JP2012522246A - Detection of fibrin and fibrinogen degradation products, and related production methods, and methods of use for cancer detection and monitoring - Google Patents

Abstract

The present invention is for detecting cancer or monitoring cancer progression by simultaneously detecting the presence of six fibrin and fibrinogen degradation products (FDP) in a biological sample in one analytical system. Methods, systems and kits.
[Selection figure] None

Description

  The present invention relates to methods and products for producing antibodies against fibrin and fibrinogen degradation products (FDP), methods for using the antibodies themselves and related to the antibodies, detection of cancer, and FDP in serum. Is widely related to the immunochemical quantification of cancer and to monitor the progress of cancer treatment.

  Despite recent advances in understanding cancer, current techniques for cancer testing and discovery leave room for improvement. In the art, attempting to detect cancer-related antigens using antibodies is a known method for cancer testing. Antigens are macromolecules such as proteins, nucleic acids or polysaccharides that cause an immune response in the body. The immune system of mammals and other animals has a function of detecting foreign substances such as antigens associated with cancer, and responds to antigens by producing antibodies. The antibody is specific for the target and reacts with a cancer associated antigen. Thus, there is a strong association between detecting the detection of cancer-associated antigens or blood antibodies that target antigens from within the circulating blood of an animal and the presence of cancer in that individual. As a result, tests that detect the presence of such antigens or antibodies are useful in cancer testing and diagnosis.

  One possible cancer-associated antigen as a target is a pool containing both fibrin and fibrinogen degradation products (FDP). In healthy individuals, production of fibrin and fibrinogen degradation products are both limited, but in cancer patients, FDP is excessive when proteolytic enzymes such as plasmin and thrombin are released from cancer cells. Produced. FDP is also produced as a by-product in other cancer-related processes such as angiogenesis and metastasis. Since FDP leaks from the tumor into the surrounding body fluid, an increase in FDP concentration is observed in the urine of bladder cancer patients, in the serum of lung cancer patients, and in the plasma or serum of other cancer patients. The usefulness of quantifying FDP in cancer diagnosis has been questioned for many years. This is because an analytical method improved so as to quantitatively measure FDP with the required sensitivity has not been developed. Current FDP analysis is usually limited to measuring one of the components of a particular FDP, eg, a D-2 mer representing this group.

  Cancers of fibrin and fibrinogen degradation products (FDP) are via tissue factor (TF) and urokinase-type plasminogen activator (uPA) that regulates the pathways that produce fibrin and fibrinogen degradation products. Increase both concentrations (Figure 1). When fibrinogen is degraded by plasmin, fragments D and E are produced as the final product of the first stage. Thrombin converts fibrinogen to fibrin in response to signals from the coagulation cascade. When fibrin is degraded by plasmin, a D-2 mer is produced as the final product of the first stage. The composition of FDP produced via cancer-induced degradation by plasmin is affected by the relative amounts of the two substrates, fibrin and fibrinogen.

  Three essential types of fragments (fragment D, fragment E and D-2 mer) of fibrin and fibrinogen degradation products (FDP), and optionally added three types of fragments (fragment Y and Disclosed herein is a method for detecting cancer comprising simultaneously detecting a total of six FDP components from a defined mixture comprising two distinct types of early plasmin digestion products—IPDP) . The method includes simultaneously detecting three essential FDP antigens contained in a biological sample with a group of specific polyclonal antibodies, wherein the polyclonal antibody is specific for three FDP fragments.

  In one embodiment of the present disclosure, a method of detecting cancer in a subject is provided, the method comprising obtaining a biological sample from the subject, and said biological sample from fibrin and fibrinogen degradation products ( A reaction with an antibody preparation that binds to fragment D, fragment E, and D-2 mer, which are at least three antigens related to FDP), to form an antibody-FDP complex, and said antibody-FDP complex And detecting a subject's cancer. In another embodiment, the antibody preparation further optionally binds to at least one of fragment Y and the initial plasmin digestion product (IPDP). In another embodiment, the antibody preparation is a polyclonal antibody preparation.

  In other embodiments, the method comprises an enzyme immunoassay.

  In other embodiments, the diagnostic step further includes at least one additional diagnostic test.

  In one embodiment of the present disclosure, a method of monitoring a patient's cancer is provided, the method comprising: (a) obtaining a first biological sample from a subject at a first sampling time point; and (b). Obtaining a second biological sample from the subject at a second sampling time after the first sampling time; and (c) converting the biological sample into a fibrin and fibrinogen degradation product (FDP). Reacting with an antibody preparation that binds to at least three relevant antigens, fragment D, fragment E, and D-2 mer, to form an antibody-FDP complex; and (d) the antibody-FDP complex. (E) determining the ratio of the concentration of FDP in the second biological sample to the concentration of FDP in the first biological sample; and (f) whether the cancer has progressed. Process to decide Hints, as appropriate, first, repeating the steps for additional biological sample obtained at a later point in time the second time point (a) to (e).

  In another embodiment, the antibody preparation further optionally binds to at least one of fragment Y and the initial plasmin digestion product (IPDP). In another embodiment, the antibody preparation is a polyclonal antibody preparation.

  In other embodiments, the method comprises an enzyme immunoassay.

  In another embodiment, it is assumed that cancer has progressed when the ratio is 1.15 or more. In another embodiment, when the ratio is less than 1.15, the cancer is regressed or stable.

  In one embodiment of the present disclosure, a kit for detecting cancer in a subject is provided, the kit comprising fragment D, fragment E, and at least three antigens associated with fibrin and fibrinogen degradation products (FDP). An antibody preparation that binds to the D-2 mer to form an antibody-FDP complex, a detection system, and instructions for measuring FDP and relating the presence of FDP to cancer.

  In another embodiment, the detection system comprises detection antibodies specific for fragment D, fragment E and D-2 mer, which are at least three antigens associated with fibrin and fibrinogen degradation products (FDP). . In another embodiment, the antibody preparation further binds to at least one of fragment Y and an initial plasmin digestion product (IPDP), as appropriate.

  In another embodiment, the antibody and the detection system comprise an enzyme immunoassay.

