membrane, the cytoplasm of the cell and/or in the soluble compartments of the sample.
  • the binding partner is an antibody or antibody fragment containing the binding domain thereof.
  • the sample is preferably an original sample, i.e., in the form substantially as it was obtained, and is treated with a non-ionic detergent prior to contact with its binding partner.
  • the total target molecule is made available, it is measured using any detection method known in the art, and preferably using any of a number of immunoassay techniques, most preferably by sandwich enzyme immunoassay (EIA) .
  • EIA sandwich enzyme immunoassay
  • a method for typing blood by detecting an erythrocyte blood group antigen in a sample from a subject.
  • the blood group antigen is the A antigen.
  • the above methods may also be used to measure the total amount of the blood group antigen.
  • the methods are used to determine in a sample the total amount of a target molecule, in which the target molecule is a cytokine, a tumor-specific or tumor-associated molecule, a cell adhesion molecule, a multidrug resistance marker, complement receptor, or cellular DNA (the latter preferably to detect polyploid cells) .
  • the target molecule is a cytokine, a tumor-specific or tumor-associated molecule, a cell adhesion molecule, a multidrug resistance marker, complement receptor, or cellular DNA (the latter preferably to detect polyploid cells) .
  • the present invention is also directed to a method for detecting the presence of malignant or premalignant cells in gastrointestinal mucosal tissue, the tumor associated with a decrease in the blood group A antigen, wherein a decrease in the amount of specific binding or blood group A antigen is indicative of the presence of malignant or premalignant cells.
  • an improvement in patient condition e.g., remission
  • positive response to therapy is accompanied by a decrease or increase in the total amount of the target molecule, or a disappearance thereof.
  • the present invention is directed to the detection and/or measurement of the total amount of a molecule (hereinafter "target molecule") in a sample and the use of such detection or measurement in the detection, diagnosis, staging, determination of severity, and therapy of diseases and disorders, and in the enumeration of cells expressing the target molecule.
  • target molecule a molecule
  • Use of a detergent in the assay methods provided by the invention allows detection and/or measurement of target molecules heretofore not readily accessible for detection by binding to a binding partner such as an antibody.
  • the target molecule may be an "endogenous" target molecule, which as used herein, refers to a molecule that is encoded by the genome of the host cell or is produced by the host cell not due to the presence of an exogenous organism.
  • endogenous target molecules are erythrocyte antigens, platelet antigens, cytokines, cell-adhesion molecules, multidrug resistance markers, cellular DNA and the like, which are discussed in more detail below.
  • the target molecule may also be an "exogenous" target molecule.
  • exogenous target molecule refers to a target molecule (a) encoded by a genome not of the host cell; or (b) otherwise produced by an exogenous agent.
  • Typical exogenous target molecules which may be measured according to the present invention are molecules associated with microbial pathogens (see below) .
  • total amount of a target molecule refers to the total amount of the target molecule in a sample, including target molecules which may be present in or bound to the cell membrane, in any of a number of intracellular compartments including the cytoplasm and nucleus, and in the extracellular soluble compartments of the tissue, fluid or biological sample, which are made available for detection by use of the assay methods provided by the invention.
  • the soluble compartment may include an endogenous, spontaneously released, soluble target molecule or an exogenous, soluble target molecule.
  • the exogenous target molecule may be one which was purposely administered to the subject, for example, a therapeutic cytokine.
  • the total amount of target molecule includes the amount of target molecule present in the membrane, intracytoplasmic and cell culture media compartments of the sample.
  • the present invention provides an assay method of a sample treated with non-ionic detergent, that measures the total amount of a target molecule present in all three of these compartments simult neously.
  • the method of the present invention measures the total amount of a target molecule in the intracytoplasmic and membrane compartments, where the cells have been rendered free of the released/soluble compartment, for example by washing the cells free of the serum or cell culture supernatant prior to solubilization with detergent.
  • the method is used to measure the total amount of a target molecule in the membrane compartment, wherein the intracytoplasmic and soluble compartments have first been removed leaving just the cell membranes to be analyzed.
  • the method is used to measure the total amount of a target molecule in the released/soluble compartment, for example, the serum, body fluid or culture medium, from which the cells have first been removed prior to solubilization with detergent.
  • the total amount of target molecule may include the amount of the target molecule present in any one compartment or in any combination of compartments depending upon the nature of the sample. For example, if a sample of cells has been washed free of culture medium or serum/plasma (or other body fluid in which the cells were obtained) , then the total amount of target molecule would include the membrane-associated and cytoplasmic compartments only.
  • Non-limiting examples of target molecules for which the present invention is intended to apply are proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, and the like, or fragments thereof.
  • Preferred target molecules according to the present invention are blood group antigens, microbe-associated molecules, cytokines, adhesion molecules, hormones (e.g., insulin), and the like.
  • the invention provides a method for detecting and/or measuring the total amount of a target molecule which is intracellular (i.e., a molecule which is not accessible on the cell surface, and thus, not, for example, a surface antigen) .
  • the target molecule is a cell surface molecule.
  • the target molecule is not a leukocyte cell surface marker (a "leukocyte cell surface marker" being an antigen or polypeptide found on the cell surface of a leukocyte) .
  • the measurement of a total amount of target molecule as described herein can substitute for direct enumeration of cells expressing the target molecule, and can be valuable in cell typing, monitoring the effect of a therapeutic treatment on a subject, detecting, diagnosing or staging a disease in a subject, in predicting therapeutic outcome or disease prognosis and in evaluating and monitoring immune status of patients.
  • two or more distinct target molecules can be measured separately, preferably in a concurrent fashion.
  • the measured amount of the target molecule in a sample from a patient is compared to a baseline level of target molecule that is the level established to be present in a comparable sample not affected by the patient's disorder or from a patient without the disorder or from the patient at an earlier time.
  • Binding partners for use in the assays of the invention include but are not limited to receptors for and antibodies to the target molecules.
  • binding partners which are not antibodies include cell surface receptors for the target molecules. Additional and specific examples of non- antibody binding partners are described in the subsections below.
  • other non-antibody binding partners include those used as binding partners in the following assays: for assay of CD4
  • the target molecule is an antigen
  • detection and/or measurement of the target molecule is carried out by a method comprising immunospecific binding of the target molecule to at least one antibody. All antigens which carry an antigenic determinant or epitope which can be detected or quantitated in an immunoassay are intended to be within the scope of the present invention.
  • a preferred subject for the methods of the present invention is a vertebrate, including but not limited to a mammal, fish, amphibian, reptile, bird, marsupial, and most preferably, a human.
  • the methods and kits of this invention are applicable to human clinical and veterinary uses.
  • a sample which is subjected to testing according to the present invention is a sample derived from a biological cell or organism, and is preferably an "original sample" obtained from the subject and, in particular, which is not subjected to processing such as any fractionation steps (e.g., fractionation to separate cells from extracellular fluid or one type of cell from another type, or to remove certain subcellular components (e.g., membrane fraction, cytoplasmic fraction, nuclear fraction) ) .
  • the sample may be subjected to one or more processing steps, such as washing of and enrichment of cells, or isolation of the fluid portion of the sample.
  • the sample includes, but is not limited to, any biological fluid, preferably a body fluid.
  • body fluids include, but not limited to, whole blood, serum, plasma, urine, synovial fluid, cranial or spinal fluid, saliva, tissue infiltrate, cervical or vaginal exudate, tissue infiltrate, pleural effusions, bronchoalveolar lavage fluid, gastric lavage fluid, small or large bowel contents, fecal preparations, and the like.
  • a sample may be a swab specimen from mucosal surface or a body orifice, for example, oral, anal, urogenital, endocervical, conjunctival or nasopharyngeal.
  • the biological fluid may be a cell culture medium or supernatant of cultured cells.
  • the sample can be a biopsy specimen or other tissue sample.
