Classifications

• • 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
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
  • G01N33/57585—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds identifiable in body fluids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/745—Assays involving non-enzymic blood coagulation factors
  • G01N2333/75—Fibrin; Fibrinogen

Definitions

• This invention relates generally to ifnmunoassays for the detection of cancer.
• cancer antigens have been discovered, for example: cancer antigens CEA, CAl 9-9 and CA242 are used in the diagnosis and treatment of gastrointestinal cancer; cancer antigen CAl 25 is used in the diagnosis and treatment of ovarian cancer; cancer antigen AFP is associated with testicular and liver cancers; the CAl 5-3 and HER2/neu antigens are associated with breast cancers; and the PSA and PAP antigens have been shown to be associated with prostate cancer.
• Some antigens such as the carcinoembryonic antigen, are found in patients with a number of different cancers, such as lung, liver, pancreas, breast, head or neck, bladder, cervix and prostate, in addition to those suffering from adenocarcinoma of the colon. However, in these cases only 30% of the patients test positive. This amount is too low for this antigen to be useful as a diagnostic tool.
• Cancer associated markers may arise from a variety of sources including those associated with common oncogenic processes. For example, it is known that a wide variety of tumor cells of different lineages release proteases into interstitial fluid at a higher rate than normal cells. Sylven B., "Lysosomal Enzyme Activity in the Interstitial Fluid of Solid Mouse Tumour Transplants," Eur. J. Cancer, 4:463-414, (1968); Sylven B., “Cellular Detachment by Purified Lysosomal Cathepsin J'Eur. J. Cancer, 4:559-562, (1968). A number of lines of evidence support the concept that this increased protease activity contributes directly to the invasiveness of tumor cells and to the destruction of the adjacent host tissue.
• tissue metalloproteinase inhibitors block cell invasion in vitro.
• the administration of either natural or synthetic metalloproteinase inhibitors has been shown to prevent metastasis in a simple lung colonization model.
• Goldberg et al. "Extracellular Matrix Metalloproteinases in Tumor Invasion and Metastasis," in Regulatory Mechanisms in Breast Cancer, Lippman ME, and Dickson RB (eds), Boston, Kluwer Academic Publishers, pp. 421-440, (1990).
• Protease release by tumor cells can also result in the proteolysis of plasma proteins. Theoretically the extent of proteolytic degradation of these proteins can be correlated with the activity of the tumor cells and used indirectly to evaluate their tumor burden or degree of malignancy. Therefore the identification of antigens associated with the proteolytic activity associated with malignancy should yield new markers that are associated with oncogenic processes.
• pan-marker or universal marker antigen(s) will be useful for the routine screening of patients to determine if they have cancer. After an initial screening, patients with elevated concentrations of the pan-marker, when compared to a "normal" population, would be further screened to determine if they do in fact have cancer and the specific type of cancer from which they are suffering. Additionally, it is desirable that such a pan-marker is present in blood, or other biological fluids, so that testing can be performed on easily obtainable samples.
• the present invention is directed to immunoassay s for the detection of cancers.
• the invention provides a method for detecting cancer in a subject by contacting a biological sample obtained from the subject with an antibody that binds an epitope on a blood protein degradation peptide that is masked in the blood protein and determining the presence of an antibody-peptide complex.
• the blood protein is human flbrinogen and the antibody recognizes an epitope comprising the amino acids 15 to 21 of the ⁇ -chain of human fibrinogen.
• the assay is an enzyme-linked immunoadsorbent (ELISA) assay.
• the assay is a sandwich type ELISA immunoassay.
• Biological samples which are assayed in the present invention may be obtained from a variety of sources.
• the biological sample consists of human blood.
• the assay includes the additional step of screening a biological sample isolated from the subject for the presence of a second tumor marker.
• the second tumor marker consists of either PSA, CEA, CA 15-3, CA 19-9 or CA 125, or a combination thereof.
• a significant feature of the invention is the identification of cancer markers which comprise epitopes on endogenous proteins that are usually inaccessible to immunodetection in normal subjects.