FIG. 1 shows a schematic diagram illustrating the production of fibrin and fibrinogen degradation products (FDP) induced in cancer. FIG. 2 shows non-reduced (FIG. 2A) and reduced (FIG. 2B) SDS-PAGE gels of FDP immunogen lots (8 lots 03, 13, 15, 17, 19, 21, 24 and 25). UD indicates undegraded fibrinogen, D indicates fragment D, E indicates fragment E. FIG. 3 shows human serum samples from colorectal cancer (CRC) patients (n = 5) and standard control serum (n = 5) (FIG. 3A exposed to film for about 5 seconds, FIG. FIG. 2 shows a Western blot analysis of anti-FDP antibody binding to 30 seconds exposed). FIG. 3C shows the coomassie blue staining of the accompanying gel representing the total amount of protein loaded per lane. The location of the FDP subtype is indicated on the gel. FIG. 4 shows antigen immunoprecipitation (IP) in human serum (normal and CRC patients) using anti-FDP polyclonal antibody group (+) and rabbit IgG (−) binding beads. Three samples were tested: FDP calibration control (FDP), healthy human serum (N4) and CRC serum (C15). FIG. 5 shows immunoprecipitated FDP antigen in 5 CRC patients and 5 normal patients using anti-FDP polyclonal antibody group (FIG. 5A) or rabbit IgG (control) -conjugated beads (FIG. 5B).

  Detection of fibrin and fibrinogen degradation products (FDP) can be a useful clinical test tool in many cancers. The concentration of FDP correlates with cancer development, stage, progression, and prognosis. Current analysis of FDP is usually limited to the measurement of one specific FDP component, for example the D-2 mer that is representative of this group. On the other hand, since the presence or absence of FDP and the presence or absence of cancer or the progression of cancer are related to each other, the present inventor thought that a plurality of fibrin and fibrinogen degradation products should be measured simultaneously. For purposes of this disclosure, the term “cancer progression” includes cancer progression, regression or recurrence in a patient. The analysis disclosed herein uses an antibody preparation that specifically recognizes at least three FDP antigens, and the antibody preparation is particularly useful for detecting and monitoring cancer progression. These three essential FDPs are D fragment, E fragment and D-2 mer (also known as X fragment). Three additional FDPs useful for detecting and monitoring cancer are the two early plasmin digests (IPDP) identified by sequence and fragment Y. In one embodiment of the method, the three essential FDP antigens are detected almost simultaneously in one analysis. In other embodiments, 4, 5, or 6 FDP antigens are detected simultaneously in the same assay.

  In certain embodiments, the disclosed analysis measures fragment D, fragment E, and D-2 mer. In other embodiments, the disclosed analysis measures Fragment D, Fragment E, D-2 mer and one or two IPDPs. In another embodiment, the disclosed analysis measures fragment D, fragment E, and fragment Y. Furthermore, in another embodiment, the disclosed analysis measures fragment D, fragment E, D-2 mer, fragment Y and one or two IPDPs.

  Immunoassays are well known in the art, and the affinity-purified anti-FDP antibodies disclosed herein can be used in a variety of assays, including but not limited to enzyme immunoassays. Method (ELISA), dot or slot plot, sepharose bead collection system or various rapid test methods such as lateral flow system or flowing water system.

  One particular feature of the methods described herein is that the capture and / or detection antibody can specifically bind to three, four, five or six FDP antigens. Since an antibody preparation agent has a plurality of antibody specificities, the antibody preparation agent is referred to herein as an antibody group. In certain embodiments, the antibody preparation is a polyclonal antibody preparation against an immunogen comprising 3, 4, 5, or 6 FDP antigen species. Polyclonal antibodies are produced in a variety of organisms, including but not limited to rabbits, goats, horses, donkeys, sheep, chickens or sharks. In other embodiments, antibody preparations generated against 3, 4, 5 or 6 FDP antigens are individually prepared and mixed to form an anti-FDP antibody population.

  Further, in other embodiments, the antibody preparation can comprise a group of monoclonal, single specific polyclonal, chimeric or humanized antibodies or antibody fragments, wherein the groups of antibodies are substantially simultaneously in a single analysis. As long as it can react with 3, 4, 5 or 6 FDP antigens, each antibody reacts with one or more of the 6 FDP antigens. Such antibody preparations are well known in the art and will not be described in detail herein.

  The disclosure also includes systems and kits for detecting or monitoring the binding to FDP and the presence or progression of cancer in a subject. In one embodiment, the system binds to and detects three, four, five or six FDPs contained in the antibody population and the biological sample, and the presence or progression of cancer in the subject, Includes detection reagents to correlate with the presence of 4, 5 or 6 FDPs.

  In one embodiment, the kit binds to and detects three, four, five or six FDPs contained in the group of antibodies and biological sample and detects the presence or progression of cancer in the subject, Includes detection reagents to correlate with the presence of 4, 5 or 6 FDPs. In other embodiments, the kit can include one or more of the following in a package or container. (1) a group of one or more antibodies that bind to three, four, five, or six FDPs to capture FDP from a biological sample, (2) detection antibodies, (3) (a) Analysis using antibody group or (b) solid support for immobilizing at least one of detection antibodies, (4) detection reagent, (5) washing solution, and (6) components of kit and biological sample Instructions for doing. Embodiments in which two or more of components (1) to (6) are in the same container are also used.

  Where a kit is provided, different reagents may be packaged in separate containers and mixed directly prior to use or attached to a solid support. Packing the components separately in this way enables long-term storage without compromising the strength of the function of the components.

  The compositions included in a particular kit are provided in containers that maintain the state of these various compositions and are not absorbed or altered by the material of the container.

  As described above, the kit may be provided with the teaching material. The instructions are printed on paper or other media and / or electronically readable media such as floppy disks, CD-ROMs, DVD-ROMs, Zip disks, video tapes, recording tapes, It may be provided by a flash memory device or the like. The detailed description is not physically attached to the kit, so the user can view the Internet website specified by the kit manufacturer or distributor or provide it by email.

  In addition, the claimed methods can be used to detect cancer-related FDP in systems and kits in biological samples from various sources, including but not limited to serum , Blood, plasma, saliva, sputum, urine, ascites, tumor tissue, cerebrospinal fluid, vaginal or rectal secretions, and tears.