  • the sample can comprise any type of cell including but not limited to a blood cell (erythrocyte, neutrophil, eosinophil, monocyte, macrophage, T lymphocyte, B lymphocyte, etc.), epithelial cell, endothelial cell, neuron, glial cell, etc.
  • the method provided by the present invention overcomes many of the limitations of prior art methods, in particular as regards the measurement or detection of cell-surface target molecules, which heretofore required cell isolation followed by direct or indirect immunofluorescence analysis by microscopy or flow cytometry.
  • Limitations of the prior art procedures include the requirement for: (1) fresh samples, (2) fairly large sample sizes or a large number of cells, (3) enriched cell populations rather than whole tissue or blood, (4) extensive preparation time, and (5) expensive equipment, such as a flow cytometer.
  • the methods provided herein overcome these limitations.
  • the present method is well-suited for the measurement of the total amount of an endogenous target molecule in a sample.
  • the prior solubilization of the cells in a sample in order to make available the total amount of a cellular target molecule, such as but not limited to a cell-surface antigen, in a sample is a major improvement over existing approaches.
  • the fact that the methods disclosed herein do not require fresh samples is also highly advantageous for measuring a cell-surface target molecule.
  • Each patient sample can be treated with the detergent preparation, as described herein, and stored frozen. This is especially useful for analysis of a 5 series of samples obtained from the same patient over a period of time, as in a longitudinal study. All samples can then be thawed and analyzed concurrently. This is a definite improvement over flow cytometric analysis where fresh intact cells are generally
  • the target molecule is not a leukocyte cell surface marker such as CD4, CD8, T cell antigen receptor, etc.
  • the present invention provides a method to detect or measure the total
  • the invention provides a method of typing blood by detecting blood group markers on red blood cells by use of an assay of the invention.
  • blood group markers include but
  • the total amount of a blood group antigen from the ABO group is not limited to A, B, H, Lewis antigens, Rh system antigens, and others known in the art, for example, Duffy, Kidd, Lutheran, Kell, MN, Gerbich, P, I, Sda, and Pr (see Section 2.4 ⁇ upra) .
  • the total amount of a blood group antigen from the ABO group is not limited to A, B, H, Lewis antigens, Rh system antigens, and others known in the art, for example, Duffy, Kidd, Lutheran, Kell, MN, Gerbich, P, I, Sda, and Pr (see Section 2.4 ⁇ upra) .
  • the total amount of a blood group antigen from the ABO group is not limited to A, B, H, Lewis antigens, Rh system antigens, and others known in the art, for example, Duffy, Kidd, Lutheran, Kell, MN, Gerbich, P, I, Sda, and Pr (
  • antigen A may be detected or measured by detergent treatment of a whole blood sample, for example, followed by assay using one or more binding partners to the blood group antigen, e.g., anti-A antibodies.
  • transferase enzyme associated with premalignant changes in gastric mucosa may be measured by providing to the detergent treated blood or cell or tissue sample the A precursor and assaying for the A antigen product formed by the action of A transferase in the sample. Assay of sequential samples for the A antigen or the A transferase, preferably samples of mucosal tissue from the gastrointestinal tract, is used to detect changes leading to carcinoma formation, or to monitor the therapy of a premalignant or malignant lesion associated with a fall in A antigen.
  • the methods of the present invention are useful for detecting changes in cells in their progression from a normal to a neoplastic or malignant state.
  • the measurement in a sample of the total amount of a tumor-specific or tumor-associated antigen allows early detection of precancerous or cancerous stages.
  • the method of the present invention can measure the total amount of an antigen in the cell- membrane, intracellular and extracellular fluid compartments simultaneously, in contrast to convention methods, less antigen need be present to pass the detection threshold. This would allow earlier diagnosis than is currently available.
  • new classes of tumor-specific or tumor-associated molecules for example, those that are only expressed intracellularly, can be identified and detected in a cell, tissue or body fluid sample using the present methods.
  • the present methods can therefore detect cells in the process of progression to neoplasia or cancer, including stages of non-neoplastic cell growth including hyperplasia, metaplasia, or most particularly, dysplasia, where expression of a new or normally unexpressed target molecule is associated with these steps.
  • stages of non-neoplastic cell growth including hyperplasia, metaplasia, or most particularly, dysplasia, where expression of a new or normally unexpressed target molecule is associated with these steps.
  • hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • endometrial hyperplasia often precedes endometrial cancer.
  • Metaplasia is a form of controlled cell growth in which one type of adult, or fully differentiated, cell substitutes for another type of adult cell. Metaplasia occurs in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. The most disordered form of non-neoplastic growth is dysplasia, a frequent precursor of cancer, which occurs mainly in the epithelium, is the most disordered form of non- neoplastic cell growth. A dysplastic change involves the loss of individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply staining nuclei, and exhibit pleomorphism.
  • Dysplasia characteristically occurs at sites of chronic irritation or inflammation, typically in the cervix, respiratory passages, oral cavity, and gall bladder.
  • abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia
  • the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or in vitro by a cell sample from a patient, can indicate the desirability of prophylaxis or therapy.
  • Characteristics of a transformed phenotype in a cell include morphological changes, looser attachment to the substratum, loss of contact inhibition, loss of anchorage-dependence, protease release, increased sugar transport, decreased serum requirement for in vitro growth, expression of fetal antigens, disappearance of particular cell surface proteins, etc.
  • characteristics associated with a transformed or malignant phenotype see, for example, Robbins and Angell ( ⁇ upra) , pp. 84-90.
  • Such characteristics can be detected using the methods of the present invention when the change involves the appearance of a new tumor-specific antigen, the inappropriate or increased expression of a tumor-associated antigen, or the loss or diminution of a normal cellular antigen.
  • Non-limiting examples of pre-neoplastic lesions include leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, Bowen's disease, a carcinoma in ⁇ itu , fibrocystic disease (cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia) ) .
  • Tumor antigens which can thus be detected or measured according to the invention include but are not limited to carcinoma-associated antigen, breast cancer-associated antigen, melanoma-associated antigen, melanoma-associated pigmentation antigen, melanoma-associated proteoglycan; an erb, ras, myc, neu, or rel oncoprotein; oncoprotein p53, etc. (see, e.g., Klein, J. , supra , pp. 419-428; see also Section 2.3 supra) .
  • Monoclonal antibodies to the above-listed antigens, and others, are commercially available (see Lin ⁇ cott ' ⁇ Director of Immunological and Biological Reagents, 1992, 7th Ed., Santa Rosa, CA) and are envisioned for use in immunoassays of the invention.
  • the present invention is useful in a method for determining the propensity of blood to clot in response to injury by measuring the total amount of a platelet-associated target molecule, or assessing the number of platelets, in a sample using an immunoassay.
  • Current methods rely on counting platelets in blood, either microscopically, which is highly tedious, or in an automated counter.
  • a blood sample, or other platelet-containing sample is treated with non-ionic detergent to solubilize and uniformly disperse platelet-associated target molecules.
  • the target molecule is a platelet antigen
  • antibodies to platelet antigens which are well-known in the art (see, for example, Lin ⁇ cott ' ⁇ Director of Immunological and Biological Reagents, supra, at p. 18) are used to measure the total platelet-associated antigen in an immunoassay. Binding partners other than antibodies can also be used.
  • the platelet glycoprotein GPIIb-IIIa binds to the sequence Arg-Gly-Asp-Ser (see Bacon-Baguley et al . , 1987, J. Biol . Chem . 262 : 1921-20) .
  • the Arg-Gly-Asp-Ser sequence or proteins comprising the same can be used as binding partners to detect and/or measure platelet GPIIb-IIIa.
  • Methods of the invention for measuring platelet-associated target molecules are useful, for example, in the diagnosis of thrombocytopenia (decreased number of platelets) and in diagnosing, staging or monitoring therapy of any disease or disorder associated with altered platelet numbers relative to a baseline level.
  • thrombocytopenia decreased number of platelets
  • diagnostics of any disease or disorder associated with altered platelet numbers relative to a baseline level.
