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
• • 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
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
• • 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
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
• • 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
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor 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
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present application relates generally to methods of producing and to the production of antibody populations against fibrinogen and fibrin degradation products (FDP), to the antibody populations themselves and to related methods of use, to the detection of cancers and for monitoring the progress of cancer treatment by immunochemically measuring the quantity of FDP in serum.
• FDP fibrinogen and fibrin degradation products
• Antigens are macromolecules, such as proteins, nucleic acids or polysaccharides, which are capable of eliciting an immune response in the body.
• the immune systems of mammals and other animals have the ability to detect foreign agents, such as the antigens associated with cancer, and to respond to these antigens by producing antibodies which specifically target and react with those cancer-associated antigens.
• FDP fibrin and fibrinogen degradation products
• FDP fibrin and fibrinogen degradation product
• TF tissue factor
• uPA urokinase-type plasminogen activator
• FIG. 1 fibrinogen degradation products and fibrin degradation products
• Cleavage of fibrinogen by plasmin produces fragments D and E, as the primary end-products.
• Thrombin converts fibrinogen to fibrin in response to signals from the coagulation cascade.
• Cleavage of fibrin by plasmin produces D-dimer as a primary end-product.
• the composition of FDP produced through cancer-induced plasmin cleavage is influenced by the relative amounts of the two substrates; fibrin and fibrinogen.
• Disclosed herein are methods for the detection of cancer comprising the simultaneous detection of a defined mixture of fibrin and fibrinogen degradation products (FDP) containing three essential fragment types (fragment D, fragment E, and D-dimer) and optionally an additional three fragment types (fragment Y and two distinguishable forms of initial plasmin digest product - IPDP) for a total of six FDP components.
• the method comprises the simultaneous detection of the three essential FDP antigens in a biological sample with a specific polyclonal antibody pool in which the polyclonal antibodies are specific for the three FDP fragments.
• a method for detecting cancer in a subject comprising the steps of: obtaining a biological sample from the subject; reacting the biological sample with an antibody preparation which binds at least three antigens associated with fibrin and fibrinogen degradation products (FDP) to form antibody-FDP complexes wherein the three FDP-associated antigens are fragment D, fragment E and D-dimer; detecting the antibody-FDP complexes; and diagnosing cancer in the subject.
• the antibody preparation additionally optionally binds to at least one of fragment Y and initial plasmin digest products (IPDP).
• IPDP initial plasmin digest products
• the antibody preparation is a polyclonal antibody preparation.
• the method comprises an enzyme-linked immunosorbent assay.
• the diagnosing step further comprises at least one additional diagnostic test.
• a method is providing for monitoring cancer in a patient comprising the steps of: (a) obtaining a first biological sample from the subject, the first sample collected at a first sampling time point; (b) obtaining a second biological sample from the subject, the second sample collected after the first sampling time point at a second sampling time point; (c) reacting the biological samples with an antibody preparation which binds at least three antigens associated with FDP to form antibody-FDP complexes wherein the three FDP- associated antigens are fragment D, fragment E and D-dimer; (d) detecting the antibody-FDP complexes; (e) determining the ratio of the level of FDP in the second biological sample to the level of FDP in the first biological sample; (f) determining that cancer has progressed; and optionally repeating steps (a)-(e) with additional biological samples taken at time points after the first and the second time points.
• the antibody preparation additionally optionally binds to at least one of fragment Y and initial plasmin digest products (IPDP).
• IPDP initial plasmin digest products
• the antibody preparation is a polyclonal antibody preparation.
• the method comprises an enzyme-linked immunosorbent assay.
• the cancer has progressed if the ratio is greater than or equal to 1.15. In yet another embodiment, the cancer has regressed or is stable if the ratio is less than 1.15.
• a kit for detecting cancer in a subject, the kit comprising an antibody preparation which binds to at least three antigens associated with FDP wherein the three FDP-associated antigens are fragment D, fragment E and D-dimer; a detection system; and instructions for measuring the FDP and correlating the presence of the FDP with cancer.
• the detection system comprises a detection antibody specific for at least three antigens associated with FDP wherein the three FDP- associated antigens are fragment D, fragment E and D-dimer.
• the antibody preparation optionally additionally binds to at least one of fragment Y and initial plasmin digest products (IPDP).