  When using the above analytical method for cancer testing, each laboratory must determine their normal and abnormal ranges based on the study of regional populations. Example 11 provides a typical distribution of healthy and cancer patient samples. The concentration of FDP in a biological sample from a patient suspected of having cancer correlates with the presence of cancer, which identifies samples that exceed the normal range based on predetermined numbers by.

When the above analysis method is used to monitor a patient diagnosed with cancer, the reference value should be determined prior to the evaluation of the FDP concentration. The value of FDP obtained with the first serum is a reference value. The value obtained with the following serum is evaluated by calculating the following ratio (R). In the formula, a is the first FDP value, and b is the current FDP value.
R = b / a
If the ratio of the current FDP value to the baseline FDP value is greater than 1.15, the patient is expected to progress disease, but the FDP antigen test results monitor disease progression in these cancer patients. Should be used in conjunction with other standard clinical methods.

  Available commercial reagents cited in the examples were used according to manufacturer's instructions unless otherwise specified.

Example 1
Preparation of FDP immunogens FDP immunogens were prepared by purchasing purified human fibrinogen containing both fibrin and fibrinogen. In this description, fibrin and fibrinogen are referred to as fibrin / ogen. Fibrin / nogen was then reacted with plasmin to form fibrin and fibrinogen degradation products (FDP) by the method of Haverkate and Timan (Thromb. Res. 10: 803-812, 1977). Fibrin / nogen plasmin degradation was controlled by running the reaction for a specific time and stopped with a cocktail of protease inhibitors.

  In particular, the following method was used. Using a 50 ml conical tube, 20 ml of MOPS buffer (5 mM 3- (N-morpholino) propane-sulfonic acid, pH 7.4 containing 0.1 M sodium chloride and 20 mM calcium chloride) was brought to 37 ° C. Incubated at 37 ° C. incubator until reached (approximately 20-25 minutes), purified human fibrin / nogen was added to the warmed MOPS buffer and stirred at 37 ° C. until dissolved. 1 ml of pH 7.4 PBS was added to activate plasmin and 5 units of plasmin was added to the fibrin / nogen in the MOPS solution. And this liquid mixture was stirred with the 37 degreeC incubator for 3 hours. The degraded FDP was recovered from the agitator and 200 μl protease inhibitor cocktail was added and the solution was mixed well. Next, the total protein concentration of the concentrated stock FDP solution was measured, and evidence of FDP degradation products was verified by SDS-PAGE with a 4-20% gradient under denaturing and reducing conditions (see FIG. 2). . The obtained FDP solution was stored at −40 ° C.

  This FDP immunogen was also used as a control in antibody purification and FDP calibration and FDP detection systems.

(Example 2)
Production of Rabbit Anti-FDP Antibody Rabbits were immunized with the FDP immunogen prepared according to Example 1. Each rabbit was injected with an emulsion containing 1.0 to 1.5 ml phosphate buffered saline containing 1 mg of immunogen and the same amount of complete Freund's adjuvant as the primary immunization. Three weeks after injection, each rabbit was bled and the serum was analyzed for antibodies to FDP. One week after blood collection, each rabbit was given a boost by injection into an emulsion containing 1.0 to 1.5 ml phosphate buffered saline containing 1 mg of immunogen and the same amount of non-complete Freund's adjuvant. It was. Rabbits were raised according to the schedule of booster immunization and blood was collected until they could not survive.

  Rabbit sera obtained at various time intervals after immunization were analyzed for antibody concentration against FDP by performing a standard titer analysis using a 96-well plate system. In a typical titer analysis of serum, 10 μg of immunogen is fixed in a well of a 96-well microtiter plate, blocked with FBS, further reacted with a 1: 1 dilution series of various rabbit sera, washed, Detected with horseradish peroxidase (HRP) combined with goat anti-rabbit antibody (Sigma), washed, incubated with 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution, stop solution added, A value of OD 450 nm was measured. The titer was determined by graphing the OD 450 nm value against the dilution factor and determining the inflection point of the curve.

  Rabbit anti-FDP antibody was purified by immunoaffinity as in Example 6.

(Example 3)
Generation of chicken anti-FDP antibodies Chickens were immunized with the FDP immunogen prepared according to Example 1. Each chicken was injected as a primary immunization with an emulsion containing 1.0 to 1.5 ml phosphate buffered saline containing 1 mg of immunogen and the same amount of complete Freund's adjuvant (day 0). Each chicken received additional immunizations on days 35, 56, 77 and 98. Chickens were bled on days 26, 40, 60, 81 and 100 (serum collection). Serum response testing began on day 45. The first IgY recovery was performed on day 53. Initial affinity purification began on day 56.

  Chicken sera obtained at various time intervals after immunization were evaluated for antibody concentration against FDP by performing a standard titer analysis using a 96-well plate system. A subset of chicken anti-FDP IgY antibodies were conjugated with HRP prior to analysis. In a typical titer analysis of serum, 10 μg of immunogen is fixed in a well of a 96-well microtiter plate, blocked with FBS, further reacted with a 1: 1 dilution series of various rabbit sera, washed, Incubated with TMB solution, stop solution was added, and OD 450 nm value was measured. The titer was determined by graphing the OD 450 nm value against the dilution factor and determining the inflection point of the curve.

  Chicken anti-FDP antibody was purified by immunoaffinity as in Example 6.

Example 4
Generation of goat anti-FDP antibody Goats were immunized with the FDP immunogen prepared according to Example 1. Each goat was injected with an emulsion containing 1.0 to 1.5 ml phosphate buffered saline containing 1 mg of immunogen and the same amount of complete Freund's adjuvant as the primary immunization. Three weeks after injection, each goat was bled and the serum was analyzed for antibodies to FDP. One week after blood collection, each goat was given a boost by injection into an emulsion containing 1.0 to 1.5 ml phosphate buffered saline containing 1 mg of immunogen and the same amount of non-complete Freund's adjuvant. It was. The goats were bred according to the boost and blood collection plan until they could not survive.