  • such disorders include but are not limited to side effects of medical treatments.
  • the baseline level is the level in a healthy individual or a standard level determined to be associated with disease absence or remission.
  • the total amount of a cell adhesion molecule in a sample is detected or measured according to the methods described herein. Any known cell adhesion molecule for which a specific binding partner is available can be detected or measured, as the case may be.
  • Examples of such molecules include molecules of the ⁇ 2 integrin family, including the leukocyte cell adhesion molecules, the complement receptor family such as CR3 and CR4, ICAM-1 and ICAM-2, and members of the ⁇ l integrin family including the homing receptors such as CD44 and MEL-14, the VLA proteins, LFA-1, MAC- 1, P150,95, and the like.
  • selectins which represent another cell adhesion molecule family present on various cell types. Examples of selectins on endothelial cells include CD62 (also termed GMP140 or P-selectin) , LECCAMs, ELAM-1 (also termed E-selectin) , lymph node addressin (also termed MECCA-79) , etc.
  • Target molecule-binding partner pairs other than antibody-antigen pairs which can be used to detect or measure an adhesion molecule according to the invention include but are not limited to those set forth in Table IV (see Butcher, 1991, Cell 67:1033-36) :
  • the cell types listed in Table IV above or subpopulations thereof can be used as sources of the respective member of the binding pair for purification of said member, e.g., by immunoaffinity chromatography of blood cell extracts.
  • immunoaffinity chromatography See also Larsen et al . , 1989, Cell 59:305-312 for a CD62 purification method; Lobb et al . , 1991, J. Immunol . 147:124, for ELAM-1; and Berg et al . , 1991, J. Cell Biol . 224:343, for lymph node addressin
  • Chemical synthesis, recombinant methods, or commercial sources may also be used to obtain the desired adhesion molecule binding partner.
  • ICAM-1, ICAM-2, and VCAM-1 have been cloned and sequenced (see Staunton et al . , 1988, Cell 52:925-933) , European Patent Application Publication 387,688, published September 19, 1990; Osborn et al. , 1989, Cell 59:1203; Polte et al . , 1990, Nucl . Acid ⁇ Re ⁇ . 28:5901) .
  • measurement of adhesion molecules according to the invention can be used to diagnose, stage, determine the severity of, or monitor various diseases or disorders.
  • diseases or disorders in which an increased amount of an adhesion molecule is detected or measured relative to a baseline level include but are not limited to disorders involving an undesirable inflammatory response such as: inflammatory arthritis (rheumatoid arthritis, seronegative spondyloarthritides, juvenile rheumatoid arthritis, vasculitis, psoriatic arthritis, polydermatomyositis) , systemic lupus erythematosus (SLE) , asthma, inflammatory dermatoses (psoriasis, dermatitis herpetiformis, eczema, necrotizing and cutaneous vasculitis, bullous diseases) , reperfusion injury, septic shock (sepsis) , adult respiratory distress syndrome, tissue damage relating to tissue transplantation, thermal injury (burn) , other autoimmune disorders [in addition to SLE and rheumatoid arthritis: glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic
  • such diseases or disorders in which a decreased amount of an adhesion molecule is detected or measured relative to a baseline level include but are not limited to leukocyte adhesion deficiency (Anderson and Springer, 1987, Ann. Rev. Med . 38:175-194), which involves an inherited deficiency in the integrins LFA-1, Mac-1, and pl50,95; and diabetes mellitus, granulocytasthenia, and recurrent pyogenic infections, which have been reported to involve cell adherence defects (Gallin et al . , 1980, Ann . Int . Med. 92:520-538).
  • the baseline level is that level established to be present in a comparable sample not affected by the disorder or from a patient without the disorder or from the patient at an earlier, healthy time.
  • detection and/or measurement of a cell adhesion molecule in a sample according to the invention can be carried out in order to assess the metastatic potential of a tumor.
  • the invention provides methods of determining metastatic or invasive potential of a malignant tumor in a subject by detecting and/or measuring the total amount of a cell adhesion molecule in a sample from a tumor-bearing subject.
  • the detection or measurement of abnormal levels of a cell adhesion molecule relative to a baseline level in normal cells from the subject or to a standard level established for non- metastatic tumor cells or normal cells indicates that the tumor can metastasize.
  • measurements of the following adhesion molecules are envisioned to be useful for determining metastatic potential of the indicated tumor types: LFA-1, the ⁇ l integrin subunit CD29, CD31 (PECAM-1) , and CD44, which are implicated in metastatic spread of lymphomas (Roos, E. , 1991, Cancer-Meta ⁇ ta ⁇ i ⁇ Rev.
  • the amount of double stranded and single stranded DNA (deoxyribonucleic acid) in a cell is a useful marker for the presence of malignant or pre-malignant cells.
  • Normal euploid cells have only a single "copy" of cellular DNA and, in the human, 46 chromosomes, whereas malignant cells are often polyploid (diploid, tetraploid, etc.) having, for example, doubled or quadrupled the normal chromosome complement.
  • polyploidy is assessed by flow cytometric analysis of total cellular DNA using DNA binding dyes.
  • the present invention provides an assay for polyploidy by measuring the total amount of DNA present in a sample, preferably from a tissue or a suspension of single cells, in an assay following pretreatment of the cells with non-ionic detergent as described herein, for example with TRAxTM reagent, to make the DNA available for binding by a DNA binding partner.
  • DNA binding partners include but are not limited to DNA binding proteins and anti-DNA antibodies, which are well-known in the art (see, for example, Lin ⁇ cott ' ⁇ Directory, supra, at p. 50). This method allows a laboratory lacking a flow cytometer to detect polyploidy or measure the amount of DNA in a cell or cells by direct assay, thus providing a way of detecting a tumor in a cellular sample.
  • the present invention provides a method for rapid detection of a cytokine present at the cell surface and/or intracellularly and/or in the extracellular fluid compartment of a biological sample.
  • the method is used to detect a cytokine in a sample of whole blood, including a stored sample of blood.
  • the present method allows much greater reliability in measuring cytokine and flexibility in scheduling assay procedures to allow concurrent assay of multiple samples from the same or different subject, thereby decreasing interassay variability.
  • the method of the present invention overcomes the shortcomings of existing immunoassays or bioassays of cytokines, discussed above, by measuring the total amount of cytokine in a sample, which contains cells (including the membrane-bound and intracytoplasmic content) as well as the fluid, such as the blood, in which the cell is collected.
  • the method of the present invention to measure a cytokine can provide information of cytokine level associated with the producing cells, allowing the approximation of the number of cytokine-producing cells in a sample.
  • the present invention also provides a method to estimate the number of cells producing a cytokine; a panel of control cells can be used as standards to determine a conversion factor for converting cytokine levels measured according to the invention into cell equivalent units.
  • the methods of the present invention are useful for measuring the total amount of any cytokine, including, but not limited to interleukin 1 (IL-1) , interleukin 2 (IL-2) , interleukin 3 (IL-3) , interleukin 4 (IL-4) , interleukin 5 (IL-5) , interleukin 6 (IL-6) , interleukin 7 (IL-7) , interleukin 8 (IL-8) , interleukin 9 (IL-9) , interleukin 10 (IL-10) , interleukin 11 (IL-11) , interleukin 12 (IL-12) , monocyte chemoattractant protein-I (MCP-I) , granulocyte-macrophage colony stimulating factor (GM-CSF) , granulocyte colony stimulating factor (G-CSF) , macrophage colony stimulating factor (M-CSF) , tumor necrosis factor ⁇ (TNF ⁇ ) , tumor necrosis factor ⁇ (TNF
  • the present method of the present invention is used to measure total IL-4 in a blood sample to monitor the level of IL-4 producing lymphocytes in an allergy patient undergoing allergen im unotherapy; for example, in which the presence of or increased levels of IL-4 indicates an allergic response, or increased severity thereof and vice versa.
