KR20060102592A - Immunological complexes and kits for cancer diagnostics using autoantibody detection, and the uses thereof - Google Patents

Abstract

The present invention relates to a detection method for anti-ECPKA detection in a biological sample, and also to an immunocomplex and kit for cancer diagnosis using anti-ECPKA detection, and a method for diagnosing cancer in mammals using the same.

The anti-ECPKA IgG antibody-EIA method according to the present invention is superior in sensitivity and specificity than the ECPKA enzyme assay and shows that autoantibody assays are much more useful for diagnosing cancer than conventional antigen assays.

Anti-ECPKA Autoantibodies, Cancer Diagnosis

Description

Immunocomplex and kit for cancer diagnosis using autoantibody detection method and method using same {IMMUNOLOGICAL COMPLEXES AND KITS FOR CANCER DIAGNOSTICS USING AUTOANTIBODY DETECTION, AND THE USES THEREOF}

1 is an assay of ECPKA autoantibodies obtained using the format of the preferred immunoassay of the present invention in normal and cancer patients.

Fig. 2 shows ECPKA activity values of normal and cancer patients obtained using the ECPKA enzyme assay as a control of the sensitivity and specificity of the format of the immunoassay of the present invention.

Figure 3 shows the correlation between the assay value of the anti-ECPKA antibody isolated from the serum of normal and cancer patients and the ECPKA enzyme assay.

Figure 4 shows the titers of anticancer antigen antibodies in the serum of normal and cancer patients using HCT-15 tumor extract as cancer antigen.

5 shows the titers of anticancer antigen antibodies in serum of various cancer patients.

The present invention relates to an immunocomplex and detection method for anti-ECPKA detection in a biological sample, and also to an immunocomplex and kit for cancer diagnosis using anti-ECPKA detection and a cancer diagnosis method for a mammal using the same.

Tumor markers are synthesized by malignant tumor cells and secreted into blood vessels. These markers are the result of metabolic changes due to infiltration or tumors, and are made by host tissue. Tumor marker concentrations in blood and tissue fluids are very useful for diagnosing, screening, and tracking tumor progression and degeneration. An ideal tumor marker would be cancer detection with a simple blood test and the concentration would be proportional to the stage of tumor progression. However, due to lack of sensitivity and specificity, until now, a single label could not screen healthy people and cancer patients. The use of tumor markers for cancer detection is well described in the literature below.

1. Sluss, PM et al . [2004] "ESTABLISHMENT OF A CENTRAL LABORATORY SERUM TUMOR MARKER SERVICE ON A CONSOLIDATED IMMUNODIAGNOSTIC PLATFORM: DEVELOPMENT OF PRACTICE STANDARDS, SERVICE IMPROVEMENTS, AND OPERATIONAL EFFICIENCY," Clin. Leadersh. Manag. Rev. 18 [1]: 25-31;

2. Gion, M. [2000] "SERUM TUMOUR MARKERS: FROM QUALITY CONTROL TO TOTAL QUALITY MANAGEMENT," Breast 9 [6]: 306-11;

3. Wiesner, A. [2004] "DETECTION OF TUMOR MARKERS WITH PROTEINCHIP ^® TECHNOLOGY," Curr. Pharm. Biotechnol. Feb; 5 [1]: 45-67;

4. Crawford, N.P. et al. [2003] "TUMOR MARKERS AND COLORECTAL CANCER: UTILITY IN MANAGEMENT," J. Surg. Oncol. 84 [4]: 239-48;

5. Agnantis, N.J. et al. [2003] "TUMOR MARKERS. AN UPDATE APPROACH FOR THEIR PROGNOSTIC SIGNIFICANCE. PART I. IN VIVO," 17 [16]: 609-18;

6. Riley, R.D. et al. [2004] “A SYSTEMATIC REVIEW OF MOLECULAR AND BIOLOGICAL TUMOR MARKERS IN NEUROBLASTOMA,” Clin. Cancer Res. 10 [1 Pt 1]: 4-12;

7. Given, M. et al. [2000] "THE PREDICTIVE OF TUMOUR MARKERS CA 15-3, TPS AND CEA IN BREAST CANCER RECURRENCE," Breast. 9 [5]: 277-80.

Currently available cancer markers are cancer antigens. For example, prostate cancer can be diagnosed by assaying prostate-specific antigen (PSA) specific to the prostate gland (Gretzer, MB et al . [2003] "PSA MARKERS IN PROSTATE CANCER DETECTION," Urol. Clin. North Am 30 [4]: 677-86). In addition, Carcino-Embryonic Antigen (CEA) markers have been shown to be diagnostically useful for detecting colon cancer (Crawford, NP et al. [2003] "TUMOR MARKERS AND COLORECTAL CANCER: UTILITY IN MANAGEMENT," J. Surg. Oncol. 84 [4]: 239-48). As a cancer antigen, CA15-3 is directly related to breast cancer (Cheung, KL et al. [2003] "OBJECTIVE MEASUREMENT OF REMISSION AND PROGRESSION IN METASTATIC BREAST CANCER BY THE USE OF SERUM TUMOUR MARKERS," Minerva. Chir. Jun; 58 [3]: 297-303). Cancer antigen CA19-9 has been used to diagnose gastrointestinal cancer (Grotowski, M. [2002] "ANTIGENS [CEA AND CA 19-9] IN DIAGNOSIS AND PROGNOSIS COLORECTAL CANCER," Pol Merkuriusz Lek. 12 [67]: 77-80; Trompetas, V. et al . [2002] "GIANT BENIGN TRUE CYST OF THE SPLEEN WITH HIGH SERUM LEVEL OF CA 19-9," Eur. J. Gastroenterol. Hepatol. 14 [1]: 85-8) , Cancer antigen CA125 has been used for the diagnosis of ovarian cancer (Anderiesz, C. et al. [2003] "SCREENING FOR OVARIAN CANCER," Med. J. Aust. 178 [12]: 655-6).