  Goat sera obtained at various time intervals after immunization were evaluated for antibody concentration against FDP by performing a standard titer analysis using a 96-well plate system. In a typical titer analysis of serum, 10 μg of immunogen is fixed in a well of a 96-well microtiter plate, blocked with FBS, further reacted with a 1: 1 dilution series of various goat sera, washed, Detected with HRP combined with rabbit anti-goat antibody (Sigma), washed, incubated with TMB solution, stop solution was added, and OD450nm value was measured. The titer was determined by graphing the OD 450 nm value against the dilution factor and determining the inflection point of the curve.

  Goat anti-FDP antibody was purified with immunoaffinity as in Example 6.

(Example 5)
Preparation of FDP bound to Sepharose CL 4B Periodate oxidized Sepharose CL 4B (20 ml) was washed with phosphate buffered saline (PBS) at pH 7.5, 10 times the volume of Sepharose gel (eg 20 ml 10 times that of Sepharose gel is equivalent to 200 ml). Washed and drained Sepharose CL 4B was mixed with 20 ml (1 gel volume) of a 2.0 mg / ml solution of pooled FDP antigen (FDP in MOPS, see Example 1).

  3.5 ml (1/6 of the gel volume) 0.1 M aqueous sodium cyanoborohydride deionized (DI) is added to this suspension and the suspension is shaken for 16-20 hours at room temperature. It was. Then 40 ml (twice the volume of the gel) 0.1 M glycine and 0.1 M sodium cyanoborohydride solution dissolved in pH 7.5 PBS was added to the suspension and the suspension was continuously Stir at room temperature for an additional 2 hours. The Sepharose CL-4B gel to which FDP was bound was degassed for 15 minutes and packed in a chromatographic column by gravity flow. The gel was washed successively with the following solutions: DI water (10 times the volume of the gel), PBS pH 7.5 (10 times the volume of the gel), 0.5 M sodium chloride dissolved in PBS (gel PH 0.15M sodium chloride (10 times the gel volume), pH 7.5 PBS (10 times the gel volume). When not in use, the gel was stored at 4 ° C. in 0.1% sodium azide dissolved in PBS for up to 1 week.

(Example 6)
Purification by immunoaffinity of polyclonal antibodies against FDP Polyclonal antisera was filtered through a 0.2 μm syringe filter prior to running over a Sepharose CL 4B gel column coupled with FDP prepared as in Example 5. Filtered polyclonal antiserum was added and its volume was approximately the same as the gel volume (1 gel volume). After the polyclonal antiserum is added, the Sepharose CL 4B gel column coupled with FDP is again washed with PBS (5 times the volume of the gel), 0.5 M sodium chloride (5 times the volume of the gel) solution in PBS. It was. The anti-FDP antibody purified by immunoaffinity bound to the FDP of the column was eluted with 0.15 M sodium chloride at 0.1 M sodium acetate / pH 3. The eluate was collected in a fraction tube for monitoring, and fractions for which protein concentrations (OD 280 nm greater than 0.2) were later detected were transferred to PETG bottles for long-term storage. The pH of the resulting antibody solution was neutralized by adding 1M Tris buffer at pH 8.5.

(Example 7)
Characterization of Rabbit Polyclonal Antibody to FDP In an embodiment, the capture antibody is an affinity purified polyclonal rabbit antibody to FDP. Rabbits were immunized and antibodies were isolated as in Example 3.

  SDS-PAGE showed high purity of the antibody purified by affinity. The major protein band in the non-reducing anti-FDP preparation has a molecular weight of approximately 152 kDa, which corresponds to the molecular weight of rabbit IgG. Under reducing conditions, the 152 kDa protein band disappeared and at the same time two protein bands of approximately 59 kDa and 30 kDa appeared, which were close to the molecular weights of the heavy and light chains of IgG, respectively. Thus, anti-FDP antibodies are primarily IgG and not IgM (approximately 900 kDa molecular weight) or IgA (approximately 320 kDa dimer). The purity of the FDP antibody group purified by affinity was estimated to be about 90% qualitatively based on SDS-PAGE analysis.

Binding of the anti-FDP antibody to the FDP antigen follows a typical Langmuir adsorption isotherm. From this data, the binding constant of the antibody was calculated to be 1.25 × 10 ⁻⁹ M using a double reciprocal plot. Since the antibody preparation was based on polyclonal, the binding constant obtained in this study was a composite binding constant, eg, the average of the binding constants contributed by antibodies produced by different clones.

  Capture and detection antibodies ensure the specificity of any ELISA based test. In order to confirm that the FDP antibody group captures FDP in human serum samples, the FDP antibody group was detected in both Western blot (FIG. 3) and immunoprecipitation (IP) experiments (FIGS. 4, 5 and Table 2). Cross-reacted with human serum samples. In addition, proteins immunoprecipitated by the FDP antibody group were sequenced and identified using mass spectrometry.

  Western blot analysis of human serum samples from both colorectal cancer (CRC) and standard control patients was used to determine the number of FDP antigens in human serum and their estimated molecular weight. Figures 3A and 3B show western blots of the same gel exposed to film for 5 seconds and 30 seconds, respectively. FIG. 3C shows the corresponding Coomassie stained gel. In FIG. 3, six major FDP antigens were detected in CRC serum samples and the estimated molecular weights were 340, 260-320, 220, 150, 80 and 50 kDa. These bands correspond to undegraded fibrinogen, early plasmin digestion product (IPDP), D-2mer fragment / fragment X, fragment Y, fragment D and fragment E. In contrast, only one major band at approximately 220 kDa was detected in the standard control serum. For all serum samples tested, the intensity of response of serum samples from CRC patients was sufficiently greater than from standard controls.

  As shown in Table 1, the average signal density of all 6 FDP components was significantly higher in serum samples from 5 CRC patients than from 5 standard control patients (p value ≦ 0.00000005). ). Zion image density software (Sion) was used for densitometric analysis of the cross-reaction level in the Western blot (FIG. 3A). FIG. 1 shows that the anti-FDP group can distinguish between cancer and standard serum samples based on individual FDP concentrations.

  To confirm the number and size of FDP antigens captured from human serum samples, immunoprecipitation experiments were performed using either rabbit anti-FDP IgG or control rabbit IgG bound to Sepharose beads (FIG. 4). And 5). Immunoprecipitation also enriched for FDP antigens, which were sequenced by Coomassie stained SDS-PAGE.