  • IL-2 can also be measured to detect T cell anergy (non-responsiveness) , wherein a decrease in IL-2 is indicative of T cell anergy.
  • the method is used to monitor the level of lymphocytes producing one or more interferons in an infected patient.
  • the present method is used to monitor a transplant patient for the level of lymphocytes producing one or more interleukins, in particular during rejection episodes (correlating with increased expression of interleukins) and following immunosuppressive therapy.
  • total cytokine is measured as a means of monitoring cytokine-producing blood cells in patients with a hematological diseases in relation to therapy and/or disease relapse, or to monitor immune activation.
  • Any binding partner specific to the cytokine target molecule can be employed in the assay methods of the invention.
  • a cell surface receptor for the cytokine can be used.
  • the IL-2 receptor can be used to assay for IL-2 (see Rubin et al . , 1986, J. Immunol . 137:3841-3844) .
  • an antibody binding partner is employed.
  • a multidrug resistance marker such as P-glycoprotein, the product of the MDRl gene (Twentyman et al . , 1992, J. Natl . Cancer Inst . 84:58-60; Holzmayer et al . , 1992, J. Natl . Cancer Jnst. 84:1486-1491; Clarke et al . , 1992, J. Natl . Cancer In ⁇ t . 84:1506-1512) can be detected or measured by the assay of the invention.
  • the invention provides a method for detecting multidrug resistance (i.e., lack of sensitivity to drugs commonly used to treat cancer such as anthracyclines, vinca alkaloids, and epipodophyllotoxins) or a poor prognosis for a patient with a malignancy, by detecting or measuring the amount of P-glycoprotein in a sample from the patient containing or suspected of containing cancer cells.
  • Such cancer cells include but are not limited to cells of leukemias, lymphomas, myelomas, breast cancers, esophageal carcinomas, childhood sarcomas, neuroblastomas, lung cancers, in particular small cell lung cancers, ovarian carcinomas, and other tumors, and, in.particular, metastatic cancers.
  • An increase in the measured amount of P-glycoprotein in the sample relative to the level of P-glycoprotein present in comparable cells which are not multidrug resistant or which are not cancer cells indicates that the cells in the sample from the patient are multidrug resistant.
  • Comparable cells are preferably cells of the same type as the cells present in the patient sample.
  • the method of the invention has utility in monitoring the response of cancer patients to chemotherapy, or in determining the course of therapy.
  • Antibodies to P-glycoprotein are known in the art, e.g., C219, JSB-1, and MRK16 (see e.g., Wishart et al . , 1990, Br. J. Cancer 62:758-761; Thiebaut et al . , 1989, J. Hi ⁇ tochem . Cytochem . 37:159-164;
  • the present invention is particularly suitable for the detection or measurement of total amount of a complement receptor that may be found in a fluid, cell or tissue sample. Especially useful in this regard is the detection of CRl (CD35) or CR2 (CD21) on the surface of blood cells or other cells.
  • CRl complement Receptor Type II
  • B cells and T cells are important sources of CRl.
  • An important source of CRl is the red blood cell, which is notably quite fragile when compared to other blood cells.
  • the present invention provides an alternative assay which enables one to determine and measure the presence of CD35 in a safe and efficient manner. Measurement of CD35 is useful in many research and disease associated situations, such as those set forth in Section 2.4 above.
  • the CD35 detection is relevant to HIV infections, wherein alterations in amount of red blood cell CRl could be measured as a function of the progression of the disease. Appropriate selection of a course of therapy and the success of such therapeutic intervention may also be tracked by measuring this red blood cell marker.
  • Binding partners suitable for use in the methods of the invention for detection of complement receptor are those capable of binding to all or a portion of the target complement receptor molecule.
  • Especially preferred are antibodies to CD35, opsonins capable of binding to complement receptor, as for instance C3b, C4b, (binds CRl), iC3b (binds CRl and CR2) , C4d, C3d, C3dg (binds CR2) , and the like.
  • EXOGENOUS TARGET MOLECULES For detection of an exogenous target molecule, preferably a molecule associated with a pathogenic microorganism (e.g. an antigen of a microbial pathogen) , any clinical specimen may be used, typically a sample of body fluid, a tissue specimen, or a swab from a mucosal surface.
  • a target molecule "associated with" a pathogenic microorganism shall mean a molecule whose presence is indicative of the presence of the microorganism, e.g., a pathogen-specific surface or envelope antigen, core protein, nucleic acid binding protein, enzyme, etc.
  • Swab specimens are obtained from sites suspected of containing the microorganism or containing cells within which the microorganism is growing.
  • sites suspected of containing the microorganism or containing cells within which the microorganism is growing include oral, nasopharyngeal, conjunctival, urogenital, endocervical, anorectal, and the like.
  • Such a swab may then be placed in a transport tube containing a buffer or, preferably, the lysing reagent (detergent) .
  • the swab is moistened with the lysis reagent and transported in this form to the laboratory for assay.
  • the lysis reagent, containing the released antigens may be assayed directly or stored frozen, as described above.
  • the method of the present invention is especially advantageous for detecting the presence of, and thus diagnosing infection by, intracellular pathogens, since molecules associated with such pathogens may be sequestered within the cell and thus unavailable for detection by prior art assay methods.
  • the assay methods of the invention can also be used to determine the severity of, stage, or monitor therapy of an infection by a microbial pathogen. For example, measurement of the amount of a microorganism- associated target molecule in a sample can be used as an indication of the severity of infection; the number of infectious units/pathogens in the sample can be calculated from such amount by using a conversion factor previously determined using known amounts of infectious units, which reflects the relationship between the amount of target molecules and number of infectious units.
  • the organism to be detected is an intracellular pathogen
  • the number of cells required is a function of the nature of the microorganism, the quantity of the target molecule per cell, the type and affinity of the binding partner used in the assay, and the like, readily determinable by one skilled in the art. If the microbe-associated target molecule is not secreted or available on the infected cell surface, the cells must be treated to release the organism or its target molecule from the cell in a manner in which it is available for binding to a binding partner, and thus detectable, with preferably no further manipulation or with minimal need for further sample handling.
  • the target molecules for example, free in the cytoplasm, or in vacuoles, vesicles or phagosomes, it is preferred to release the maximal amount of target molecule from such compartments.
  • Treatment with detergent as described herein is the preferred method for making the target molecule available to a binding partner for detection.
  • One of ordinary skill in the art will appreciate the applicability of the present invention to measurement of any target molecule for which an appropriate binding partner is available.
  • antibodies for a wide variety of infectious microorganisms, or for antigens associated with these microorganisms are commercially available and readily adaptable for use according to the methods disclosed herein.
  • a non-limiting list of the organisms which can be detected (or whose toxic products can be detected) using the method of the present invention appears in Table V, below, and in Tables I, II, and III, ⁇ upra .
  • Antibodies which allow detection of the above organisms can be found listed in Lin ⁇ cott ' ⁇ Directory of Immunological and Biological Reagent ⁇ , Seventh Edition, 1992, Santa Rosa, CA, pp. 43-48. Also listed therein are the commercial sources for such antibodies.
  • exemplary immunoassay configurations and antibodies for detection and/or measurement of a hepatitis B virus antigen are found in U.S. Patent No, 4,474,878 and U.S. Patent No. 4,642,285.
  • Binding partners which are not antibodies or derivatives or fragments thereof can also be used in the assays of the invention.
  • cell- surface receptors for microbial pathogen-associated target molecules can be used as binding partners for such target molecules to detect and/or measure the amount of the target molecules.
  • ICAM-1 is a binding partner which can be used to detect and/or measure a rhinovirus-associated target molecule
  • CD4 is a binding partner for gpl20 or gpl60 associated with Human Immunodeficiency Virus (HIV), etc.
  • One major advantage of the present method is particularly relevant for infected samples.
  • the minimum sample preparation required in the method of the present invention substantially lowers risk of creating aerosols that are hazardous to the laboratory worker.