• the antibody and detection system comprise an enzyme-linked immunosorbent assay.
• FIG. 1 depicts a schematic representation of cancer-induced generation of fibrin and fibrinogen degradation products (FDP).
• FIG. 2 depicts non-reducing (FIG. 2A) and reducing (FIG. 2B) SDS-PAGE gels of FDP immunogen lots (8 lots designated 03, 13, 15, 17, 19 21 , 24 and 25).
• UD refers to undigested fibrinogen
• D refers to fragment D
• E refers to fragment E.
• FIG. 3C depicts Coomassie Blue staining of the companion gel depicting the total amount of protein loaded per lane. The location of FDP subtypes are indicated on the gels.
• FIG. 4 depicts immune precipitation (IP) of antigens from human serum (normal and CRC patients) using anti-FDP polyclonal antibody pool (+) and rabbit IgG (-) bound beads. Three samples were tested: FDP calibrator control (FDP), a normal human serum (N4) and a CRC serum (C15).
• FIG. 5 depicts FDP antigens immune precipitated from the sera of five CRC and five normal patients using anti-FDP polyclonal antibody pool (FIG. 5A) or rabbit IgG (control) bound beads (FIG. 5B).
• Detection of fibrin and fibrinogen degradation products can be a valuable clinical diagnostic tool in a number of cancers. FDP levels are correlated with cancer occurrence, stage, progression and prognosis. Current assays for FDP are usually restricted to measuring one specific FDP component, such as D-dimer, as a representative of this group. In contrast, the present inventor has determined that a plurality of the fibrin and fibrinogen degradation products must be measured simultaneously in order to correlate the presence of, or a change in the levels of, FDP with the presence or progression of cancer, respectively.
• the phrase "progression of cancer” includes the progression, regression or re-occurrence of cancer in a patient.
• the presently disclosed assay uses an antibody preparation which specifically recognizes at least three FDP antigens that are particularly useful in detecting, and monitoring, the progression of cancer. These three essential FDP are fragment D, fragment E and the D-dimer (also known as fragment X). Three additional FDP which are useful for detection and monitoring cancer are two initial plasmin digest products (IPDP), defined by sequencing, and fragment Y.
• IPDP initial plasmin digest products
• the three essential FDP antigens are detected approximately simultaneously in the same assay. In other embodiments, four, five or six FDP antigens are detected simultaneously in the same assay.
• the disclosed assay measures fragment D, fragment E and D-dimer. In another embodiment, the disclosed assay measures fragment D, fragment E, D-dimer and one or both of the IPDP. In yet another embodiment, the disclosed assay measures fragment D, fragment E, D-dimer and fragment Y. In yet another embodiment, the disclosed assay measures fragment D, fragment E, D-dimer, fragment Y and one or both of the IPDP.
• Immunoassays are well known in the art and the affinity-purified anti-FDP antibodies disclosed herein can be used in different assay methods including, but not limited to, enzyme-linked immunosorbent assays (ELISA), dot or slot blots, sepharose bead trapping analyte systems, or various rapid test formats, such as lateral-flow or flow-through systems.
• ELISA enzyme-linked immunosorbent assays
• dot or slot blots include dot or slot blots, sepharose bead trapping analyte systems, or various rapid test formats, such as lateral-flow or flow-through systems.
• the capture and/or detection antibody is able to specifically bind to three, four, five or six FDP antigens.
• the antibody preparation is referred to herein as an antibody pool to reflect that the antibody preparation contains multiple antibody specificities.
• the antibody preparation is a polyclonal antibody preparation raised against an immunogen preparation which contains three, four, five or six FDP antigen species.
• Polyclonal antibodies can be generated in a variety of species including, but not limited to rabbit, goat, horse, donkey, sheep, chicken or shark.
• antibody preparations produced against each of the three, four, five or six FDP antigens can be prepared independently and pooled together to form the anti-FDP antibody pool.
• the antibody preparation can comprise a pool of monoclonal, monospecific polyclonal, chimeric or humanized antibodies or antibody fragments in which each antibody reacts with one or more of the six FDP antigens as long as the antibody pool can react with three, four, five or six FDP antigens in the same assay approximately simultaneously.