  Using Affi-Gel® Hz Immunoaffinity Kit (BIO-RAD) according to the manufacturer's instructions, the rabbit anti-FDP IgG or control rabbit IgG antibody was conserved in the Fc portion of the IgG heavy chain. Covalently bound to Sepharose beads via the moiety. Human serum samples were randomly selected and eluted from # 1 to # 10 with PBS pH 7.4. Eluted serum samples were incubated with either anti-FDP conjugated beads or control rabbit IgG conjugated beads. In addition, two types of beads were incubated with a prepared 0.25 mg / ml solution of FDP and served as positive controls for the IP reaction. The IP reaction between the eluted serum and control rabbit IgG binding beads served as a control for recognizing false positive reactions between non-specific rabbit IgG and serum antigen. The IP reaction was carried out overnight at 4 ° C. in a rotator. The IP beads were washed 5 times with PBS and the antigen was extracted in non-reducing Laemli sample buffer at 95 ° C. for 8 minutes. Antigens extracted from anti-FDP and control rabbit IgG beads were then performed with 4-20% SDS-PAGE. FIG. 4 shows all of the experimental conditions for two representative serum samples, N4 from standard patient 4 and C15 from colorectal cancer patient 15. FIGS. 5A and 5B show the reproducibility of the IP response by showing 5 additional standards in the IP analysis and 5 additional colorectal cancer patients.

  The results of the IP experiment shown in FIG. 4 show that four major FDP-specific antigens and one non-specific antigen were captured from the serum of colorectal cancer patients by anti-FDP beads (+). . From this sample (C15 (+), second lane from the right), the estimated molecular weights (MW) of the four main FDP-specific antigens are 340, 300, 220, and 50 kDa, respectively, in descending order. One non-specific antigen in this lane is about 25 kDa. In addition, control lanes N4 (−) and C15 (−) (third and fifth lanes from the right) in the serum sample showed a reaction with a 25 kDa protein, and the proteins were N4 + and C15 (+) (from the right). Corresponds to the 25 kDa protein in N4 and C15 samples exposed to anti-FDP beads (+) as shown in the second and fourth lanes). The presence of a 25 kDa protein in all these N4 (−), N4 (+), C15 (−), and C15 (+) lanes indicates an IP reaction of non-specific interaction between the serum sample and any of the IgG-bound beads. That 25 kDa protein was extracted from The positive control reactions in IP analysis are designated FDP (−) and FDP (+) (6th and 7th lane from the left). The bead control lane (FDP (-)) showed no interaction between the FDP calibration solution without serum and negative control beads. The FDP (+) reaction contains one small band and three large bands. The estimated molecular weight of one small band is 340 kDa, which is consistent with the molecular weight of undegraded fibrinogen. The estimated molecular weights of the three main FDP-specific antigens in the FDP control antigen solution are 220 kDa (matches the molecular weight of fibrinogen fragment X or D-2 mer), 80 kDa (matches the molecular weight of fibrinogen fragment D monomer) ) And 50 kDa (corresponding to fragment E monomer of fibrinogen). Anti-FDP beads captured four major FDP antigens from human serum of colorectal cancer patients. On the other hand, in the standard serum, the same bead showed one small (faint band) antigen of about 53 kDa. By comparing the anti-FDP beads and the control beads in the IP reaction of the human serum sample (and FDP), the specificity of the anti-FDP antibody group can be confirmed.

  FIG. 5 shows that the FDP antigen pattern is reproducible between multiple patient samples. In this experiment, sera from an additional 5 CRCs and 5 standard patients (randomly selected from clinical trial samples) were tested in immunoprecipitation reactions with both control rabbit IgG and anti-FDP beads, respectively. In addition, FDP immunizing antigen (calibration solution) was tested on two types of beads. The SDS-PAGE of FIG. 5A contains extracted FDP-specific antigens from FDP (positive control), CRC patient serum samples, and standard serum samples. The SDS-PAGE shown in FIG. 5B shows antigen binding to control rabbit IgG beads from the same sample in the same order as in FIG. 5A.

  The experiment shown in FIG. 5 confirms that there are four major FDP-specific antigens and one non-specific antigen of 25 kDa in human serum. In FIG. 5A, the FDP control lane is obtained from fibrin and fibrinogen plasmin degradation in vitro under conditioned conditions and contains three major bands and one thin band. Based on the approximate molecular weight of the non-reduced sample, the major band is captured by the FDP antibody group from the FDP control sample and in descending relative molecular weight order, fibrinogen fragment D-2 mer, fragment D monomer, and It corresponds to fragment E monomer. The thin band of 340 kDa is probably undegraded fibrinogen and is also based on its approximate molecular weight. FDP-specific antigens derived from human serum are approximately 340, 300 (280-320), 210, and 50 kDa. These bands correspond to undegraded fibrinogen, early plasmin digest (IPDP), fragment D-2 mer / fragment X, and fragment E, respectively. IP reactions here (and all other IP experiments) were standardized by adding each serum sample at an equivalent concentration to the IP reaction. In FIG. 5B, the major antigen is about 25 kDa. As mentioned above, this suggests that the 25 kDa serum protein reacts nonspecifically with all of the rabbit IgG bound to the beads. Also, the 25 kDa protein interacts non-specifically with rabbit anti-FDP IgG, probably bound by reaction with the beads shown in FIG. 5A.

  In the present experiment shown in FIG. 5, based on the concentration measurements in Table 2, FDP-specific antigens from CRC patient sera were 6.6 to 9.8 times more than that from standard patient sera. However, the non-specific antigen (approximately 25 kDa) had a cancer to non-cancer ratio corresponding to 0.9 to approximately 1. As shown in Table 2, the average signal density of all FDP-specific antigens was significantly higher in serum samples from 5 colorectal cancer patients than from 5 standard control patients (p value). ≦ 0.000035). Non-specific antigen bands from cancer patient sera were not significantly different from those of the standard control (p = 0.53). Table 2 confirms that the anti-FDP antibody group can distinguish between cancer and standard serum samples based on their respective FDP concentrations.