Most solid cancers cause abnormalities in chromosome segregation during cell division due to chromosomal instability. Some enzyme kinases are involved in maintaining proper chromosome segregation or regulating cell cycle progression. cAMP-dependant protein kinase (PKA) plays a functional role in many places, including cellular signaling, metabolism, and proliferation (Matyakhina, L. et al . [2002] "PROTEIN KINASE A AND CHROMOSOMAL") STABILITY, "Ann. NY. Acad. Sci. 968: 148-57; Tortora, G. et al . [2002]" PROTEIN KINASE A AS TARGET FOR NOVEL INTEGRATED STRATEGIES OF CANCER THERAPY, "Ann. NY. Acad. Sci. 968: 139-47).

There are two classes of PKA in mammalian cells (Krebs, EG et al. [1979] "PHOSPHORYLATION-DEPHOSPHORYLATION OF ENZYME," Annu. Rev. Biochem. 48: 923-39), which is type I (PKA-I). ) And type II (PKA-II). They are distinguished by different control subunits (R subunits) RI and RII and have a common catalyst subunit (C subunit) (Beebe, SJ et. Al . [1986] "CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES, "In: The Enzymes: Control by Phosphorylation, EG Krebs et al. [Eds] Academic Press: Orlando and London. Pp. 43-111).

Conventionally, the enzymatic activity of protein kinase has been assayed using the method of transferring the radioactive phosphate group ( γ - ³² P) of ATP to residues of a suitable protein or peptide substrate (Witt, JJ et al . [1975] Anal Biochem. 66: 253-8; Casnellie, JE [1991] Methods Enzymol. 200: 115-20; US Pat. No. 6,498,005). PKA enzyme assay methods have been described in several documents (Cohen, CB et al . [1999] "A MICROCHIP-BASED ENZYME ASSAY FOR PROTEIN KINASE A," Anal. Chem. [1999] 273: 89-97; Cho, YS et al. [2000] "EXTRACELLULAR PROTEIN KINASE A AS A CANCER BIOMARKER: ITS EXPRESSION BY TUMOR CELLS AND REVERSAL BY A MYRISTATE-LACKING C _ALPHA AND RII _BETA SUBUNIT OVEREXPRESSION," Proc. Natl. Acad. Sci. USA. ]: 835-40).

Biochemical studies and gene cloning have identified four isoforms of regulatory subunits, RIα, RIβ, RIIα, and RIIβ (Amieux, PS et al . [2002] "THE ESSENTIAL ROLE OF RI ALPHA IN THE MAINTENANCE OF) REGULATED PKA ACTIVITY, "Ann. NY. Acad. Sci. 968: 75-95; McKnight, GS et al . [1988]" ANALYSIS OF cAMP-DEPENDENT PROTEIN KINASE SYSTEM USING MOLECULAR GENETIC APPROACHES, "Recent Prog. Honn. Res. 44: 307-35; Levy, FO et al . [1988] "MOLECULAR CLONING, COMPLEMENTARY DEOXYRIBONUCLEIC ACID STRUCTURE AND PREDICTED FULL-LENGTH AMINO ACID SEQUENCE OF THE HORMONE-INDUCIBLE REGULATORY SUBUNIT OF 3 ', 5'-CYCEPENDOSINE PROTEIN KINASE FROM HUMAN TESTIS, "Mol. Endocrinol. 2: 1364-73).

It is very important that the PKA-I and PKA-II ratios can change dramatically during cell growth, differentiation and transformation (Lohmann, SM et al . [1984] "REGULATION OF THE CELLULAR AND SUBCELLULAR CONCENTRATIONS AND DISTRIBUTION". OF CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES, "In: Advances in Cyclic Nucleotide and Protein Phosphorylation Research, P. Greengard et al . [Eds] Raven Press: New York. Pp. 63-117; Cho-Chung, YS [1990]" ROLE OF CYCLIC AMP RECEPTOR PROTEINS IN GROWTH, DIFFERENTIATION, AND SUPPRESSION OF MALIGNANCY: NEW APPROCHES TO THERAPY, "Cancer Res. 50: 7093-100; Cho-Chung, YS [2003]" cAMP SIGNALING IN CANCER GENESIS AND TREATMENT, "Cancer Treat Res. 115: 123-43).