• the preparation of such antibodies is well known in the art and will not be described in detail in this disclosure.
• the present disclosure also includes systems and kits for binding to FDP and detecting, or monitoring, the presence or progression of cancer in a subject.
• the systems comprise antibody pools and detection reagents for binding and detecting three, four, five or six FDP in biological samples and correlating the presence of the three, four, five or six FDP with the presence or progression of cancer in the subject.
• kits comprise antibody pools and detection reagents for binding and detecting three, four, five or six FDP in biological samples and for correlating the presence of the three, four, five or six FDP with the presence or progression of cancer in the subject.
• a kit can contain one or more of the following in a package or container: (1 ) one or more antibody pools which bind three, four, five or six FDP to capture FDP from the biological sample; (2) detection antibody; (3) a solid support for immobilizing at least one of (a) the antibody pool or (b) the detection antibody; (4) detection reagents; (5) wash solutions; and (6) instructions for performing an assay using the kit components and biological samples.
• a kit can contain one or more of the following in a package or container: (1 ) one or more antibody pools which bind three, four, five or six FDP to capture FDP from the biological sample; (2) detection antibody; (3) a solid support for immobilizing at least one of (a) the antibody pool or (b) the detection antibody; (4) detection reagents;
• kits When a kit is supplied, the different reagents can be packaged in separate containers and admixed or attached to the solid support immediately before use. Such packaging of the components separately can permit long-term storage without losing the active components' functions.
• compositions included in particular kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container.
• kits can also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, flash memory device, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
• the claimed methods, systems and kits can be used to detect cancer-associated FDP in biological samples from a variety of sources including, but not limited to, serum, whole blood, plasma, saliva, sputum, urine, peritoneal fluid, tumor tissue, cerebrospinal fluid, vaginal or rectal secretions, and tears.
• Example 11 provides a typical distribution of normal and cancer patient samples. Levels of FDP in biological samples from patients suspected of having cancer are correlated with the presence of cancer by identifying those samples that exceed the normal range by a predetermined number.
• a baseline reading should precede the evaluation of their FDP levels.
• the FDP value of the initial serum draw serves as the baseline reading.
• the FDP immunogen was prepared by purchasing a purified human fibrinogen product that contained both fibrin and fibrinogen; which is referred to as fibhn/ogen for the purposes of this procedure.
• the fibrin/ogen was then reacted with plasmin to form fibrin and fibrinogen degradation products (FDP) by the Haverkate and Timan procedure (Thromb. Res. 10:803-812, 1977).
• the plasmin degradation of the fibrin/ogen was controlled by running the reaction for a specific period of time and then stopping the reaction with a protease inhibitor cocktail.
• the digested FDP were removed from the shaker and 200 ⁇ l protease inhibitor cocktail was added and the solution mixed thoroughly.
• the total protein concentration of the concentrated stock FDP solution was then measured, and the FDP breakdown product signature was verified on a 4-20% gradient SDS-PAGE under denaturing and reducing conditions (see FIG. 2).
• the resultant FDP solution was stored at -40 0 C.
• the FDP immunogen was also used as in the immunoaffinity purification of the antibody and as an FDP calibrator and control in the FDP detection system.
• Rabbits were immunized with FDP immunogen prepared according to Example 1. Each rabbit received injection of an emulsion consisting of 1 mg immunogen in 1.0 to 1.5 ml phosphate buffer saline and equal volume of Complete Freund's Adjuvant for the first immunization. Three weeks after the injection, each rabbit was bled and the serum was assayed for antibodies against FDP. One week after the bleeding, a booster was given to each rabbit by injection of an emulsion consisting of 1 mg immunogen in 1 to 1.5 ml phosphate buffer saline and equal volume of Incomplete Freund's Adjuvant. The rabbits were maintained on a schedule of boosters and bleeds until they were no longer viable.
• the rabbit anti-FDP was immunoaffinity purified as in Example 6.
• Example 3 Production of Chicken Anti-FDP Antibodies
• Chickens were immunized with FDP immunogen prepared according to Example 1. Each chicken received injections of an emulsion consisting of 1 mg immunogen in 1.0 to 1.5 ml phosphate buffer saline and equal volume of Complete Freund's Adjuvant for the first immunization (day 0). Each chicken was given additional immunizations on days 35, 56, 77, and 98. The chickens were bled (serum collection) on days 26, 40, 60, 81 , and 100. Serum response testing was begun on day 45. The first harvest of IgY occurred on day 53. The first affinity purification began on day 56.