(Example 8)
Characterization of anti-FDP capture antibodies In one embodiment described in Examples 2 and 7, capture antibody groups were produced and their properties were evaluated.

Example 9
FDP antibody complexed with horseradish peroxidase (HRP) for detection The detection antibody was enzyme labeled with horseradish peroxidase (HRP) so that it could be detected in the assay. That is, in this example, the detection antibody was a polyclonal rabbit anti-human FDP antibody produced according to the method of Example 2. The antibody is a purified immunoglobulin fragment of rabbit antiserum, conjugated with horseradish peroxidase, which has a very high intrinsic enzyme activity.

  The detection antibody immunogen contained both fibrin and fibrinogen degradation products. Antibodies react with natural human fibrinogen in addition to the FDP fragment subtypes D, E, X / D-2 mer, and Y. Trace substances contaminating the antibody were removed by solid phase adsorption along with human plasma proteins. The specificity of the antibody was confirmed by Western blot analysis. As detected by the capture antibody, the detection antibody was confirmed to be cross-reactive with the same six FDP fragments in human serum of cancer patients.

(Example 10)
Enzyme immunoassay for FDP (ELISA)
The FDP ELISA is an enzyme-labeled sandwich immunoassay. Capture antibody (polyclonal, rabbit anti-FDP) was obtained from rabbit serum as described in Examples 2, 7 and 8.

  In an exemplary embodiment, the FDP immunoassay includes the use of a removable piece in a 96-well microliter plate configuration. The holes in the microliter plate were coated with a rabbit anti-FDP antibody purified by affinity. Human serum samples were diluted (1: 200) and dispensed into wells. FDP was captured from the serum sample by an anti-FDP antibody immobilized in a microliter plate hole. After the washing step, an anti-FDP antibody bound to HRP (detection antibody) was added to the well. The anti-FDP-HRP complex binds to the captured FDP to form an immunological sandwich with the immobilized anti-FDP antibody. Through the second washing step, TMB, an enzyme substrate, was added to the holes. The evaluation item was a value of 450 nm in a microplate reader after the reaction was stopped with 0.1 N hydrochloric acid. The intensity of the color produced is proportional to the amount of FDP in the serum. The amount was quantified by interpolation based on a standard curve using a purified FDP calibration.

(Example 11)
Distribution of FDP in serum from healthy subjects and patients with benign diseases To examine the general distribution of FDP values in the healthy group, samples of 209 women and 212 men who were aware that there was no disease were used on that day. It was. The subjects were divided into two groups, gender and 40-64 years and over 65 years. Analytical values were determined in duplicate. Cumulative distribution has been demonstrated. An order statistic for each 5th percentile was determined.

  The diversity of samples from subjects with benign disease is summarized to examine the distribution of serum FDP levels in benign disease, which may also be present in patients with confirmed cancer. Samples were collected from geographically diverse locations throughout the United States with appropriate informed consent and with IRB approved procedures. Table 3 below shows the number of samples and the composition of the benign disease group. A total of 400 patients were tested and this group included 74 healthy samples.

  Table 4 shows the characteristics of each benign group tested as well as healthy individuals. Significant dispersion was seen in all groups except the healthy group. The mean value tends to be asymmetric with samples that showed high FDP values. However, the median value in each of these groups is good within the reference range for deemed healthy individuals. As with all other experiments in this study, FDP values were determined in duplicate in all subjects, who accepted according to IRB approved procedures.

  Analysis shows that the median of the benign disease group is as large as the median of healthy subjects. All benign diseases except benign diseases of the pancreas have similar distribution and 95% confidence limits as the healthy group.

(Example 12)
Distribution of FDP in sera from malignant subjects This study was performed on 439 patients who were diagnosed with primary malignancy and were being treated. Subjects were divided into five groups: lung / liver cancer, breast / ovary / cervical cancer, gallbladder / biliary tract / pancreatic cancer, gastric cancer, and colorectal cancer. FDP analysis was performed in duplicate on the sample. One-way analysis of variance was performed with Tamhane's post hoc test. Results were not significantly different between groups. Details are shown in Table 5 for each cancer.

(Example 13)
Distribution of FDP value ratio in each disease group For each of the above healthy, benign and disease groups, FDP values were analyzed in order to examine differences in FDP concentration in each disease group. StatXact® software was used in this analysis to determine an accurate 95% confidence interval in statistics. The distribution table is shown in Table 6.

(Example 14)
Continuous (long-term) monitoring of patients diagnosed with colorectal cancer Samples for this part of the study were obtained from two existing sample banks. Forty-eight sets were obtained from the Geffin Cancer Center in Vero Beach, Florida, and 64 sets were obtained from a serum bank at the MD Anderson Cancer Center in Houston, Texas.

  Among a total of 445 evaluable findings, there were 112 evaluable patient sets. The samples in the series of surveillance studies were not biased because they were already accumulated samples and were collected indiscriminately and included all patients diagnosed with colorectal cancer in the bank at the time of collection. The average number of findings per patient is 4.0.

  In short, a series of samples were collected from 112 colorectal cancer patients, resulting in 445 combined findings that yielded FDP measurements and disease progression determined. It was. Continuous observation lasted for at least as long as 9 months on average. Progression of FDP values in a series of monitoring sets was evaluated as a percentage change between current and past values (Y), and 15% was determined to be the smallest percentage of progression of a particular disease in the FDP analysis . Clinical disease progression (D) was determined by the subject's physician based on medical procedures and analysis in the clinical laboratory that was the standard treatment during the monitoring period. The primary purpose of this analysis is to show that FDP analysis is beneficial by showing that the sum of sensitivity and specificity exceeds 1.

  The response to treatment uses information provided in a doctor's record based on the results of clinical examination and image analysis (eg, bone scan, CT scan, nuclear magnetic resonance image analysis, X-ray examination, ultrasonography). It was evaluated. Response to treatment is defined as follows.

  No complete remission or evidence of disease (CR or NED): complete of all clinical and image measurable diseases based on laboratory tests and image analysis, or other diagnostic methods directed by a physician Disappearance.