  • the solubilization procedure using concentrated detergent also inactivates enveloped viruses, such as HIV-1, thereby making subsequent analysis safer.
  • the p24 antigen of HIV (the causative agent of AIDS) is detected or measured.
  • diagnosis of infection by Chlamydia trachomati ⁇ is carried out by detecting Chlamydial antigen (Schachter and Dawson, 1978, Human Chlamydial Infection ⁇ , PSG Publishing Co., Inc., Littleton, MA) by the assay of the invention using samples which are urogenital, endocervical, conjunctival, or nasopharyngeal swab specimens.
  • the amount of total target molecule is determined in an assay wherein the target molecule is bound to a specific binding partner.
  • the assay is an immunoassay wherein one or more epitopes of the target molecule (antigen) is recognized by a specific antibody or antibodies.
  • two different epitopes of the antigen are bound by two different antibodies in the course of the immunoassay.
  • the sample being analyzed is first treated to solubilize the cellular components (step 1) . It is important that the cells be solubilized without damaging the integrity of the target molecule being measured, which would interfere with binding to its binding partner.
  • a preferred solubilization method is to treat the cells with concentrated non-ionic detergent to efficiently lyse the cells, followed by dilution of the detergent- treated sample prior to assay. Other methods of solubilizing cells, such as repeated freeze-thaw cycles, sonication, hypotonic shock, or the addition of lower concentrations of detergents, are not as effective. Ionic detergents such as sodium dodecyl sulfate (SDS) are not effective, because they interfere with the subsequent target molecule-binding partner binding.
  • SDS sodium dodecyl sulfate
  • Non-ionic (includes zwitterionic) detergents for use in present invention include but are not limited to Triton X-100 (Octylphenol-polyethlene glycolther) , Nonidet P-40 (NP-40, "Octylphenol-ethylene oxide condensate”) , Tween-20 (Polyoxyethlene sorbiton monolaurate), CHAPS (3-[ (C3-cholamido propyl-dimethyl- ammonio]l-propane sulfate, inner salt), CHAPSO (3-[(3- Cholamido propyl-dimethyl-ammonio]2 hydroxypropane sulfonate) , BIGCHAP (N,N-Bis(3-D-gluconamido propyl)deoxycholamide) , Deoxy-BIGCHAP (N,N-Bis(3-D- gluconamido propyl)deoxychlamide) , (3-
  • Sulfopropyl)dimethyl (3-methacrylamido-propyl ammonium inner salt) to mention a few.
  • One skilled in the art may refer to publications such as various reagent catalogs for additional non-ionic detergents that may be appropriate for use in accordance with the present invention.
  • One such reagent supplier is Sigma Chemical Company, located in St. Louis, Missouri, USA.
  • the sample is solubilized with Triton X-100, Nonidet P-40, Tween-20 and/or CHAPS.
  • the sample is solubilized with concentrated detergent to give a final detergent concentration of about 2% to about 4% in the detergent-treated sample.
  • the total volume of non-ionic detergent added to the sample should not dilute out the target molecule in the sample.
  • 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.
  • 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.
  • more than one non-ionic detergent is used to treat the sample.
  • a relatively high concentration of Tween- 20 and Triton X-100, or Triton X-100 and NP-40, or NP- 40 and Tween-20 can be used.
  • a prepared concentration of each detergent ranges from about 1% to about 2% after addition to the sample; where the total preferred concentration of detergent in the detergent- treated sample would then range 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%.
  • the final detergent concentration in the sample is 1.5% Triton X-100 and 1% NP-40.
  • the detergent concentration is a concentration that inactivates bacteria, viruses, or other pathogens. It is a particular advantage of the invention that the lytic concentrations of non-ionic detergent, such as the preferred ranges set forth above, are also typically pathogen-inactivating concentrations.
  • the sample is preferably diluted with buffer (step 2) prior to analysis for detection of target molecule.
  • the dilution can be 2-fold; preferably it is 5-fold or greater.
  • the buffer is chosen to be compatible with the assay for detecting the target molecule.
  • Dilution of the sample may be performed with any of a number of buffers well-known in the art.
  • a preferred buffer is phosphate buffered saline, preferably supplemented with protein, with no stabilizers added.
  • an important component of the methods of the present invention is the simple direct technique used to solubilize cells in a sample using a non-ionic detergent, making the target molecule available to its binding partner while conserving recognition by its binding partner.
  • the target molecule is made available to an antibody or a combination of a capture antibody and a detection antibody, while maintaining antigenic integrity of the antigen to be detected.
  • a sample comprising 100 ⁇ l of whole blood is assayed for total amount of a target molecule.
  • the target molecule is a blood cell antigen
  • a sample of 10-25 ⁇ l of whole blood is used.
  • the amount of sample used is a function of the type of target molecule and its abundance in the preparation, and can be determined by one of ordinary skill in the art without undue experimentation.
  • a blood sample is spotted on filter paper, and the filter paper is dried and then placed in a vial containing detergent for cell solubilization.
  • a solubilized sample may be stored frozen after detergent treatment, for example, at -20°C, preferably at -70°C. This has the advantage of allowing the concurrent analysis of multiple samples taken at different times in a single assay, thus reducing interassay variability.
  • a sample can be lyophilized by methods known in the art (see, e.g., U.S. Patent No. 5,059,58) and then stored prior to detergent addition. At the desired time, the lyophilized sample can be retrieved, and detergent added directly to the sample for assaying according to the invention.
  • an immunoassay is carried out such as a sandwich or a competitive EIA (enzyme immunoassay) (see Examples, below) .
  • the immunoassay utilizes a CELLFREE® assay kit.
  • a modification of the above-described method can also be used to assay the total amount of a plurality of two or more target molecules.
  • the relative amounts of two or more target molecules may be compared.
  • Measurement of the total amount of a target molecule is an improvement over measuring only the cell bound target molecule, the target molecule in a cell lysate or the soluble target molecule, for the following reasons.
  • the measurement can provide information of the total amount of target molecule present in all three compartments, not just the amount present in one or two compartments.
  • the measurement of total target molecule is simpler than other procedures that involve more cumbersome steps of sample preparation, complex equipment and time.
  • small quantities of sample e.g., 100 ⁇ l, and as little as 5-10 ⁇ l of whole blood, can be directly analyzed in a simple assay format without prior enrichment of particular fractions of the sample.
  • the small volume of whole blood necessary has major benefits in use of the assays in the pediatric setting where the patients are infants and small children.
  • the present methods represent a significant savings in cost per sample analyzed. Furthermore, elimination of a need for expensive equipment makes the method widely available to many laboratories or clinics, and improves its adaptability to non-laboratory conditions, such as field conditions.
  • ASSAY FORMATS AND KITS This invention provides a method to prepare a sample to measure total target molecule. Many different assays and assay formats can be used to detect the target molecule of interest in the treated sample. Both competitive and non-competitive binding assays can be used. In preferred aspects, immunoassays are used, employing an antibody as a binding partner; these include but are not limited to the immunoassays used for measuring soluble antigens.
  • a kit for carrying out the assay for total target molecule includes a kit with components that allow the measurement of multiple target molecules in one sample, for example the measurement of total antigen A and total Le b , or total chlamydial antigen and total cytokine, etc.
  • a kit of this invention allows the measurement of total target molecule and measurement of soluble target molecule in the same sample, such as a blood sample.
  • Any procedure known in the art for the measurement of analytes can be used in the practice of the instant invention. Such procedures include but are not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, enzyme immunoassays (EIA) , preferably the enzyme linked immunosorbent assay (ELISA) , "sandwich” immunoassays, precipitin reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays, to name but a few.
  • EIA enzyme immunoassays
  • ELISA enzyme linked immunosorbent assay
  • sandwich immunoassays precipitin reactions, gel diffusion reactions, immunod
  • one or more binding partners used in an assay to bind a target molecule according to the invention is labeled; in another embodiment, such a first binding partner is unlabeled, and a labeled, second binding partner of the first binding partner is used to detect the bound first binding partner.