• Chicken serums obtained at various time intervals after immunization were assayed for the concentration of antibodies against FDP by performing a standard titer assay using an 96-well plate system. A subset of the chicken anti-FDP IgY antibodies were conjugated to HRP prior to the assay.
• 10 ⁇ g of immunogen was immobilized in the wells of a 96-well microtiter plate; blocked with FBS; then reacted with a 1 :1 dilution series of the various rabbit serums; washed; incubated with a TMB solution; stop solution, and read at OD 450nm. The titer was determined by graphing the OD 450nm value versus the dilution factor; then determining the inflection point of the curve.
• the chicken anti-FDP was immunoaffinity purified as in Example 6.
• Goats were immunized with FDP immunogen prepared according to Example 1. Each goat received injections of an emulsion consisting of 1 mg immunogen in 1.0 to 1.5 ml phosphate buffer saline and equal volume of Complete Freund's Adjuvant for the first immunization. Three weeks after the injection, each goat was bled and the serum was assayed for antibodies against FDP. One week after the bleeding, a booster was given to each goat by injection of an emulsion consisting of 1 mg immunogen in 1 to 1.5 ml phosphate buffer saline and equal volume of Incomplete Freund's Adjuvant. The goats were maintained on a regime of boosters and bleeds, until the animal was no longer viable.
• Goat serums obtained at various time intervals after immunization were assayed for the concentration of antibodies against FDP by performing a standard titer assay using an 96-well plate system.
• 10 ⁇ g of immunogen was immobilized in the wells of a 96-well microtiter plate; blocked with FBS; then reacted with a 1 :1 dilution series of the various goat serums; washed; detected with a HRP-coupled rabbit anti-goat antibody (Sigma); washed; incubated with a TMB solution; stop solution, and read at OD 450nm.
• the titer was determined by graphing the OD 450nm value versus the dilution factor; then determining the inflection point of the curve..
• the goat anti-FDP was immunoaffinity purified as in Example 6.
• Pehodate-oxidized Sepharose CL 4B (20 ml) was washed with 10 times the Sepharose gel volume (i.e., ten times 20 ml of Sepharose gel which equals 200 ml) of Phosphate Buffered Saline (PBS) pH 7.5. The washed and drained Sepharose CL 4B was mixed with 20 ml (1 gel volume) of a 2.0 mg/ml solution of the pooled FDP antigens (FDP in MOPS, see Example 1 ).
• PBS Phosphate Buffered Saline
• the gel was washed sequentially with the following solutions: Dl water (10 times the gel volume), PBS pH 7.5 (10 times the gel volume), PBS containing 0.5M NaCI (10 times the gel volume), 0.1 M sodium acetate/ 0.15M NaCI pH 3 (10 times the gel volume), PBS pH 7.5 (10 times the gel volume).
• Dl water 10 times the gel volume
• PBS pH 7.5 10 times the gel volume
• PBS containing 0.5M NaCI 10 times the gel volume
• 0.1 M sodium acetate/ 0.15M NaCI pH 3 10 times the gel volume
• PBS pH 7.5 10 times the gel volume
• Polyclonal antiserum was filtered through a 0.2 ⁇ m syringe filter prior loading it onto the FDP-coupled Sepharose CL 4B gel column prepared as in Example 5.
• the volume of filtered polyclonal antiserum that was added is approximately equal to the volume of the gel (1 gel volume).
• the FDP-coupled Sepharose CL 4B gel column was washed again with PBS (5 times the gel volume) and then PBS containing 0.5M NaCI (5 times the gel volume).
• the immunoaffinity purified anti-FDP antibodies that bind to the FDP on the column were eluted with 0.1 M sodium acetate/ 0.15M NaCI pH 3.
• the eluent was collected in the fraction tubes for monitoring and later those fractions with detectable protein concentrations (OD 280nm greater than 0.2) were transferred to a PETG bottle for long term storage.
• the pH of the resultant antibody solution was neutralized by adding 1 M Tris buffer pH 8.5.