The cAMP signaling system has been proposed as a therapeutic target for malignant tumors of the lymphatic system (Lerner, A. et al . [2000] "THE cAMP SIGNALING PATHWAY AS A THERAPEUTIC TARGET IN LYMPHOID MALIGNANCIES," Leuk Lymphoma. 37 [1-2] Cho-Chung, YS et al . [1995] "cAMP-DEPENDENT PROTEIN KINASE: ROLE IN NORMAL AND MALIGNANT GROWTH," Crit. Rev. Oncol. Hematol. 21 [1-3]: 33-61; Cho-Chung, YS et al . [1993] "THE REGULATORY SUBUNIT OF cAMP-DEPENDENT PROTEIN KINASE AS A TARGET FOR CHEMOTHERAPY OF CANCER AND OTHER CELLULAR DYSFUNCTIONAL-RELATED DISEASES," Pharmacol. Ther. 60 [2]: 265-88). . In human cancer cell lines and primary tumors, granule-macrophage colony-stimulating factors or pobolesters after transformation with chemicals, virus carcinogens, and Ki- ras oncogenes, or transforming growth factor-α (Cho-Chung, YS [1990] "ROLE OF CYCLIC AMP RECEPTOR PROTEINS IN GROWTH, DIFFERENTIATION, AND SUPPRESSION OF MALIGNANCY: NEW APPROACHES TO THERAPY) , "Cancer Res. 50: 7093-100; Miller, WR et al . [1993]" TYPES OF CYCLIC AMP BINDING PROTEIN IN HUMAN BREAST CANCERS, "Eur. J. Cancer. 29A: 989-91), RI / PKA- It was confirmed that the expression of I was increased (Cho-Chung, YS [1990] "ROLE OF CYCLIC AMP RECEPTOR PROTEIN GROWTH, DIFFERENTIATION, AND SUPPRESSION OF MALIGNANCY: NEW APPROACHES TO THERAPY," Cancer Res. 50: 7093-100; Cho Chung , YS et al [2002] "DISSECTING THE CIRCUITRY OF PROTEIN KINASE A AND cAMP SIGNALING IN CANCER GE NESIS: ANTISENSE, MICROARRAY, GENE OVEREXPRESSION, AND TRANSCRIPTION FACTOR DECOY, "Ann. NY. Acad. Sci. 968: 22-36). In contrast, a decrease in the expression of RIα / PKA-I results in growth inhibition caused by a wide range of human cancer cell lines grown in nude mice, site-selective cAMP analogs targeting the RIα subunit of PKA in human tumors, and antisense oligonucleotides. Cho-Chung, YS et al . [1989] "SITE-SELECTIVE CYCLIC AMP ANALOGS AS NEW BIOLOGICAL TOOLS IN GROWTH CONTROL, DIFFERENTIATION AND PROTO-ONCOGENE REGULATION," Cancer Inv. 7: 161-77; Cho-Chung , YS et al . [1999] "ANTISENSE DNA-TARGETING PROTEIN KINASE A-RI α SUBUNIT: A NOVEL APPROACH TO CANCER TREATMENT," Front Biosci. 4: D898-D907).

It has previously been shown that various cancer cell species secrete PKA into the medium (Cho, YS et al . [2000] "EXTRACELLULAR PROTEIN KINASE A AS A CANCER BIOMARKER: ITS EXPRESSION BY TUMOR CELLS AND REVERSAL BY A MYRISTATE-LACKING C _ALPHA) AND RII _BETA SUBUNIT OVEREXPRESSION, "Proc. Natl. Acad. Sci. USA. 97 [2]: 835-40). This extracellular PKA (ECPKA) is present in an active state in the form of a free catalytic subunit (C subunit) ("PKA Cα"), whose activity is specifically inhibited by PKI, a PKA inhibitory protein.

In expression vectors that increase PKA-I intracellularly, overexpression of Cα or RIα of PKA significantly increases ECPKA expression. Conversely, overexpression of the RIIβ subunit that eliminates PKA-I and increases PKA-II to reverse the transgenic phenotype reduces ECPKA. Cα mutations that prevent myristylation increase intracellular PKA but inhibit ECPKA increase. This suggests that the NH ₂ -terminal myristyl group of Cα is required for ECPKA expression. In cancer patient serum, ECPKA expression was markedly increased in contrast to that of normal subjects (Cho, YS et al . [2000] "EXTRACELLULAR PROTEIN KINASE A AS A CANCER BIOMARKER: ITS EXPRESSION BY TUMOR CELLS AND REVERSAL BY A MYRISTATE- LACKING C _ALPHA AND RII _BETA SUBUNIT OVEREXPRESSION, "Proc. Natl. Acad. Sci. USA. 97 [2]: 835-40).

The development of monoclonal antibodies has enabled the discovery of numerous tumor-binding antigens in the serum and tissue of malignant tumor patients. Protein products of oncogenes and tumor suppressor genes can be detected extracellularly and can act as potential markers for carcinogenesis in vivo. Some of the growth factors were encoded by oncogenes, for example, the p21-ras protein was encoded by the ras oncogene found in patient blood. Antibodies to p53 tumor suppressor proteins have been found in the serum of breast cancer, lung cancer patients and infants with B-lymphoma (Winter, SF et al . [1992] "DEVELOPMENT OF ANTIBODIES AGAINST p53 IN LUNG CANCER PATIENTS APPEARS TO BE DEPENDENT ON THE TYPE OF p53 MUTATION. "Cancer Res. 52: 4168-74; Lubin, R., et al . [1993]" ANALYSIS OF p53 ANTIBODIES IN PATIENTS WITH VARIOUS CANCERS DEFINE B-CELL EPITOPES OF HUMAN p53: DISTRIBUTION ON PRIMARY STRUCTURE AND EXPOSURE ON PROTEIN SURFACE. "Cancer Res. 53: 5872-6; Crawford, LV, et al . [1982]" DETECTION OF ANTIBODIES AGAINST THE CELLULAR PROTEIN p53 IN SERA FROM PATIENTS WITH BREAST CANCER. "Int. J. Cancer. 30 : 403-8). Antibodies to oncogenes such as c-myc and c-myb have been found in colon and breast cancer serum (Sorokine, I., K. et al . [1991] "PRESENCE OF CIRCULATING ANTI-C-MYB ONCOGENE PRODUCT ANTIBODIES IN HUMAN SERA. "Int. J. Cancer. 47: 665-9; Ben-Mahrez, K., et al . [1988]" DETECTION OF CIRCULATING ANTIBODIES AGAINST C-MYC PROTEIN IN CANCER PATIENT SERA. "Br. J. Cancer 57: 529-34; Ben-Mahrez, K., et al . [1990] "CIRCULATING ANTIBODIES AGAINST C-MYC ONCOGENE PRODUCT IN SERA OF COLORECTAL CANCER PATIENTS." Int. J. Cancer. 46: 35-8).