  • a first binding partner is unlabeled
  • a labeled, second binding partner of the first binding partner is used to detect the bound first binding partner.
  • labeled goat anti-rat immunoglobulin can be used to detect the bound monoclonal antibody.
  • polyclonal and/or monoclonal antibodies can be used in sandwich immunoassays according to the invention.
  • a first binding partner which is not an antibody or antibody fragment or derivative is used, and a second binding partner which is an antibody or antibody fragment or derivative is used.
  • the enzyme which can be used to detectably label an antibody include, but are not limited to, horseradish peroxidase, alkaline phospha- tase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, staphylococcal nuclease, ⁇ -V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, / 9-galac- tosidase, ribonuclease, urease, catalase, glucoamylase and acetylcholinesterase.
  • the detection antibody can also be labeled with a fluorescent compound.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the detecting antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepenta- acetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepenta- acetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of lumines ⁇ cence that arises during the course of a chemical reaction.
  • particularly useful chemi- luminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibody. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction.
  • biolumi- nescent protein The presence of a biolumi- nescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. Any other label known in the art may be used, e.g., a radionuclide, etc.
  • an antigen or an antibody is preferably bound to a solid phase support or carrier.
  • solid phase support or carrier any support capable of binding an antigen or antibodies.
  • supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configu ⁇ ration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads.
  • an antibody-antigen- antibody sandwich immunoassay is done, i.e., antigen is detected or measured by a method comprising binding of a first antibody to the antigen, and binding of a second antibody to the antigen, and detecting or measuring antigen immunospecifically bound by both the first and second antibody.
  • the first and second antibodies are monoclonal antibodies.
  • the second monoclonal antibody must bind to a site different from that of the first antibody (as reflected e.g., by the lack of competitive inhibition between the two antibodies for binding to the antigen) .
  • the first or second antibody is a polyclonal antibody.
  • both the first and second antibodies are polyclonal antibodies.
  • a "forward" sandwich enzyme immunoassay is used, as described schematically below.
  • An antibody capture antibody, Abl
  • the sample is brought in contact with the Abl-coated matrix and such that any antigen in the sample to which Abl is specific binds to the solid-phase Abl. Unbound sample components are removed by washing.
  • An enzyme- conjugated second antibody detection antibody, Ab2 directed against a second epitope of the antigen binds to the antigen captured by Abl and completes the sandwich.
  • a chromogenic substrate for the enzyme is added, and a colored product is formed in proportion to the amount of enzyme present in the sandwich, which reflects the amount of antigen in the sample.
  • the reaction is terminated by addition of stop solution.
  • the color is measured as absorbance at an appropriate wavelength using a spectrophotometer.
  • a standard curve is prepared from known concentrations of the antigen, from which unknown sample values can be determined.
  • Other types of "sandwich” assays are the so- called “simultaneous" and “reverse” assays.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
  • kits comprising one or more containers or vials containing components for carrying out the assays of the present invention are also within the scope of the invention.
  • a kit can comprises a detergent solution, preferably the Trax® lysing reagent (6% NP-40 and 9% Triton X-100 in IX PBS) .
  • kits are one or more binding partners, e.g., an antibody or antibodies, preferably a pair of antibodies to the same antigen, for example a leukocyte, erythrocyte or bacterial antigen, which preferably do not compete for the same binding site on the antigen.
  • a kit can comprise more than one pair of such antibodies or other binding partners, each pair directed against a different target molecule, thus allowing the detection or measurement of a plurality of such target molecules in a sample, for example, target molecules associated with several bacterial pathogens or with a bacterial and a viral pathogen.
  • one binding partner of the kit may be pre-adsorbed to the solid phase matrix, or alternatively, the binding partner and matrix are supplied separately and the attachment is performed as part of the assay procedure.
  • the kit preferably contains the other necessary washing reagents well-known in the art.
  • the kit contains the chromogenic substrate as well as a reagent for stopping the enzymatic reaction when color development has occurred.
  • the substrate included in the kit is one appropriate for the enzyme conjugated to one of the antibody preparations. These are well-known in the art, and some are exemplified below.
  • the kit can optionally also comprise a target molecule standard; i .e . , an amount of purified target molecule that is the target molecule being detected or measured.
  • a kit of the invention comprises in one or more containers: (1) a solid phase carrier, such as a microtiter plate coated with a first binding partner; (2) a detectably labeled second binding partner which binds to the same antigen as the first binding partner; (3) a standard sample of the target molecule recognized by the first and second binding partners; (4) concentrated detergent solution; and (5) optionally, diluent.
  • the target molecule is not a leukocyte cell surface marker.
  • one or more antibodies constitute the one or more binding partners used in the assays of the invention, to detect and/or measure a target molecule which is an antigen.
  • Antibodies useful in the detection of total antigen as described herein include polyclonal antibodies, monoclonal antibodies (mAbs) , and chimeric antibodies (see below) .
  • Preferred antibodies are mAbs, which may be of any immunoglobulin class including IgG, igM, IgE, IgA, and any subclass or isotype thereof.
  • the term "antibody” is also meant to include both intact molecules as well as fragments thereof which bind the antigen, such as, for example, F(ab') 2 .
  • Fab', Fab and Fv are examples of fragments thereof which bind the antigen.
  • these fragments lack the Fc fragment of an intact antibody molecule, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al . , 1983, J. Nucl . Med. 24:316-325), properties which may be desirable for particular therapeutic or diagnostic utilities. It will be appreciated that these antigen- binding or epitope-binding fragments of the antibodies useful in the present invention may be used for the detection and quantitation of the total amount of an antigen, or cells expressing or harboring the antigen, as disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments) or by reducing the disulfide bridges.
  • a host animal of any of a number of species such as rabbits, goats, sheep, horse, cow, mice, rats, etc. is immunized by injection with an antigenic preparation which may be derived from cells or microorganisms, of may be recombinantly or synthetically produced products.
  • adjuvants well-known in the art may be used to enhance the production of antibodies by the immunized host, for example, Freund's adjuvant (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substance such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, liposomes, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Propionibacterium acne ⁇
  • a mAb specific for an epitope of an antigen of interest can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • the antibody used in the methods of the present invention may also be a chimeric antibody, preferably a mouse-human chimeric antibody, wherein the heavy and light chain variable regions are derived from a murine mAb and the constant regions are of human origin.
  • MAbs or chimeric antibodies can be "humanized” by producing human constant region chimeras, where even parts of the variable regions, in particular the conserved or framework regions of the antigen-binding domain, are of human origin, and only the hypervariable regions are non-human. See for example,
  • the antibody is a single chain antibody formed by linking the heavy and light chain fragment of the Fv region via an amino acid bridge, resulting in a single chain polypeptide
  • a recombinantly cloned antibody molecule or fragment may be prepared using methods well-known in the art. Recombinant DNA methodology (Sambrook, J. et al . , 1989, MOLECULAR CLONING: A LABORATORY MANUAL, 2d
  • nucleic acid sequences which encode a mAb molecule, chain or epitope-binding fragment.
  • Antibody molecules may be purified by known techniques, e.g. immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC
  • Epitope specificity can be ascertained, for example, by observing the ability of a second antibody to inhibit binding of a first antibody to its antigen.
  • the amount of target molecule present in a sample may serve as a useful measure for the success or failure of the treatment.
  • the sample may be derived from a body fluid, a cell sample or a tissue sample of the subject.
  • the total target molecule is compared to a "baseline” or “control” value which, depending on the target molecule, the disease, and the treatment, may be the amount of target molecule in a similar sample from a normal subject, from the patient prior to disease onset or during remission of disease, or from the patient prior to the initiation of therapy.
  • baseline value may be the amount of target molecule in a similar sample from a normal subject, from the patient prior to disease onset or during remission of disease, or from the patient prior to the initiation of therapy.
  • a fall in the amount of a microbe-associated target molecule or its disappearance in a cell, tissue or body fluid sample is indicative of effective therapy.