• the capture antibody is an affinity purified, polyclonal rabbit antibody to FDP.
• the rabbits were immunized and the antibodies isolated as in Example 3.
• SDS-PAGE of the affinity purified antibodies demonstrated a high degree of purity.
• the major protein band in the non-reduced anti-FDP preparation had a molecular weight of approximately 152 kDa, which is consistent with the molecular weight of rabbit IgG.
• the protein band of 152 kDa disappeared and, simultaneously, two protein bands of approximately 59 kDa and of 30 kDa appeared, which are similar to the molecular weights of the heavy and the light chains of an IgG molecule, respectively. Therefore, the anti-FDP antibodies were primarily IgG, not IgM (M.W. approx. 900 kDa), nor IgA (a dimer of about 320 kDa).
• the purity of the affinity-purified FDP antibody pool was qualitatively estimated at about 90%, based on SDS-PAGE analysis.
• the binding of anti-FDP antibodies to FDP antigens followed a typical Langmuir adsorption isotherm. From this data, using a double reciprocal plot, the binding constant of the antibodies was calculated to be 1.25x10 "9 M. Because the antibody preparation was of polyclonal origin, the binding constant obtained in this study was a composite binding constant, i.e. an average of binding constant contributed by antibodies produced by different clones.
• the capture and detection antibodies ensure the specificity of any ELISA based test.
• the FDP antibody pool was cross-reacted with human serum samples in both Western Blots (Fig. 3) and in immune precipitation (IP) experiments (FIGs. 4 and 5 and Table 2) in order to verify that the FDP antibody pool captures FDP in human serum samples.
• the proteins that were immunoprecipitated by the FDP antibody pool were sequenced and identified using mass spectrometry.
• FIGs. 3A and 3B depict Western blots of the same gel exposed to film for 5 sec and 30 sec respectively.
• FIG. 3C depicts the corresponding Coomassie-stained gel.
• six major FDP antigens were detected in CRC serum samples with estimated molecular weights of 340, 260-320, 220, 150, 80 and 50 kDa. These bands correspond with undigested fibrinogen, initial plasmin digest products (IPDP), fragment D-dimer/fragment X, fragment Y, fragment D and fragment E, respectively.
• IPDP initial plasmin digest products
• fragment D-dimer/fragment X fragment Y
• fragment D and fragment E fragment D and fragment E
• the rabbit anti-FDP IgG or the control rabbit IgG antibodies were covalently linked to Sepharose beads via a conserved carbohydrate moiety in the Fc portion on the IgG heavy chain.
• Human serum samples were selected at random and diluted 1 to 10 in PBS, pH 7.4. The diluted serum samples were incubated with either the anti-FDP-bound beads or control rabbit IgG-bound beads. In addition, both types of beads were incubated with a 0.25 mg/ml solution of prepared FDP to serve as a positive control for the IP reactions.
• the IP reactions between the diluted sera and the control rabbit IgG bound beads serve as a control for recognizing false positive reactions between the non-specific rabbit IgGs and serum antigens.
• the IP reactions were incubated overnight at 4°C on an end-over-end rotator.
• the IP beads were washed five times with PBS, and the antigens were extracted in non-reducing Laemmli sample buffer at 95°C for 8 minutes. Then the extracted antigens from the anti-FDP and control rabbit IgG beads were run on a 4-20% SDS-PAGE.
• FIG. 4 shows a complete set of experimental conditions for two representative serum samples; N4 is from normal patient 4 and C15 is from colorectal cancer patient 15.
• FIGs. 5A and 5B reveal the reproducibility of these IP reactions by showing five additional normal and five additional colorectal cancer patients in the IP assay.
• the results of the IP experiment, presented in FIG. 4, reveal that four major FDP-specific antigens and one non-specific antigen have been captured from the serum of the colorectal cancer patient serum by the anti-FDP beads (+). From this sample (C15(+), 2 nd to last lane from the right), the estimated molecular weights (MW) of the four major FDP-specific antigens are 340, 300, 220, and 50 kDa, respectively, in descending order. The one non-specific antigen in this lane is approximately 25 kDa.