• the invention disclosed herein offers a number of performance advantages over assays in the prior art. First, they enable immunochemical measurements of proteolytic degradation products in the presence of, and without interference by the endogenous normal protein molecules. Second, these embodiments detects multiple cancers with a high degree of specificity and sensitivity. Third, when such assays are used together with additional established organ-specific markers, the overall clinical performance is improved.
• FIG. 1 shows a graph of the standard curve for fibrinogen digestion products
• FIG. 2. shows a graph derived from dilution of a high titer patient sample.
• FIG. 3. shows a scatterplot of normalized FDP ratios of serum samples from normal subjects and patients having cancer of the breast, colon, lung, ovary or prostate.
• FIG. 4. shows a scatterplot of CA 15-3 levels and FDP levels in serum samples from breast cancer patients.
• FIG. 5. shows a scatterplot of CA 19-9 levels and FDP levels in serum samples from colon cancer patients.
• FIG. 6. shows a scatterplot of CEA levels and FDP levels in serum samples from colon cancer patients.
• FIG. 7 shows a scatterplot of CEA levels and FDP levels in serum samples from lung cancer patients.
• FIG. 8. shows a scatterplot of CA 125 levels and FDP levels in serum samples from ovarian cancer patients.
• FIG. 9. shows a scatterplot of PSA levels and FDP levels in serum samples from prostate cancer patients.
• FIGs. lOa-d show western blots made from SDS-PAGE gels of pleural effusate from a patient with lung cancer.
• FIG. 10a is derived from a reduced gel and the probe was derived from a ring shaped particle extract.
• FIG. 10b is derived from a reduced gel and probed with a monoclonal antibody of the invention.
• FIG. 10c. is derived from a non-reduced gel and the probe was derived from a ring shaped particle extract.
• FIG. lOd. is derived from a non-reduced gel and probed with a monoclonal antibody of the invention.
• FIGs. 1 la-c show the selectivity of the assay through graphs of the standard curves for flbrinogen fragment D, flbrinogen and flbrinogen fragment E as a function of absorbance at 450 nm.
• antibody is used in the broadest sense and specifically covers monoclonal antibodies and variations thereof including antibody fragments, chimeric or other recombinant molecules that are known in the art.
• monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
• tumor marker as used herein is broadly defined as any one of a wide variety of peptides, nucleic acids and related molecules of which the presence or levels of are used to assess the status of oncogenic processes.
• masked as used herein, for example in the context of degradation peptides, is broadly defined as peptide sequences that are not appreciably recognized or accessible by detection systems, such as antibodies, in normal endogenous proteins.
• a "masked" peptide may exist within the interior of a native protein, but is not exposed until the protein is degraded and an internal peptide is released or exposed.
• degradation peptide as used herein is broadly defined as a peptide fragment of a larger protein which has been degraded, for example, as occurs with the proteolytic degradation of blood proteins that is observed in oncogenic processes.
• mammal refers to any mammal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention, the mammal is a human.
• the present invention is directed at a method of screening for cancer by detecting an epitope in a protein peptide not generally accessible on the full length protein but which becomes so upon proteolytic degradation. Such peptides are generated by the action of proteases which are involved oncogenic processes.
• the present invention illustrates the association between common oncogenic processes such as proteolysis and novel cancer antigens. Proteases are associated with oncogenesis and are released at a higher rate into the interstitial fluid of growing tumor cells than normal cells. Several lines of evidence support the hypothesis that this increase in the quantity of protease released by the cancer cells contributes directly to the invasiveness of tumor cells and to the destruction of the adjacent host tissue.
• proteases In the case of breast cancer metastases, four classes of proteases appear to be involved in disease progression. These four classes of proteases include cathepsins B and L (cysteine proteases), cathepsin D (aspartyl protease), collagenases (metallo- proteases) and urokinase and plasminogen (serine proteases). Proteases have been implicated in a number of malignant conditions and researchers have observed increased secretion of proteases into the interstitial fluid around growing tumors. These proteases inevitably act on proteins, including those in the coagulation cascade leading to the formation of fibrin. Furthermore fibrin is very frequently observed at the invading periphery of malignant neoplasms.