  • a cure of the disease is accompanied by a decrease to undetectable levels of the microbe- associated target molecule. Lack of change, or a rise, in the target molecule is indicative that the therapy is not successful.
  • a subject being treated for Chlamydia trachomati ⁇ infection is tested sequentially after initiation of therapy using the present method to detect the amount of a chlamydia-associated target molecule present in sequential samples taken from the subject.
  • the therapeutic treatments the response to which may be evaluated according to the present invention include but are not limited to radiotherapy, chemotherapy and drug therapy, vaccine administration, immunosuppressive, immuno-restorative or immunostimulatory regimens, and the like.
  • measurement of the total amount of a target molecule is used to detect, diagnose or stage a disease or disorder in a subject.
  • a disease or disorder, associated with a particular target molecule is detected, diagnosed or staged by measuring the total amount of the target molecule in a sample, and comparing that to a baseline level, as described above.
  • a microbial infection including but not limited to those listed in Tables I-III and Table V is diagnosed by the detection of the target molecule associated with a microbial pathogen.
  • Thrombocytopenia is detected by the decrease in the amount of a platelet-associated target molecule in a blood sample compared to a baseline amount.
  • a pre-malignant lesion or cancer is detected by the appearance of a tumor-specific or tumor-associated target molecule (e.g., antigen) in cells of a sample.
  • a pre ⁇ malignant change in the gastrointestinal mucosa is detected by measuring the total amount of the blood group A antigen or the level of the A-transferase enzyme in a sample.
  • the progression of the disease, or the regression in response to therapy is monitored by measuring changes in the appropriate target molecule, up or down, over time.
  • the present invention also provides for the detecting or staging of disease, or the monitoring of treatment by measuring the total amount in a sample of a plurality of at least two target molecules.
  • a target molecule associated with a microbial pathogen may be measured in conjunction with one or more T cell surface markers, for example, CD35, CD4 and CD8, either in soluble form or in total form, to diagnose, detect, stage or monitor treatment of a disease or disorder such as those discussed above.
  • T cell surface markers for example, CD35, CD4 and CD8
  • the measurement of the soluble and/or total levels of a T cell marker serves as a measure of the function of the immune system, which may be modified in parallel with the disease course or treatment efficacy.
  • a disease caused by HIV-1 or HIV-2 infection may be monitored by measurements of one or more leukocyte surface markers, preferably total CD4, as well as detecting the total HIV antigen in a blood sample, a blood cell sample or in a select population of T cells and/or monocytes.
  • the response to an AIDS therapeutic for example, azidodeoxythymidine, dideoxyinosine, y interferon, ⁇ interferon, soluble CD4, an HIV vaccine, etc., can be monitored using such a method.
  • both (1) the p24 antigen of HIV, and (2) an erythrocyte blood group antigen, are detected and/or measured according to the invention.
  • a clinical specimen suspected of containing the organism is obtained on a swab from a site thought to be infected, including urogenital, endocervical, conjunctival and nasopharyngeal sites. In males, urine is also tested.
  • a swab is placed in a transport tube containing the TRAxTM lysing reagent (6% NP-40 and 9% Triton X-100 in lx PBS) diluted 1:6 in a volume of 200 ⁇ l. The swab is moistened with the lysis reagent and transported to the laboratory for assay.
  • a volume of sample diluent about 800 ⁇ l is added to the tube, the contents mixed, and the swab is discarded.
  • the contents of the tube are then subjected to the immunoassay.
  • the quality of the sample is important for detection of this organism by immunoassay. Since Chlamydia are obligate intracellular parasites, it is essential that a sample contain a sufficient number of infected cells, and that the organisms can be released from these cells for detection.
  • the TRAxTM lysing reagent disrupts epithelial cells and their internal vesicles which contain chlamydial reticulate bodies, which increases the yields of antigen for the immunoassay. Standard TRAxTM immunoassay methods are then used.
  • the immunoassay is performed as follows: 1. 100 ⁇ l of sample is added to duplicate wells of a 96-well icroplate coated with antibody to a
  • Chlamydial antigen such as the antibodies in the CHLAMYDIAZYME® assay kit from Abbott Laboratories; positive control wells receive 100 ⁇ l of a known concentration of chlamydial antigen; negative control wells receive 100 ⁇ l of the assay buffer. 2. 100 ⁇ l of an enzyme-conjugated antibody to chlamydial antigen, or, as in the CHLAMYDIAZYME® assay kit, an unlabeled anti-chlamydial antibody followed by an enzyme-conjugated anti- immunoglobulin reagent, is added to all wells. A horse radish-peroxidase enzyme is preferred.
  • the plate is incubated for 1-3 hours at room temperature.
  • the cut-off value for designating a sample as being positive for the antigen is 0.1 O.D. unit greater than the mean of the negative control values. Any value less than or equal to this is considered negative. Samples which fall between the cut-off value and the positive range are considered equivocal and may be retested.
  • an antigen especially a low molecular weight antigen such as blood group A antigen
  • competitive immunoassay is preferred to a sandwich assay because of the lack of multiple antigenic binding sites on the target molecule.
  • the level of antigen A is declining.
  • a competitive assay is selected.
  • a synthetic blood group A determinant is conjugated to bovine serum albumin (BSA) , using conventional methods (for example, 0'Sullivan et al., Anal . Biochem .
  • Synthetic BSA conjugates of oligosaccharide blood group antigens are commercially available from Chembiomed, Ltd. , Edmonton, Alberta, CANADA, or from ELA Technologies, Inc.
  • TRAxTM detergent solution containing 6% NP-40 and 9% Triton X-100 in lx PBS
  • 5 volumes of fresh EDTA-treated whole blood or another body fluid or cell sample is mixed with 5 volumes of fresh EDTA-treated whole blood or another body fluid or cell sample, followed by gentle mixing.
  • This method of cell lysis is superior to solubilization or disruption of cells by hypotonic treatment or sonication.
  • the treated sample is diluted 1:5 in the standard TRAxTM sample diluent (4% BSA, 1% sucrose, 0.01% thi erosal in lx PBS, pH 7.5) to reduce the possible effects of detergent on the assay performance. Samples are assayed fresh or stored at -70°C prior to analysis.
  • the samples are assayed in a one step format.
  • PLATE COATING Microplates are coated with 300 ⁇ l of synthetic blood group A determinant, Fuc ⁇ l ⁇ 2GalNAc ⁇ l ⁇ 3Gal?--, conjugated to BSA in a solution of phosphate buffered saline (PBS), 5 mM MgCl 2 , 0.01% thimerosal. The mixture is incubated for 16 hours at 4°C, following which the plates are blocked for 2 hours at 37°C with 5% BSA in PBS, 0.01% thimerosal. The plates are then washed 3 times with PBS, 0.05% Tween-20, 0.01% thimerosal, air dried for 1 hour at room temperature and stored at 4°C. Plates are used directly with no additional washing required prior to use.
  • PBS phosphate buffered saline
  • ASSAY To assess A-transferase activity by immunoassay, a substrate for the enzyme which is linked by the enzyme to A precursor material in the sample is provided. As A antigen is produced as a result of the enzymatic reaction, it competes with the immobilized A antigen for binding to labeled antibody.
  • the immunoassay is performed by adding to the coated wells a test sample and a "conjugate solution" containing a substrate for A-transferase, UDP-GalNAc (Sigma Chemical Co.) and a horse radish peroxidase (HRP)-conjugated mAb specific to the blood group A antigen or to the A-transferase enzyme, such as the anti-A-transferase mAb from Chembiomed, Inc., Edmonton, CANADA, in a solution of 100 mM sodium cacodylate (pH 6.8), 50 mM MnCl 2 , 1% BSA, 0.01% thimerosal.