• control lanes N4(-) and C15(-) (3 rd and 5 th lanes from the right) for the serum samples also show a reaction with a 25 kDa protein, which corresponds with the 25 kDa protein in the N4 and C15 samples exposed to anti-FDP beads (+), as shown in N4(+) and C15(+) (2 nd and 4 th lanes from the right).
• the presence of the 25 kDa protein in all of these lanes, N4(-), N4(+), C15(-), C15(+) confirms that the 25 kDa protein extracted from the IP reactions of serum samples non-specifically interacts with any IgG bound bead.
• the positive control reactions for the IP assay are designated FDP(-) and FDP(+) (6 th and 7 th lanes from the left).
• the bead control lane (FDP(-)) reveals no interactions between the FDP calibrator solution, which is not serum based, and the negative control beads.
• the FDP(+) reaction contains one minor band and three major bands.
• the estimated MW of the one minor band is 340 kDa, which corresponds with the MW of undigested fibrinogen.
• the estimated molecular weights of the three major FDP-specific antigens in the FDP control immunogen solution are 220 kDa (which corresponds with the MW of the fibrinogen fragment X or D-dimer), 80 kDa (which corresponds with the MW of fibrinogen fragment D monomer) and 50 kDa (which corresponds with the fibrinogen fragment E monomer).
• the anti-FDP beads captured four major FDP antigens from the human serum of a colorectal cancer patient; whereas, the same beads only brought one minor (faint band) in the normal serum at approximately 53kDa. Comparisons between the IP reactions of the human serum samples (and FDP) with the anti-FDP beads versus the control beads confirms the specificity of the anti-FDP antibody pool.
• FIG. 5 reveals that the pattern of FDP antigens is reproducible among multiple patient samples.
• sera from an additional five CRC and five normal patients were tested in immune precipitation reactions, separately, with both the control rabbit IgG and the anti-FDP beads.
• the FDP immunogen (calibrator solution) was tested with both types of beads.
• the SDS-PAGE in FIG. 5A contains the extracted FDP-specific antigens from the FDP (positive control), the CRC patient serum samples, and the normal serum samples.
• the SDS-PAGE shown in FIG.5B shows the antigens adhering to the control rabbit IgG beads from the same samples and in the same order as in FIG. 5A.
• the experiment shown in FIG. 5 confirms that there are four major FDP- specific antigens in human sera, and one non-specific antigen at 25 kDa.
• the FDP control lane which is derived from an in vitro plasmin digestion of fibrin and fibrinogen under controlled conditions, contains three major bands and one minor band. Based on the approximate molecular weights of these non-reduced samples, the major bands captured by the FDP antibody pool from the FDP control sample correspond to fibrinogen fragment D-dimer, fragment D monomer, and fragment E monomer, in descending order relative to the molecular weights.
• the minor band at 340 kDa is presumably undigested fibrinogen, also based on its approximate molecular weight.
• the FDP-specific antigens from human sera are at approximately 340, 300 (280-320), 210, and 50 kDa. These bands correspond with undigested fibrinogen, initial plasmin digest products (IPDP), fragment D-dimer/fragment X, and fragment E, respectively.
• IPDP initial plasmin digest products
• the IP reactions in this (and all of the other IP experiments) were normalized by adding equivalent concentrations of each serum sample to the IP reactions.
• the predominant antigen is at approximately 25 kDa. As discussed previously, this indicates that the serum protein at 25 kDa reacts non-specifically with all rabbit IgG bound beads. The 25 kDa protein also apparently interacts non-specifically with the rabbit anti-FDP IgG bound bead reactions shown in FIG. 5A. Table 2. Quantification of DR-70 ⁇ k® (FDP)-specific antigens from CRC patient sera
• the FDP specific antigens from the CRC patient sera were between 6.6 and 9.8 times more abundant than that from the normal patient sera, based on densitometry readings presented in Table 2.
• the nonspecific antigen (approximately 25 kDa) had a ratio of cancer relative to non-cancer that was equal to 0.9 or approximately 1.
• the average signal densities of all of the FDP-specific antigens were significantly (p-values ⁇ 0.000035) higher in the serum samples from the five colorectal cancer patients than from the five normal control patients.
• Table 2 confirms that anti-FDP antibody pool can distinguish between cancer and normal serum samples based on their respective FDP levels.