• the invasiveness of some tumor cells has been correlated with an increased expression of collagenase. Genetic manipulation of such tumor cells, in culture, to reduce the activity of the collagenase results in a decrease in the invasiveness of the cell and metastases caused by the cells, in vitro. Furthermore, the addition of tissue metalloproteinase inhibitors to tumor cells results in blocking of the cell's invasiveness in vitro. Similarly, the administration of either natural or synthetic metalloproteinase inhibitors prevents metastasis of lung cancer cells.
• One consequence of the release of proteases by tumor cells into the bloodstream is the proteolysis of serum proteins such as flbrinogen. Therefore, the extent of proteolytic degradation of serum proteins can be correlated with the activity of the tumor cells. Quantitatively the degree of proteolysis can be determined by measuring the quantity of the degradation products generated by the action of the proteases. This measurement is, therefore, an indirect estimate of the degree of malignancy of the tumor cells.
• a significant feature of the invention is the identification of cancer markers which comprise epitopes on endogenous proteins that are generally inaccessible to immunodetection. Specifically, while these epitopes are usually masked by the factors such as the 3 dimensional structure of the protein, they become unmasked and accessible to immunodetection for example, upon proteolytic degradation that occurs in oncogenesis. With this knowledge, methods which measure unique epitopes that are either sterically or immunochemically unreactive in the native flbrinogen molecule and are manifested secondary to proteolytic degradation of flbrinogen are of particular interest.
• the measurement of serum flbrinogen degradation product (FDP) levels may represent a useful measure of malignancy.
• FDP serum flbrinogen degradation product
• methods to detect proteolytic degradation products of flbrinogen and other plasma proteins with minimal interference from the parent protein (the protease substrate) are of particular interest for use in a cancer detection assay.
• Oncochek an immunoassay
• peptides associated with oncogenic processes may be found in detectable concentrations in the biological samples of warm-blooded animals, including humans, possessing a disease which disrupts epithelial tissue.
• unmasked peptides may be indicative of a variety of diseases and are detectable in a variety of samples, with or without purification of such peptides.
• degradation peptides are shown to be associated with invasive cancers. Invasive cancers include cervical, urogenital (e.g., bladder and prostate), lung, colorectal, and head and neck cancers.
• Such peptides are also associated with epithelial disorders (i.e., non-invasive or pre-invasive cancers and disorders unrelated to cancer) including epithelial inflammations and collagen degenerative diseases.
• Biological samples containing peptides associated with oncogenic processes may come a variety of sources.
• Representative types of biological samples include urine, cervical secretions, bronchial aspirates (including bronchial washings), sputum, saliva, feces, serum, synovial and cerebrospinal fluid.
• the type of biological sample in which peptides are present may depend chiefly on the location of the particular disease. For example, urine is preferred for the detection of invasive urogenital cancers and urogenital epithelial disorders. Cervical secretions are preferred for the detection of invasive cervical cancers and cervical epithelial disorders. Bronchial aspirates and sputum are preferred for the detection of invasive lung cancers and lung epithelial disorders.
• a bronchial aspirate is taken further permits one to identify the location of a disease within a lung.
• Saliva is preferred for head and neck cancers.
• Feces are preferred for invasive colorectal cancers and colorectal epithelial disorders.
• Cerebrospinal fluid is preferred for brain cancers.
• serum may be used for the detection of complexes as a "pan" marker (i.e., a general screening technique) from which follow-up tests would be recommended to identify the particular disease. It would be evident to those of ordinary skill in the art how to associate other biological samples with a particular disease location.
• the presence or amount of a peptide may be determined in a variety of ways, including non-immunological and immunological.
• Non-immunological methodologies include the use of protein stains such as Coomassie blue or silver stains.
• a sample suspected of containing a peptide of interest is subjected to SDS-PAGE and identified using a protein stain.
• Other non- immunological methodologies include the use of radioisotopes and the like as reporter groups. Such methods are amenable to quantification where it is desired to determine the amount.
• the presence or amount of a peptide associated with oncogenic processes may be detected by immunological means.
• Detection may be, for example, by Western blot analysis utilizing immobilized complexes or components thereof on nitrocellulose, or Immobilon or similar matrix in conjunction with specific antibodies to the peptides. Detection can also be achieved by immunoassay.
• a peptide is isolated from a sample and contacted with an appropriate detection antibody.