In addition to the new labels mentioned above, certain other proteins, hormones and enzymes have been used as labels for the past 30 years. Notable among these are carcinoembryonic antigen (CEA), α-fetoprotein (AFP), human chorionic gonadotropin (HCG), and prostatic acid phosphatase (PAP).

Most of the labeling materials including the above labeling materials lack specificity. The concentration may increase in benign tumors or during pregnancy. All of these markers are based on antigen detection methods, but are less specific or sensitive. Therefore, it is necessary to discover new biomarker molecules and make them available for clinical use. The present invention is to meet this need.

Summary of the Invention

The present invention relates to a highly sensitive enzyme immunoassay (EIA) for IgG antibody assays against extracelluar protein kinase A (ECPKA). Experiments with 295 patients with various types of cancer and 100 normal patients found that the concentration of anti-ECPKA IgG antibodies was significantly higher in cancer patients (92%) than in normal patients (14%). There was no significant correlation between the anti-ECPKA IgG antibody assay using EIA and the ECPKA antigen assay using PKA enzyme assay. The anti-ECPKA IgG antibody-EIA method was superior in sensitivity and specificity than the ECPKA enzyme assay. The results of the present invention show that autoantibody assays are much more useful for cancer diagnosis than conventional antigen assays.

Specifically, the present invention provides an immunoassay comprising the following steps, assaying for the presence or concentration of anti-ECPKA autoantibodies in a biological sample from a mammal:

(a) contacting a biological sample with an antigen specific to an anti-ECPKA autoantibody, wherein the step is combined with the antigen when the anti-ECPKA autoantibody is present in the biological sample. Carried out under conditions sufficient to form the antibody complex form;

(b) contacting the anti-ECPKA autoantibody binding molecule to the antigen-anti-ECPKA autoantibody complex formed above, wherein said anti-ECPKA autoantibody binding molecule is the antigen-anti-ECPKA formed above. Carried out under conditions sufficient to bind the anti-ECPKA autoantibody of the autoantibody complex to form an expanded complex; And,

(c) determining the presence and concentration of anti-ECPKA autoantibodies in the biological sample by assaying the presence and concentration of the expanded complex formed above.

In one embodiment of the immunoassay of the invention, the specific antigen for the anti-ECPKA autoantibody is an extracellular PKA protein.

In another embodiment of the immunoassay of the present invention, the anti-ECPKA autoantibody is an antibody of a mammalian species different from that of the mammal, and is specific for an antibody produced in the mammal.

In another embodiment of the immunoassay of the invention, the mammal is a human and the anti-ECPKA autoantibody is a human IgG antibody.

In another embodiment of the immunoassay of the invention, the anti-ECPKA autoantibody binding molecule is labeled for detection (in particular, chemical labeling or enzymatic effect).

In another embodiment of the immunoassay of the invention, said antigen specific for the anti-ECPKA autoantibody of step (a) is immobilized to a solid retardation prior to contact with the biological sample.

In another embodiment of the immunoassay of the invention, said antigen specific for the anti-ECPKA autoantibody of step (a) is immobilized on a solid support after contact with said biological sample.

In another embodiment of the immunoassay of the present invention, the immunoassay used is an immunochromatographic immunoassay,

In step (a) above, the biological sample is placed in contact with the first porous carrier, the first porous carrier containing an unfixed, labeled antigen specific for the anti-ECPKA autoantibody;

In step (b) above, the formed antigen-anti-ECPKA autoantibody complex is placed in contact with the second porous carrier, the second porous carrier is connected with the first porous carrier, and the immobilized anti-ECPKA autoantibody Contains a binding molecule; And,

In step (c) above, the presence or concentration of anti-ECPKA autoantibodies in the biological sample is assayed by detecting the presence of labeled antigens specific for anti-ECPKA autoantibodies in a second porous carrier.

The present invention also relates to an immunocomplex containing an antigen specific for an anti-ECPKA autoantibody bound to an anti-ECPKA autoantibody, wherein the anti-ECPKA autoantibody is additionally bound to an anti-ECPKA autoantibody binding molecule. will be.

In one embodiment of the immunocomplex of the invention, the antigen specific for the anti-ECPKA autoantibody is an extracellular PKA protein.

In another embodiment of the immunocomplex of the invention, the anti-ECPKA autoantibody binding molecule is labeled for detection (especially chemical labeling or enzymatic effect).

In another embodiment of the immunocomplex of the invention, the anti-ECPKA autoantibody binding molecule is an immunological molecule.

In another embodiment of the immunocomplex of the invention, the anti-ECPKA autoantibody is a human autoantibody and the anti-ECPKA autoantibody binding molecule is an anti-human IgG antibody.

The present invention also relates to a kit consisting of the following configurations for assaying the presence and concentration of anti-ECPKA autoantibodies in a biological sample of a mammal:

Where the kit consists of an empty case consisting of a multi-filter system, a first and a second porous carrier, a second porous carrier connected to the first porous carrier, a first porous carrier connected to the multiple filter system A portion thereof can be accessed from the case;

The first porous carrier contains unfixed, labeled PKA Cα or Cα fragments; And

The second porous carrier is immobilized and contains antibodies bound to unlabeled human IgG.