  Partial Remission (PR): Bone metastases that exhibit a marked reduction in the size of the initial metastatic lesion or at least a steady state as observed by bone scan in patients with metastases at the time of observation.

  Stable (SD): little change in the size of the initial metastatic lesion, or no significant change in the size of the initial metastatic lesion, or laboratory and image analysis, or other diagnostic method directed by the physician There are no new lesions based on this.

  Progression (PD): Clinical or image analysis results clearly indicate the presence of lesions that were not seen in previous examinations, or a significant increase in the size of early or metastatic lesions.

  A series of samples were collected from 112 patients with colorectal cancer and 446 sets of findings regarding FDP analysis and disease progression were obtained. Several patients showed signs of progression even at the first examination, so we decided to evaluate the association between FDP analysis and progression at each visit. Therefore, a change was obtained from this data by taking the ratio of the value of the subsequent FDP analysis to the previous value. The purpose of this measurement was to determine an increase from the previous measurement as a means of obtaining information about the progress. Significant increase that was evidence of progression was increased by more than 15%. Therefore, if the ratio was 1.15 or higher, the value of the FDP analysis was considered positive, otherwise it was considered negative. This decision was combined with the observation of progress or non-progression at the visit.

  From the final sampling, 335 sets of findings were obtained and evaluated in two ways: visits and patients. In the initial analysis, bootstrap sampling was performed for each patient by randomly sampling one visit for each sample and recording the sensitivity or specificity at that visit. If there is progress, the sensitivity is 1, which is when the FDP analysis value has increased by 15% or more from the previous visit, otherwise it is 0. If there was no progression at the visit, no sensitivity was recorded at that visit, and the specificity was recorded as 1, if the FDP analysis was less than 15% increase at that visit, otherwise 0. In each visit analysis, there were 135 visits for sensitivity and 198 visits for specificity.

  The second analysis is performed on a patient-by-patient basis, based on the number of progressions through all visits for a given patient, and by counting the number of visits in which the FDP analysis value increased by at least 15% in the progression visits, Used to calculate the sensitivity for each patient. Similarly, the specificity for each patient was calculated by dividing the number of FDP analysis values less than a 15% increase at the visit by the number of progressions at the visit. Here, if the patient progresses every time, there is no specificity for the patient, and if the patient is not progressing at all, there is no sensitivity for the patient. As a result, the 112 patient samples had at least sensitivity, specificity, or both. The sensitivity for each patient was estimated 70, and the specificity for each patient was estimated 86.

  Positive predictive value (PPV) and negative predictive value (PNV) were also calculated on a per patient and visit basis. PPV is divided by the number of progressions when the FDP analysis value is increased by 15% or more, and NPV is divided by the number of progressions when the FDP analysis value is not increased by 15% or more, Calculated.

  If the number of samples that can be used for evaluation is small and the structure is complicated, a statistical technique called bootstrap is used to obtain a reasonable estimate of the relevant population characteristics. Such a calculation method repeatedly extracts and replaces samples from 333 findings, calculates the sum of sensitivity and specificity, and statistically calculates the resulting sample frequency distribution. Determine the sum excluding the percentile corresponding to the alpha level of the test. Such a sample automatically includes any correlation between patients, allowing an unbiased test of the null hypothesis described above. Bootstrap sampling is typically repeated multiple times until the distribution of results is consistent between trials.

  Bootstrap samples per visit were performed 2000 times by randomly sampling and replacing one visit for each of 112 patients. Average sensitivity, average specificity and sum from each sample are divided by the total sensitivity or specificity (value of 1 or 0) of all patients and the number of times the measured sensitivity or specificity was obtained. calculated. After determining the average of sensitivity and specificity for a sample, both were added to obtain the sum. This provided a 2000 bootstrap estimate of sensitivity, specificity and total for each visit. A bootstrap estimate such as one-sided and two-sided 95% confidence limits can be obtained by a frequency display of 2000 values by variables.

  The sampling method was performed by assigning a number between 1 and k for each k (1-7) visits. A sample of 112 findings was obtained by generating a random number uniformly between 0 and 1, and that number was multiplied by k to take the integer of that number and add 1. This treatment gave a random number between 1 and k for each patient. The FDP analysis ratio changed from the previous time was calculated for the patient visit corresponding to the given number. In sample extraction, a replacement was made and the process was repeated 112 times, so duplication was allowed. Sensitivity, specificity and total were calculated and recorded for each of 112 samples.

  The sensitivity for each visit calculated from the evaluation value for each visit of 333 is 100 × 88/134 = 65.19, and the specificity is 100 × 133/199 = 67.34, the sum of sensitivity and specificity. Was 132.53. The PPV was 100 × 88/153 = 57.52 and the NPV was 100 × 134/181 = 74.03.

  The results of statistical analysis to evaluate the effectiveness of FDP immunoassay to obtain information about disease progression in patients with colorectal cancer are shown in Table 7 below.

  Even if the test is conducted at α = 0.05 or α = 0.025, the sum of sensitivity and specificity from this analysis is clearly statistically significantly greater than 100. The median sum is probably about 133, the median sensitivity is about 65, and the median specificity is about 67. The total one-sided 5% confidence lower limit is about 120 and the one-sided 2.5% confidence lower limit is about 118. The median values of PPV and NPV are about 59 and 73, respectively, over 5 times. Note that these values are consistent with those determined from the visit-specific values given above in Table 7. Five iterations corresponding to 2000 samples indicate that the results are reasonable and consistent.

  With regard to analysis by patient, the sensitivity per visit calculated from the evaluation values of 112 patients is 100 × 45.68 / 69 = 66.21, and the specificity is 100 × 58.63 / 86 = 68.18. The sum of sensitivity and specificity was 134.39, the PPV was 100 × 51.83 / 97 = 53.44, and the NPV was 100 × 71.67 / 103 = 69.58.

  Even if the test is conducted at α = 0.05 or α = 0.025, the sum of sensitivity and specificity from this analysis is clearly statistically significantly greater than 100. The total median is probably about 134, the median sensitivity is about 66, and the median specificity is about 68. The total one-sided 5% confidence lower limit is about 124, and the one-sided 2.5% confidence lower limit is about 121. The median values of PPV and NPV are approximately 53 and 69, respectively, over 5 times. Note that these values are consistent with those determined from the visit-specific values given above in Table 8. Five iterations corresponding to 2000 samples indicate that the results are reasonable and consistent.