• Complexes may be isolated by capture on a solid support (e.g., heparin agarose or polystyrene or heparin coated on polystyrene) or with a "capture" antibody prior to or simultaneous with a "detection" antibody.
• peptide-antibody immunocomplexes are formed between an antibody and a peptide, without prior purification of the complex. Incubation of a sample with an antibody is under conditions and for a time sufficient to allow immunocomplexes to form. Detection of complexes or polypeptide constituents by immunological means is also amenable to quantification where it is desired to determine the amount of a peptide. Detection of one or more immunocomplexes formed between a peptide and an antibody specific for the peptide may be accomplished by a variety of known techniques, including radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISA).
• RIA radioimmunoassays
• ELISA enzyme linked immunosorbent assays
• the immunoassays known in the art include the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Pat. No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and
• the antibodies may either be labeled or unlabeled.
• unlabeled antibodies find use in agglutination assays.
• unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the antibody, such as antibodies specific for immunoglobulin.
• the antibodies can be directly labeled.
• the reporter group can include radioisotopes, fluorophores, enzymes, luminescers, or dye particles.
• the target antigen or immobilized capture antibody is adsorbed to the surface of a microtiter well. Residual protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent).
• BSA bovine serum albumin
• NGS heat-inactivated normal goat serum
• BLOTTO bufferered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent.
• the well is then incubated with a sample suspected of containing specific antibody.
• the sample can be applied neat, or, more often, it can be diluted, usually in a buffered solution which contains a small amount (0J%-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.
• a reporter group can be chosen from a variety of enzymes, including horseradish peroxidase, beta-galactosidase, alkaline phosphatase, and glucose oxidase.
• a reporter group is bound to the antibody.
• the step of detecting an immuncomplex involves removing substantially any unbound antibody and then detecting the presence or absence of the reporter group.
• a reporter group is bound to a second antibody capable of binding to the antibody specific for a peptide associated with an oncogenic process.
• the step of detecting an immunocomplex involves (a) removing substantially any unbound antibody, (b) adding the second antibody, (c) removing substantially any unbound second antibody, and then (d) detecting the presence or absence of the reporter group.
• the antibody specific for the fragment is derived from a mouse
• the second antibody is an anti-murine antibody.
• a reporter group is bound to a molecule capable of binding to the immunocomplex.
• the step of detecting involves (a) adding the molecule, (b) removing substantially any unbound molecule, and then (c) detecting the presence or absence of the reporter group.
• An example of a molecule capable of binding to the immunocomplex is protein A.
• Reporter groups suitable for use in any of the methods include radioisotopes, fluorophores, enzymes, luminescers, and dye particles.
• the invention provides a method for monitoring the course of a neoplastic condition by quantitatively determining the presence of peptides present in a biological sample over time.
• the present invention is directed at a method for measuring the quantity of proteolytic degradation products of serum proteins.
• a peptide contained within the interior of the native proteins is used.
• Such peptides are "masked” in the native protein and are not recognized or accessible by detection systems, such as antibodies, when the protein is intact. These "masked” peptides are not exposed until the protein is degraded and the internal peptides are released or exposed.
• the method measures proteolytic degradation of flbrinogen with minimal interference from intact flbrinogen.
• two different antibodies are used as the detection system.
• One of the antibodies is specific for the peptide GHRPLDK which is part of the amino acid sequence of the ⁇ -chain of flbrinogen, located near its amino terminus.
• Assay specificity is achieved by the use of two different antibodies in a two- site, solid-phase enzymometric assay.
• the more highly specific antibody, which is immobilized to the solid phase consists of a murine monoclonal to a glycine- histidine-arginine-proline-leucine-aspartate-lysine-cysteine (GHRPLDKC) octapeptide.
• GHRPLDKC glycine- histidine-arginine-proline-leucine-aspartate-lysine-cysteine
• the first seven amino acids of this peptide represent an internal sequence within the ⁇ -chain of flbrinogen, which is near the amino terminus and is exposed after initial plasminolysis (residues 15-21).
• peptide GHRPLDK has been used in one embodiment of the present invention, it will be clear to those skilled in the art that other internal flbrinogen peptides would also be of use, as would internal peptides of other proteins which are degraded by proteases produced by cancer.