In one embodiment of the kit of the invention, the labeled PKA Cα is ECPKA (especially chemical labeling or enzymatic effect).

In another embodiment of the kit of the invention, the kit detects human anti-ECPKA autoantibodies and the antibody binding to human IgG is an antibody of a mammal other than human.

The present invention also relates to a diagnostic method for diagnosing cancer in mammals other than humans using the above immunocomplex or kit.

Detailed description of the invention

Extracellular PKA (ECPKA) is secreted from cancer cells (Cho, YS et al [2000] "EXTRACELLULAR PROTEIN KINASE A AS A CANCER BIOMARKER: ITS EXPRESSION BY TUMOR CELLS AND REVERSAL BY A MYRISTATE-LACKING C _ALPHA AND RII _BETA SUBUNIT OVEREXPRESSION Proc. Natl. Acad. Sci. USA. 97 [2]: 835-40). The present invention originated from the idea that the secretion of ECPKA causes the formation of serum autoantibodies, and therefore, the assay can be used as a diagnostic and predictive marker for cancer by assaying the concentration or presence of autoantibodies. The present invention is a high sensitivity immunoassay that assays the concentration, or presence, of anti-ECPKA autoantibodies (particularly anti-ECPKA-IgG antibodies) in biological samples of suspected or confirmed cancer patients. The immunoassay based on this autoantibody provides a diagnostic method for detecting various cancer cells.

The present invention relates to antigen-antibody binding. As used herein, the "epitope", a two-dimensional or three-dimensional region of an antigen, specifically binds to the antibody, thus "specific" or "recognition" if the antigen and the antibody can immunospecifically bind to each other. Or "to combine".

The assay according to the method of the present invention may be in any format. Such a format may be heterogeneous or homogeneous, sequential or simultaneous, competitive or noncompetitive. U.S. Pat. Explain. Such assays may be of quantitative construction to assay the concentration or amount of anti-ECPKA autoantibodies, or of qualitative construction to assay the presence or absence of anti-ECPKA autoantibodies.

Heterogeneous immunoassay techniques involve the use of solid materials to which reaction products are bound and differ from the binding of non-immobilized antigens with antibodies (ie liquid phase immunoassays). The reaction product is separated from excess samples, assay reagents, and other materials by removing solids from the reaction mixture (eg, by washing). One form of solid immunoassay that has been used in connection with the present invention is a sandwich immunoassay. In this sandwich immunoassay, the more assay material present in the sample, the higher the amount of label in solid form. Such assays are generally widely used when assay material concentrations are low. This is because the solid label concentration can be detected very easily.

According to a preferred embodiment of the present invention, the antigen that specifically reacts with the anti-ECPKA autoantibody is bound to (ie fixed) the solid support, and tested with a biological sample to test for the presence of anti-ECPKA IgG antibody. Incubate by mixing. The antigen is incubated with a biological sample that is not bound and then fixed with a solid support. The support is then washed to remove non-ECPKA IgG antibodies that do not bind to the immobilized antigen. At the end of this treatment, an immunocomplex is formed between the antigen and the anti-ECPKA IgG antibody.

A detectably labeled secondary antibody (eg anti-human IgG antibody) is added and incubated under sufficient conditions to allow binding between the secondary antibody and the anti-ECPKA IgG antibody of the immunocomplex. Thereafter, washing is performed to remove secondary antibody which is not bound. If anti-ECPKA IgG antibodies are present in the sample, both antibodies will form an immunocomplex (ie, sandwich type of secondary antibody / anti-ECPKA IgG antibody / antigen). Detection of secondary antibodies in this assay suggests the presence of anti-ECPKA IgG antibodies in the sample to be tested. The sandwich assay format is described in Schuurs et al . US Pat. Nos. 3,791,932 and 4,016,043, and Pankratz, et al ., US Pat. No. 5,876,935. Secondary antibodies include naturally occurring immunoglobulins isolated from non-human primates (eg, anti-human IgG rat antibodies, anti-human IgG goat antibodies, etc.) and immunoglobulin fragments produced recombinantly and synthetically. If desired, other binding molecules (which can bind anti-ECPKA autoantibodies) can be used as the secondary antibody or can be used instead. For example, anti-ECPKA autoantibodies can be biotinylated and secondary antibodies can also be replaced with labeled avidin or streptavidin.

In order to eliminate the step of isolating non-binding portions and to reduce the equipment and detection time required for chemical bonding, homogeneous assay format can be used as an alternative. This assay has one side of the binding pair (antigen) already immobilized; However, if the other side of the binding pair (anti-ECPKA autoantibody) is present, it is detected without separation of the non-binding portion. For example, the EMIT method of Syva, Inc (Sunnyvale, CA) operating through detection of fluorescence quenching, which is a homogeneous optical method; Laser nephilometry latex particle agglomeration methods of Behringwerke, Marburg, Germany, which operate by detecting changes in light scattering; LPIA latex particle agglomeration method of Mitsubishi Chemical Corporation (Tokyo, Japan); TDX fluorescens depolarization by Abbott Park, IL; And the fluorescence energy transfer method of Cis International (Cis Bio International, Paris, France).

Any of the above assays can be applied for the purposes of the present invention.

The binding assays of the invention can be constructed according to competitive assays. In competitive assays, the more anti-ECPKA IgG antibody present in the test sample, the lesser the amount of label present in the solid phase.