  The conclusions from the above analysis of the effectiveness of the FDP immunoassay in monitoring colorectal cancer samples are as follows. These data and analysis show that FDP analysis provides useful data regarding the progression of colorectal cancer if there is a 15% or more change from the previous visit. The results of FDP immunoassays must be used in combination with standard therapeutic treatments in monitoring colorectal cancer patients.

  Unless otherwise specified, all numbers and others representing content, molecular weight, amount of characteristic of reaction conditions used in the specification and claims can be modified with the term “about” in all examples. As understood. Therefore, unless specified to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties obtained by the present invention. Can be done. Each numerical parameter shall at least be applied in terms of the reported significant digits and the usual rounding method, even if not as an attempt to limit the application of the principles equivalent to the claims. Should be obtained. Despite the numerical values and the parameters describing the scope of the invention being approximate values, the numerical values describing a particular example are reported as accurately as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation of the measurements in their respective tests.

  “A (1)”, “an (1)”, “the (its, said, etc.)” and similar indication objects (especially the following claims) used in the context of the description of the present invention Is understood to include both singular and plural. However, this is unless otherwise specified or apparently consistent with the context. The description of a numerical range herein is merely used to provide an abbreviated way of specifying individual numerical values within that range alone. Unless otherwise specified herein, individual numerical values are incorporated into the specification as individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or example terms (such as “such as”) herein is for the purpose of merely clarifying the present invention and is within the scope of the present invention, ie, the claims. It does not limit the range. Anything not mentioned in the specification is to be understood as any non-claimed element essential for the practice of the invention.

  The classification of alternative elements or embodiments of the invention disclosed herein is not to be construed as limiting. Each group of elements may be individually cited, claimed, or variously combined with other elements in the group or other elements described herein. One or more elements of the group are expected to be included or excluded from the group for convenience and / or patentability. In the event of such inclusion or exclusion, this specification is deemed to satisfy the description of all Markush groups, including that group as a variation, and thus used in the appended claims.

  Certain embodiments according to the invention are disclosed herein and include the best mode known to the inventors for carrying out the invention. Of course, variations in these described embodiments will be apparent to those of skill in the art based on the above description. The inventor expects those skilled in the art to make such modifications appropriately, and the inventor intends that the invention be practiced other than as specifically described herein. . Accordingly, this invention includes equivalent subject matter as recited in the claims appended hereto as permitted by all modifications and applicable laws. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated or clearly contradicted by context.

  Particular embodiments disclosed herein may be further limited to the claims that use the terms “consisting of” or “consisting of essential components”. When used in the claims, the term “consisting of” excludes any element, step, or content not specified in the claim, whether filed or added in amendment. The term “consisting of essential components” limits the scope of the claims to those that do not substantially affect the particular material or process and, if necessary, the new features. As claimed, embodiments of the present invention are described and can be used essentially explicitly herein.

  Furthermore, in this specification, several patent publications and publications are cited. Each of the above cited references and publications are incorporated herein by reference as they are.

  Finally, it is understood that the embodiments of the invention disclosed herein are illustrative of the nature of the invention. Other variations may be used within the scope of the present invention. Thus, by way of example and not limitation, other configurations of the invention may be utilized in accordance with the contents herein. Thus, the present invention is not limited to what has been shown and described.

Claims (15)

A method for detecting cancer in a subject,
Obtaining a biological sample from the subject;
The biological sample binds to fragment D, fragment E and D-2 mer, which are at least three antigens associated with fibrin and fibrinogen degradation products (FDP) to form antibody-FDP complexes. Reacting with an antibody preparation;
Detecting the antibody-FDP complex;
Diagnosing the subject's cancer;
A method for detecting cancer in a subject characterized by comprising: The antibody preparation is
Further optionally, it binds to at least one of fragment Y and the initial plasmin digestion product (IPDP).
The method according to claim 1. The antibody preparation is
A polyclonal antibody preparation,
The method according to claim 1. Including enzyme immunoassay,
The method according to claim 1. The diagnosis step includes:
Further including at least one additional diagnostic test,
The method according to claim 1. (A) obtaining a first biological sample from a subject at a first sampling time point;
(B) obtaining a second biological sample from the subject at a second sampling time after the first sampling time;
(C) binding the biological sample to fragment D, fragment E and D-2 mer, which are at least three antigens related to fibrin and fibrinogen degradation products (FDP), and antibody-FDP complex Reacting with an antibody preparation to form
(D) detecting the antibody-FDP complex;
(E) determining a ratio of the concentration of FDP in the second biological sample to the concentration of FDP in the first biological sample;
(F) determining whether the cancer has progressed;
Including
Optionally, repeat steps (a) to (e) for additional biological samples obtained at time points after the first and second time points.
A method of monitoring cancer in a patient, characterized by: The antibody preparation is
Further optionally, it binds to at least one of fragment Y and the initial plasmin digestion product (IPDP).
The method according to claim 6. The antibody preparation is
A polyclonal antibody preparation,
The method according to claim 6. The step of reacting and the step of detecting include
Including enzyme immunoassay,
The method according to claim 6. When the ratio is 1.15 or more, the cancer has progressed.
The method according to claim 6. If the ratio is less than 1.15, the cancer is regressed or stable,
The method according to claim 6. A kit for detecting cancer in a subject,
An antibody preparation that binds to Fragment D, Fragment E and D-2 mer, at least three antigens associated with fibrin and fibrinogen degradation products (FDP), to form an antibody-FDP complex;
A detection system;
Instructions for associating the measurement of FDP and the presence of FDP with cancer;
A kit for detecting cancer in a subject, comprising: The detection system is
A detection antibody specific for fragment D, fragment E and D-2 mer, which are at least three antigens related to fibrin and fibrinogen degradation products (FDP). kit. The antibody preparation is
Further optionally, it binds to at least one of fragment Y and the initial plasmin digestion product (IPDP).
The kit according to claim 13. The antibody and the detection system comprise an enzyme immunoassay;
The kit according to claim 12.

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