• a commercially available monoclonal antibody to the peptide GHRPLDKC can be used.
• An illustrative antibody that is useful in this assay is the murine monoclonal antibody derived from clone D1G1OVL2 and which is commercially available from Biodesign International, Kennebunkport, ME (Catalog number M42543M) and Immunotech, Inc., Westbrook, ME).
• This monoclonal antibody was generated using an immunogen prepared from the peptide GHRPLDKC conjugated to bovine serum albumin.
• the sequence of the first 7 amino acids of the octapeptide corresponds to the amino acids number 15 to 21 of ⁇ -chain of human flbrinogen.
• the monoclonal antibody recognizes fragment D of flbrinogen but does not cross react with intact flbrinogen.
• fragment D In addition to recognizing fragment D, the monoclonal antibody also reacts with flbrinogen degradation products (FDP) produced by plasminolysis. However, the monoclonal antibody does not recognize fragment E. Fragment D is the proteolytic product of flbrinogen plasminolysis. Although, in the current assay format, the immobilized monoclonal antibody to fragment D will capture fragment D or FDP, only FDP are "sandwiched" by the polyclonal anti-fibrinogen antibody, labeled with horseradish peroxidase, which is used.
• FDP flbrinogen degradation products
• the monoclonal antibody was immobilized on a solid phase and used to capture proteolytic degradation products of flbrinogen. After being captured by the immobilized mouse monoclonal antibody, the degradation products were complexed by polyclonal antibody (ovine anti-human fibrinogen-peroxidase conjugate and which is commercially available from The Binding Site, Inc., San Diego, CA) to form an immuno-sandwich. While a sandwich enzyme linked immunosorbent assay (ELISA) was used in the Examples below, relating to this invention, one skilled in the art is aware that other assay formats can also be used.
• polyclonal antibody ovine anti-human fibrinogen-peroxidase conjugate and which is commercially available from The Binding Site, Inc., San Diego, CA
• ELISA sandwich enzyme linked immunosorbent assay
• assays for the peptides described above can be combined with tests for the presence of one or more known organ-specific tumor markers to increase the clinical sensitivity and enhance the diagnostic capacity of these assays. Such combination assays may be performed at the same time or sequentially.
• organ-specific tumor markers which are associated in varying degrees with different cancer lineages and which may be utilized in conjunction with the assays described herein (see e.g. Lamerz et al., "Serum Marker Combinations in Human Breast Cancer", In Vivo 7(6B): 607-613 (1993).
• the unique epitope detected by the Oncochek immunoassay system appears to offer increased clinical sensitivity.
• the monoclonal anti-fibrinogen-peptide antibody (Clone D1G1ONL2) was dissolved and diluted to 2 ⁇ g/ml in pH 8.8 borate buffer (0J25 M Borate, pH 8.8, 0.225 M ⁇ aCl, 5 mM EDTA, 50 mM 3-amino-m-caproic acid, 10 ⁇ g/ml 4- aminobenzamidine-HCl). 120- ⁇ l aliquots of the diluted antibody solution were added to each well of each microtiter plates (96-well microtiter plates obtained from Fisher Scientific, Fair Lawn, ⁇ J) and incubated overnight (15-20 hr) at 25°C.
• Fibrinogen was plasmin-digested according to the method of Haverkate and Timan as setforth below.
• Fibrinogen obtained from Sigma Chemical Co., St. Louis, MO
• 0.05 M MOPS pH 7.4, 0.10 M NaCl and 2 mM CaCl 2
• Plasmin obtained from Sigma Chemical Co., St. Louis, MO
• the FDP was frozen until required.
• the FDP sample was diluted with phosphate buffered saline (PBS: 137 mM NaCl, 1.6 mM KCl, 8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 ) with 5 mM EDTA and 1% (w/v) BSA.
• PBS phosphate buffered saline
• TMB substrate for the horseradish peroxidase obtained from Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD
• 100 ⁇ l of TMB substrate for the horseradish peroxidase obtained from Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD
• 100 ⁇ l stop solution (0J M HC1) was added to each well.