• the capture antibody pool was produced and characterized in one embodiment as described in Examples 2 and 7.
• Example 9 FDP Antibody Conjugated to Horseradish Peroxidase (HRP) for Detection
• the detection antibody was enzyme labeled using horseradish peroxidase (HRP) for detection in the assay. More specifically, in this example the detection antibody was a polyclonal rabbit anti-human FDP antibody produced according to the methods of Example 2. The antibody is the purified immunoglobulin fraction of rabbit antiserum conjugated with horseradish peroxidase of very high specific enzymatic activity.
• HRP horseradish peroxidase
• the detection antibody immunogen included both fibrin and fibrinogen degradation products.
• the antibody reacts with native human fibrinogen as well as with the FDP fragment subtypes: D, E, X/D-dimer, and Y. Traces of contaminating antibodies were removed by solid-phase absorption with human plasma proteins. The specificity of the antibody was ascertained by Western blot analysis.
• the detection antibody has confirmed cross-reactivity with the same six FDP fragments in human serum of cancer patients as was detected by the capture antibody.
• the FDP ELISA is an enzyme-labeled, sandwich immunoassay.
• the capture antibodies polyclonal, rabbit anti-FDP
• the capture antibodies were derived from sera of rabbits as described in Examples 2, 7 and 8.
• the FDP immunoassay involves the use of removable strips in a 96-well, microtiter plate format.
• the wells of the microtiter plate were coated with affinity purified rabbit anti-FDP antibodies.
• Human serum samples were diluted (1 :200) and were applied to the wells.
• FDP was captured from the serum samples by the anti-FDP antibodies immobilized on the well of the microtiter plate.
• anti-FDP antibodies conjugated to HRP were added to the wells.
• the anti-FDP-HRP complex binds to the captured FDP to form an immunological sandwich with the immobilized anti-FDP antibodies.
• the enzyme substrate TMB was added to the well.
• the end point was read in a micro plate reader at 450 nm after the reaction was stopped with 0.1 N HCI.
• the intensity of the color formed is proportional to the amount of FDP in the serum.
• the amount was quantified by interpolation from a standard curve using purified FDP calibrators.
• Table 4 shows the characteristics of each of the benign groups tested as well as those for normal individuals. There is significant scatter within all the groups with the exception of the normal cohort. Mean values tend to be skewed by samples with high FDP values. However, the median value in each and every one of these groups is well within the reference interval for apparently healthy individuals. As in all other studies in this trial, FDP values were determined in duplicate with all subjects consented under an IRB approved protocol.
• This study was conducted in 439 patients who had been diagnosed with, and were being treated for, a primary malignancy.
• the subjects were categorized into five groups: lung/liver cancer, breast/ovarian/cervical cancer, gall bladder/biliary/pancreatic cancer, gastric cancer and colorectal cancer.
• the FDP assay was performed in duplicate on the samples.
• a one-way ANOVA was performed with post hoc Tamhane tests. The results indicate that there are no significant differences between the groups. The details are shown in Table 5 for each cancer group.
• Ovarian 31 25.8 6.5 32.3 35.5 (1 1.9, 44 .6) (0.8, 21. 4) (16.7, 51 ⁇ 4) (19.2, 54.6)
• Gall Bladder 19 42.1 26.3 31.6 0.0 (20.3, 66 .5) (9.2, 51. 2) 12 6, 56 6) (0.0, 17.7)
• Example 14 Serial (longitudinal) monitoring of patients diagnosed with colorectal cancer
• Partial Response In patients with metastases at the time of the original draw, a noticeable reduction in the size of primary metastatic lesions or bone metastases demonstrating at least stabilization as observed on the bone scan.
• Stable Disease No significant change in the size of primary metastatic lesions or no noticeable increase in the size of primary lesions or no new lesions as evidenced by the clinical exam and imaging or other diagnostic modalities as ordered by the physician.
• Progressive Disease Clinical or imaging results that clearly indicate the presence of lesions not seen on previous examinations or a significant increase in the size of primary or metastatic lesions.
• the resulting 335 paired observations from the post baseline sampling were evaluated in two ways, per-visit and per-patient evaluations.