  • a substrate for A-transferase UDP-GalNAc (Sigma Chemical Co.) and a horse radish peroxidase (HRP)-conjugated mAb specific to the blood group A antigen or to the A-transferase enzyme, such as the anti-A-transfer
  • the sample solution (50 ⁇ l) and 50 ⁇ l of conjugate solution are added to the plate, mixed and incubated, with shaking for 3 hours at room temperature.
  • the plate is then washed 3 times with PBS, 0.05% Tween-20, 0.01% thimerosal, and is blotted dry.
  • the standard TRAxTM OPD substrate in citric acid, sodium phosphate, 0.05% thimerosal, is added, 100 ⁇ l/well and allowed to incubate for 30 min at room temperature.
  • the reaction is stopped by the addition of 50 ⁇ l 2N H 2 SO A to each well.
  • the absorbance is then read at 490 nm on an EIA plate reader.
  • Standards used in the assay are dilutions of purified A-transferase from a mammalian source.
  • human serum is the source for A transferase.
  • the standard is prepared from freshly drawn blood which has been allowed to clot for about 2 hours at room temperature followed by refrigeration overnight at 4°C and centrifugation to remove the clot.
  • the A-transferase is purified from the serum by affinity chromatography on an anti-A- transferase antibody affinity column, and is stored frozen in aliquots at -70°C.
  • a antigen, or any other blood group antigen is measured in a similar assay, either competitive or sandwich type.
  • a blood sample being tested for A (or any other blood group) antigen after detergent treatment as above, is added to the coated plates described in Section 6.1.2 in the presence of the anti-A mAb conjugated to HRP.
  • Inhibition of binding of the antibody to the immobilized A antigen is an indication that A antigen is present in the sample.
  • the concentration can be obtained by running a parallel assay to generate a standard curve using known concentrations of A antigen.
  • the plate is coated with an anti-A mAb (rather than with the A antigen-BSA complex) , as above.
  • the test sample a detergent treated blood sample, is added and any A antigen is allowed to bind to the immobilized antibody.
  • each well receives HRP-conjugated anti-A antibody.
  • This "detection" antibody is either the same as the immobilized antibody or is a different mAb that binds to a different epitope of the antigen.
  • substrate is added as above, and the colored reaction product is measured. Over a certain concentration range, the amount of color generated is directly proportional to the amount of antigen in the sample.
  • the concentration of antigen in the sample is quantitated by interpolation on a standard curve generated by assaying known quantities of A antigen.
  • CD35 complement receptor
  • Plasma was obtained by centrifuging whole blood collected on EDTA and removing the plasma from the red blood cells prior to sample treatment.
  • An immunoassay was prepared by adding to the control microtiter plates, 50 ⁇ L of the test sample (plasma or lysate in dilution) and 50 ⁇ L of a conjugate solution which contained a horseradish peroxidase (HRP-conjugated) antibody specific for CD35 in a solution of Tris Buffered Saline, 25% FBS, 0.15% NP-40, 0.01% Thimerosal, 0.01% Gentamycin Sulfate, pH 7.4) .
  • HRP-conjugated horseradish peroxidase
  • Standard substrate commercially available TMB
  • Substrate Reagent was mixed in equal parts with a peroxidase substrate reagent (this combination is available as paired reagents) and added at 100 ⁇ L/well and allowed to incubate for 30 minutes at room temperature. The reaction was stopped by the addition of 0.18M H 2 S0 4 to each well.
  • CD35 (CRl)
  • EDTA Sample Status Plasma (nq/mL) Lysate fnq/mL. OD450

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Abstract

L'évaluation de la quantité totale d'une molécule cible est utilisée dans la détection, le diagnostic, la détermination de la gravité et le stade d'évolution d'une variété de maladies ou troubles et dans le contrôle de la thérapie. On effectue les mesures de la quantité totale d'une molécule cible en traitant un échantillon contenant des cellules, tel qu'un fluide biologique, avec un détergent non ionique d'une manière qui permette de lier à un ou plusieurs partenaires de laison la quantité totale d'une molécule cible présente dans la membrane cellulaire, le cytoplasme de la cellule et/ou dans les compartiments solubles du corps fluidique. Une fois que la molécule cible totale est disponible, on la mesure à l'aide d'un technique de dosage quelconque, de préférence par dosage immunologique d'une enzyme de type sandwich. Les procédés de la présente application peuvent être utilisés pour détecter et mesurer des molécules associées à des agents pathogènes microbiens, en particulier des agents pathogènes intracellulaires tels que les chlamydiae, ainsi que les antigènes du groupe sanguin des érythrocytes, des antigènes tumoraux, des cytokines et des molécules d'adhésion cellulaire en vue du diagnostic, de l'énumération des cellules exprimant ces molécules, et d'autres procédés.
PCT/US1993/010550 1992-10-30 1993-10-29 Evaluation de la quantite totale d'une molecule dans un echantillon et procedes bases sur celle-ci WO1994010571A1 (fr)

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JP6511400A JPH07509783A (ja) 1992-10-30 1993-10-29 試料中の全分子の測定及びそれに基づく方法
CA002148219A CA2148219C (fr) 1992-10-30 1993-10-29 Mesure de la quantite totale d'une molecule cible dans un echantillon; methode fondee sur ce processus
AU54571/94A AU686577B2 (en) 1992-10-30 1993-10-29 Measurement of total molecule in a sample and methods based thereon
EP93925154A EP0666987A4 (fr) 1992-10-30 1993-10-29 Evaluation de la quantite totale d'une molecule dans un echantillon et procedes bases sur celle-ci.

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US5932430A (en) * 1996-05-09 1999-08-03 Meridian Diagnostics, Inc. Immunoassay for H. pylori in fecal specimens
US6057166A (en) * 1995-12-22 2000-05-02 Universal Healthwatch, Inc. Fecal test method
WO2001048481A1 (fr) 1999-12-03 2001-07-05 Baxter International Inc. Essai de pyrogenicite a utiliser avec des systemes de dosage immunologique automatises
WO2002018931A2 (fr) * 2000-09-01 2002-03-07 Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh Solution pour la preparation d'echantillons de selles a des fins diagnostiques
EP1247094A2 (fr) * 2000-01-06 2002-10-09 Biosite Diagnostics Inc. DOSAGE POUR LA DETECTION DE i BACILLUS ANTHRACIS /i
US6607891B1 (en) 1998-07-31 2003-08-19 Mitsubishi Chemical Corporation Method of assaying insulin-like growth factor
EP1432731A2 (fr) * 2001-09-10 2004-06-30 University Of Pittsburgh Diagnostic et controle du lupus erythemateux systematique et de la sclerodermie
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WO2009005536A2 (fr) * 2006-11-22 2009-01-08 3M Innovative Properties Company Procédure de capture de cellules bactériennes entières et procédés d'analyse d'échantillons de bactéries
US7585640B2 (en) 2004-05-11 2009-09-08 University Of Pittsburgh Diagnosing and monitoring inflammatory diseases by measuring complement components on white blood cells
US7588905B2 (en) 2003-04-16 2009-09-15 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Identification and monitoring of systemic lupus erythematosus
US7807802B2 (en) 2002-11-12 2010-10-05 Abbott Lab Polynucleotides for the amplification and detection of Chlamydia trachomatis and Neisseria gonorrhoeae
CN102109525A (zh) * 2011-01-26 2011-06-29 牛刚 一种检测血液中游离乳腺癌细胞标志物的试剂盒
US8053200B2 (en) 2005-12-22 2011-11-08 Baxter International Inc. Monocyte activation test better able to detect non-endotoxin pyrogenic contaminants in medical products
US8080382B2 (en) 2003-06-13 2011-12-20 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Monitoring immunologic, hematologic and inflammatory diseases
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Also Published As

Publication number Publication date
CA2148219C (fr) 1998-12-29
AU5457194A (en) 1994-05-24
EP0666987A4 (fr) 1997-08-27
EP0666987A1 (fr) 1995-08-16
AU686577B2 (en) 1998-02-12
CA2148219A1 (fr) 1994-05-11
JPH07509783A (ja) 1995-10-26

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