• the solution in the wells of the microtiter plates was then read at
• diagnostic sensitivity is defined by equation 1 :
• the calibrators for the assay were prepared by plasminolysis of fibrinogen as described in Example 2. Intact fibrinogen (fibrinogen not subjected to prior treatment with plasmin) was unreactive in the assay of the present invention whereas immunoreactive FDP were formed from fibrinogen by plasmin treatment in a time-dependent fashion (Table I). Table I
• FIG. 1 shows a standard curve for the reaction of different concentrations of FDP (over the range of 32 to 250 ⁇ g/ml) with the assay system of the present invention.
• the results indicate that the absorbance at 450 nm is proportional to the amount of FDP added, over the range studied.
• the immunoreactive products present in the serum of a cancer patient with high levels of FDP exhibited linearity in dilutional parallelism to the FDP calibration curve over a dilution range from 5- to 80-fold (see Figure 2).
• Figure 11(A) illustrates results indicating that FD affects FDP measurements in the Oncochek assay in a pattern consistent with noncompetitive inhibition or covert cross-reactivity.
• Suelter CH. A. Practical Guide to Enzymology, New York, Wiley, p. 248, (1985).
• This inhibition pattern is consistent with the mechanism that FD binds to the solid phase of capture antibody, thus reducing the antibody sites available for binding FDPs.
• the double reciprocal plots of FE and FG inhibition studies are consistent with the absence of interaction between MAb and FE and FG (see Figures 11(B) and 11(C)). They are also consistent with the results presented in Table I, which shows the lack of response by FE and FG in the Oncochek assay.
• Table II shows the FDP levels in the sera of the 50 normal (Table Ila) control subjects and the 65 cancer patients (Table lib).
• Samples from cancer patients generally exhibited higher concentrations of FDP, using the D m /F assay format, than did control patients.
• Figure 4 is a scatterplot of CA 15-3 levels as compared to FDP normalized ratio for individual samples from breast cancer patients.
• Figure 5 is a scatterplot of CAl 9-9 levels as compared to FDP normalized ratio for 22 of the 50 individual samples from colon cancer patients.
• Figure 6 is a scatterplot of CEA levels as compared to FDP normalized ratio for 28 of the 50 individual samples from colon cancer patients.
• Figure 7 is a scatterplot of CEA levels as compared to FDP normalized ratio for individual samples from lung cancer patients.
• Figure 8 is a scatterplot of CA 125 levels as compared to FDP normalized ratio for individual samples from ovarian cancer patients.
• Figure 9 is a scatterplot of PSA levels as compared to FDP normalized ratio for individual samples from prostate cancer patients. These scatterplots demonstrate the increased sensitivity of FDP measurements relative to measurement of other cancer antigens. This increased sensitivity is particularly demonstrated by the datapoints which fall within the lower right quadrant of the plots.
• the results presented in Figure 3 show that FDP measurements detect a wide variety of cancers. Results of the Oncochek assay indicate that FDP levels in the sera of patients with various types of cancer are significantly elevated in comparison to normals. For example, FDP levels in the sera of normal control subjects were compared with those in the sera of patients with five types of cancers. Each group consisted of 50 patients and included breast, colon, lung, ovarian, and prostate cancers. The data presented in Figure 3 were subjected to a receiver-operating-characteristics (ROC) analysis to assess the relationship between the sensitivity and specificity of the assay at various threshold concentrations of FDP.
• ROC receiver-operating-characteristics
• Results shown in Table IV and Figure 3 suggest that the Oncochek immuno- assay can detect multiple cancers with a high degree of specificity and clinical sensitivity. When it is used with established organ-specific markers, improved clinical sensitivity may be achieved for breast, colon, and lung cancers.
• Samples of pleural effusate from a lung cancer patient were prepared for sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose for western blotting using standard methods well known in the art.
• Figures lOa-d show results from reduced (with mercaptoethanol; Figs. 10a-b) and non-reduced (without mercaptoethanol; Figs. 10c-d) gels.
• the probe was derived from a ring shaped particle extract. (Ring shaped particles are described in United States Patents Nos. 5,635,605, issued June 3, 1997, and 5,459,035, issued October 17, 1995.)
• the probe was the monoclonal antibodies of the invention.

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