Competitive assays, similar to the sandwich assay, provide accurate amounts of labeled anti-ECPKA IgG antibodies and determine whether the labeled antibodies and competitive anti-ECPKA IgG antibodies are contained in the tested liquid phase to ensure binding with the support. It can be done to judge. In this competitive assay, the amount of labeled antibody obtained is inversely proportional to the amount of assay present in the test sample. Smith (US Pat. No. 4,401,764) proposed a new competitive assay using mixed binding complexes capable of binding with assays or labeled assays. However, the assay and labeled assay cannot be bound to the complex at the same time. Claget (US Pat. No. 4,746,631) discloses an immunoassay using a reaction chamber. In this method, the assay / ligand / label conjugate in the test sample assay is substituted from the reaction surface and the substituted assay / ligand / label conjugate is immobilized at the second reaction site. The conjugates include biotin, bovine serum albumin and synthetic peptides as ligand components of the conjugate, and labeling components include enzymes, chemiluminescent materials, enzyme inhibitors and radioactive nucleic acids.

Lee (US Pat. No. 4,661,444) discloses a competitive immunoassay using a conjugate of an anti-idiotype antibody with a detectably labeled secondary antibody. In this method, the detectable reaction is inversely proportional to the presence of the assay in the sample. Allen (European Patent Application No. 177,191) discloses binding assays involving conjugates of ligand reagents with secondary reagents such as fluorescein. According to this assay, the binder competes with the assay (ligand) in binding to the labeled binding partner specific for the ligand, as a result of which the labeled conjugate is then separated from the reaction mixture. This binding assay format combines a competitive binding technique with a reverse sandwich assay arrangement. That is, a method of binding a binder to a solid phase and binding the binder to a labeled binding molecule before separating the binder from the mixture. However, this assay result is assayed in such a way that the amount of label bound to the solid phase is inversely proportional to the amount of assay material in the test sample, as in conventional competitive assays. Chieregatt et al . (GB Patent No. 2,084,317) described a similar assay format using a binding partner specifically labeled in a non-indirect way to the assay. Mochida et. al . (US Pat. No. 4,185,084) also described the use of dual-antigen conjugates that competitively bind and then label antigen antigens to bind immobilized antibodies. This method also detects a solid label, which is inversely proportional to the amount of label and the amount of assay material in the test sample. Sadeh et. al . (US Pat. No. 4,243,749) describes a similar enzyme immunoassay that competes with assays for binding hapten conjugates to antibodies immobilized on a solid phase. As such, various assays can be used in connection with the present invention.

In any assay format, at least one of the assay reagents will be preferentially labeled or detectable by light emission or quenching. Such component will be the antigen to which the secondary antibody, anti-ECPKA IgG antibody, or anti-ECPKA IgG antibody binds, according to the designed immunoassay mode. Radioisotope-binding assays (eg, radioimmunoassays) are where radioisotopes are used as labels. The signal can be detected by the emission of light from the phosphor or part of the phosphor (Lucas et al . [US Pat. No. 5,698,411] and Landrum et. Al . [US Pat. No. 5,976,822]). Enzyme-binding assay methods (eg, Elisha et al.) Use enzymes as labels. The signal can be detected by the emission of color or light from the color or phosphor part. Paramagnetic labels, materials used as colored particles, emulsions, colloidal metals such as selenium and gold, and other labels such as dyes can be used (see US Pat. Nos. 4,313,734, 4,373,932, and 5,501,985). The use of enzymes (particularly alkaline phosphatase, β-galactosidase, horse radish peroxidase, or urease) as detection markers is preferred.

The presence of an enzyme label is detected through the use of a chromogenic substrate that is catalyzed by the enzyme label. More preferably used are chemical labels (eg, colloidal gold, latex bead labels, etc.). Label detection can be accomplished using multiple detectors, multiple filters, gratings, or specifically other fluorescence (eg US Pat. No. 5,759,781). The use of peroxides as enzyme markers is particularly preferred, which reacts specifically with the chromogenic substrate 3,3 ', 5,5'-tetramethylbenzidine (TMB). In the case of labeling peroxides with antibodies by enzymatic labeling, periodate techniques (Nakane, PK et al . [1974] "PEROXIDASE-LABELED ANTIBODY. A NEW METHOD OF CONJUGATION," J Histochem. Cystochem. 22: 1084-90) Or incorporating a partner with a heterofunctional reagent (Ishikawa. E. et al . [1983] "ENZYME-LABELING OF ANTIBODIES AND THEIR FRAGMENTS FOR ENZYME IMMUNOASSAY AND IMMUNOHISTOCHEMICAL STAINING," J. Immunoassay. 4 [3] : 209-327).

All solid supports that are widely used in a variety of ways can be used in the immunoassay of the present invention. Suitable materials for use as solid supports are composed of synthetic materials such as polystyrene, polyvinylchloride, polyamide, or other synthetic polymers, and natural polymers such as cellulose, as well as natural polymers such as cellulose acetate, nitrocellulose, and glass. It may also be derived from. The support may take the form of a ball, branch, tube and microblack or microtiter plate. In addition, sheet-like structures such as paper strips, small plates and membranes are also suitable. The surface of the carrier may be permeable or impermeable to aqueous solutions.

Although the above description relates to assaying for the presence of anti-ECPKA-autoantibodies in biological samples that are liquids (eg, serum, blood, urine, saliva, pancreatic fluid, cerebrospinal fluid, semen, etc.), any liquid biological sample (eg, Tissue or biopsy extracts, fecal extracts, sputum and the like) may be used in the immunoassay of the present invention as well. The most suitable biological sample would be serum.