• the initial analysis performed a bootstrap sample for each patient by randomly sampling one visit at for each sample and recording the sensitivity or specificity for that visit. Note that if there was a progression and the sensitivity would be 1 if the FDP assay value increased from the previous visit by 15% or more and 0 if it did not. If there were no progression at that visit, then there would be no sensitivity reported at that visit, but the specificity would be reported as a 1 if the FDP assay value was below a 15% increase for that visit and 0 otherwise. For the per-visit analysis, there were 135 visits for sensitivity and 198 visits for specificity.
• a second analysis was done on a per-patient basis in which the number of progressions across all visits for a given patient was used to compute a patient level sensitivity by taking the number visits that FDP assay values increase by at least 15% among the number of visits that there was a progression. Similarly, the number of visits at which FDP assay values had a lower than 15% increase divided by the number of visits in which there was a non-progression allowed the computation of a per-patient specificity. Recall that if a patient had all progressions there would be no specificity for that patient and if a patient had all non-progressions, there would be no sensitivity for that patient. This resulted in a sample of 112 patients with at least one sensitivity, specificity, or both. This resulted in 70 estimates of per-patient sensitivity and 86 estimates of per-patient specificity.
• Positive (PPV) and negative (PNV) predictive values were also computed on a per-patient and per-visit basis.
• the PPV is calculated by dividing the number of times progression was present when the FDP assay value increased by 15% or more and NPV by dividing the number of times progression was absent when FDP assay values did not increase by 15% or more.
• bootstrap When small samples are available for evaluation and may have a complex structure, a statistical method called the bootstrap may be used to provide sound estimates of relevant population properties.
• a method to allow such a computation is to take repeated random samples with replacement from the 333 observations and compute the sum of the sensitivity and specificity and then determine through the frequency distribution of the resulting samples the sum that excludes the percentile corresponding to the alpha level of the statistical test.
• Such samples automatically incorporate any intra-patient correlations and yield an unbiased test of the null hypothesis above.
• Bootstrap sampling is usually repeated a number of times to be certain that the resulting distributions are consistent from run to run.
• the per-visit bootstrap sample was done 2,000 times by randomly sampling one visit for each of the 112 patients with replacement. From each sample, the mean sensitivity, means specificity, and the sum were computed by summing the sensitivity or specificity (the 1 or 0 values) over all patients and dividing by the total number of sensitivity or specificity measurements present. After the mean sensitivity and specificity for a sample was obtained, the two were added together to obtain the sum. This exercise resulted in 2000 bootstrap estimates of the per-visit sensitivity, specificity, and the sum of the two. A frequency display of these 2000 values by variable allows the boot strap estimation of the lower one- and two-sided 95% confidence limits as well as other.
• the method of sampling was done by assigning each of the k (between 1 and 7) visits per-patient a number between 1 and k.
• a sample of 112 observations was obtained by generating a uniform random number between 0 and 1 , multiplying that number by k, taking the integer of that number, and adding 1. This process provided a random number between 1 and k for each patient.
• the FDP assay ratio of change from previous was captured for the patient visit with the number obtained. Duplicates were allowed because sampling was done with replacement and the process was repeated 112 times. The sensitivity, specificity, and the sum were computed for each sample of 112 and retained.
• the sum of sensitivity and specificity from this analysis clearly is statistically significantly above 100.
• the median sum is likely to be about 133 with the median sensitivity about 65 and the median specificity about 67.
• the lower one-sided 5% confidence bound for the sum is about 120 and the lower one-sided 2.5% confidence bound is about 118.
• the median PPV and NPV are about 59 and 73, respectively, across the five samples. Note that these values are consistent with those computed from the per-visit values given above in Table 7. The five repetitions of the sample of 2,000 demonstrate that the result is robust and consistent.
• the sum of sensitivity and specificity from this analysis clearly is statistically significantly above 100.
• the median sum is likely to be about 134 with the median sensitivity about 66 and the median specificity about 68.
• the lower one-sided 5% confidence bound for the sum is about 124 and the lower one-sided 2.5% confidence bound is about 121.
• the median PPV and NPV are about 53 and 69, respectively, across the five samples. Note that these values are consistent with those computed from the per-patient values given above Table 8. The five repetitions of the sample of 2,000 demonstrate that the result is robust and consistent.

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