Ideally, the material for use in the assays of the invention is kitted. Some kits may be configured in compartments to hold a sample in a portion. Other kits also include vials and tubulars. Each container consists of separate elements. For example, in one of the containers, a suitable antigen is present in binding to the support, and the composition of the second container is liquid and detectably labeled secondary antibody is present therein. In addition, the kit may contain one or more containers containing a predetermined ECPKA or anti-ECPKA (PKA Cα or Cα fragment) autoantibody. The latter vessel can be used to create a standard curve that can insert results from samples in which the amount of autoantibodies against ECPKA is unknown.

In the use of this kit, all users should add to the container an appropriate dilution of the sample for which the amount of autoantibody to ECPKA is unknown. In the first container there is an antigen bound to the support and in the second container there is a detectably labeled antibody. After moderate incubation time, an immunocomplex is formed or separated from the supernatant, detects radioactivity against the immunocomplex or supernatant, adds an enzyme substrate and causes color development, or a chemical label (eg colloidal). Gold, latex beads, etc.) can be combined for detection.

The present invention is particularly concerned with the use of immunochromatographic assays for detecting anti-ECPKA autoantibodies. In a preferred immunochromatographic assay, two porous carriers that are in contact but spatially different are used. The first carrier consists of non-fixed, labeled PKA Cα or Cα fragments, and the second carrier contains unlabeled antibodies that are fixed and bind IgG (eg, human anti-ECPKA self In assays of antibodies, the unlabeled antibody can be an anti-human IgG antibody).

Preferably, the device will consist of an empty case, for example a plastic material. The first carrier is indirectly connected to the interior of the case via a multiple filter system that can utilize a device (eg, a device that is exposed or not completely covering the device). Test samples such as serum, plasma or complete blood can be applied directly to the filter system and penetrated therefrom within the first porous carrier. In such a device, permeation of a liquid containing anti-ECPKA autoantibodies will cause the unfixed and labeled PKA Cα or Cα fragment of the first carrier to bind to the moving antibody and then permeate the second carrier. Because the second carrier contains immobilized antibodies that bind to human IgG, any labeled PKA Cα or Cα fragments entering the second carrier can be captured therein.

Thus, detection of labeled PKA Cα or Cα fragments in a carrier containing immobilized and unlabeled antibody, which exhibits such anti-ECPKA autoantibodies, means that the anti-ECPKA autoantibodies are present in the test sample. The assay can be quantified by assaying the amount of labeled PKA Cα or Cα fragments bound in the second porous carrier.

ECPKA autoantibodies obtained using the format of the immunoassay of the present invention in normal and cancer patients are shown in FIG. 1. The incidence and mean titers of cancer patients were 92% and 2.2, respectively, which were significantly higher than those of normal patients = 14% and mean titers 0.6. Values higher than 1.0 (above the dashed line) are positive.

ECPKA activity values obtained using the ECPKA enzyme assay are shown in FIG. 2 to compare the sensitivity and specificity of the immunoassay format method of the present invention. These values, obtained from normal and cancer patients, show a lower sensitivity and specificity when compared to the frequency of normal patients = 21% and the mean = 60 mU / ml, with frequency = 83% and mean = 130 mU / ml in cancer patients. Values higher than 75 mU / ml are positive.

 The serum of cancer patients and healthy humans was separated, and the results of confirming the correlation between the anti-ECPKA antibody value and the ECPKA enzyme assay value are shown in FIG. 3. Cancer patients had a coefficient of 0.003 and normal people had a coefficient of 0.001, which was not statistically significant.

The titer of anti-cancer antigen antibody in serum when HCT-15 tumor extract was used as cancer antigen in cancer patients and normal persons is shown in FIG. 4. Values above 1.5 (dotted line) are positive.

Antigen antibody titers are shown in the serum of cancer patients with various types of cancer (lung, kidney, pancreas, ovary, colon, liver, stomach, bladder, cervix, melanoma, sarcoma, and myoma).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, embodiments of the present invention are only a part of presenting the spirit of the present invention in detail, and various modifications, changes, etc. can be made by those skilled in the art to which the present invention pertains without departing from the spirit of the present invention. It is understood that this is within the scope of the present invention.

Example

<Experimental Example 1>

Comparative Evaluation of Anti-ECPKA Autoantibody Immunoassay and PKA Enzyme Assay for Diagnosis of Human Cancer

Materials and methods

Subjects : Serum samples were obtained from 295 patients with various types of cancer cells and 100 healthy people.

ELISA : Anti-ECPKA IgG autoantibodies in serum of cancer patients and healthy people are assayed by solid ELISA assay. Diluted (50 μg per ml in PBS) of purified recombinant human PKA Cα antigen (see Purified PKA Cα, see Materials and Methods) on a round bottom polyvinylchloride microtiter plate for assay (Thermolab System, Helsinki, Finland). Concentration) to 100 μl. The plates were incubated for 30 minutes at room temperature, then washed three times with wash buffer (20 mM Hepes, 0.9% NaCl, 30 mM sucrose-0.1% bovine serum albumin [BSA], pH 7.0) and water in each well. Block in a 100 μl block case (Blockace, Serotec, http://www.serotec.com) dissolved in (4 g / 400 ml) at room temperature for 1 hour, and then remove the plate with sodium citrate washing solution (50 mM sodium citrate, 0.15 M NaCl, Wash three times with 0.1% polyoxyethylene sorbitan monolaurate Tween ^® 20, pH 5.0-5.2). Serum samples are continuously diluted with sample dilution buffer (PBS pH 7.4, 0.25% BSA [fat free fatty acid fraction V], 0.05% Tween 20), 100 μl of diluted sample is added to each vessel and incubated for 2 hours at room temperature. After washing three times with sodium citrate rinse, 100 μl of anti-human IgG-HRP antibody-enzyme conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) in 0.01 M phosphate buffered saline (PBS), 0.9% NaCl, 1% BSA The plate is then incubated for 1 hour at room temperature. The plate is washed five times with sodium citrate wash and then 100 μl of TMB substrate is added. After the reaction was stopped with 100 μl of 0.45MH ₂ SO ₄ reagent, the absorbance at 450 nm was recorded with an ELISA reader (BioRad [Hercules, CA] microplate reader). ELISA specificity is confirmed by simultaneously performing an inhibition test on serum samples incubated for 1 hour at room temperature at the final concentration of the antigen of 1 g / L.

PKA Cα purification : OT1529-Cα plasmid (Cho, YS et al . [2000] "EXTRACELLULAR PROTEIN KINASE A AS A CANCER BIOMARKER: ITS EXPRESSION BY TUMOR CELLS AND REVERSAL BY A MYRISTATE-LACKING C _ALPHA AND RII _BETA SUBUNIT OVEREXPRESSION Natl. Acad. Sci. USA. 97 [2]: 835-40) is injected with pQE31 DNA to produce pQE-Cα. pQE-Cα plasmids were expressed in E-coli and purified of the original PKA Cα protein (Paragon, Baltimore MD).

PKA assay : Enzymatic activity of PKA was determined by Rohlff, C. et al . (1993) ("8-Cl-cAMP INDUCES TRUNCATION AND DOWN-REGULATION OF THE RI ALPHA SUBUNIT AND UP-REGULATION OF THE RII BETA SUBUNIT OF CAMP-DEPENDENT PROTEIN KINASE LEADING TO TYPE II HOLOENZYME-DEPENDENT GROWTH INHIBITION AND DIFFER- 60 LEUKEMIA CELLS, "J. Biol. Chem. 268: 5774-82). For determination of serum ECPKA activity, the assay (total volume, 50 μl) was performed with or without 10 μl serum and reaction mixture (5 μM cAMP and 5 μM PKI, 50 mM Tris HCl [pH 7.5], 1 mM DTT, 10 mM MgCl _2). , 5 μM Kemptide (synthetic substrate for PKA; GIBCO / BRL), containing 1.2 μM [ γ - ³² P] ATP [25 Ci / mmol; ICN] and run at 37 ° C. for 20 minutes. After incubation, the reaction mixture is dropped into phosphocellulose discs (GIBCO / BRL) and washed three times with 0.5% phosphoric acid. Dry the filter in air and measure with a liquid scintillation counter. One unit of PKA activity is defined as the amount of enzyme that has migrated 1 pmol of ³² P from ( γ - ³² P) ATP to protein regenerated in 1 minute at 37 ° C. in a standard assay system. LDH activity was assayed using a commercial kit (Sigma).

result

Serum from patients and normal control subjects is examined using the ELISA described above, optionally showing anti-ECPKA autoantibody titers according to the ratio to the mean absorbance of the normal control serum. From anti-ECPKA autoantibody titers of normal controls, ratios greater than 1.0 are considered positive.

The coefficients of variation during and between the tests were <8.8% and <9.0%, respectively, indicating that there is reproducibility in the emotion method. Values for anti-ECPKA autoantibodies in serum of cancer patients are shown in FIG. 1. Both frequency and mean titers of patients were significantly higher than normal controls (14% frequency, 0.60 mean titers) (92% frequency, 2.2 mean titer).

In contrast, the frequency and mean titers of the ECPKA enzyme assay (in antigen concentration assays) (patient, frequency = 83%, mean value = 130 mU / ml; normal control, frequency = 21%, mean value = 60 mU / ml) were determined by cancer patients ( n = 66) and the normal control (n = 66) showed a significant overlap suggesting a lack of reactivity and specificity (FIG. 2).

The anti-ECPKA autoantibody titer of individual obtained by ELISA and the comparison of ECPKA assayed by PKA enzyme assay are shown in FIG. 3. This shows that there is no correlation between the titer of anti-ECPKA IgG antibody obtained by ELISA and ECPKA assayed by enzyme test (ECPKA antigen assay).

<Experiment 2>

Conclusion on anti-ECPKA autoantibody immunoassay and its use in human cancer

The present invention shows that the serum presence of autoantibodies against ECPKA is highly correlated with cancer. For anti-ECPKA autoantibodies, the developed ELISA has high sensitivity and specificity and shows significantly high anti-ECPKA autoantibody titers (average titer 2.2, frequency 92%) in cancer patients, but low or negative in normal subject controls. Phosphorus titer and frequency (14%). Comparing the frequency detected by ELISA with the frequency detected by enzyme assay, ELISA detecting autoantibodies against ECPKA exhibits high sensitivity and specificity when compared to enzyme assay assayed for antigenic activity.

The following experiment was carried out to investigate whether the autoantibody detection method of the present invention can be applied to other cancer antigens (secreted outside of cells).

: HCT-15 tumor (a multidrug-resistant human colon cancer raised in nude mice) is used as a source of cancer antigen. Tumors were in Buffer # 10 (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.5% sodium deoxycholate, 5 mM MgCl ₂ , Protease Inhibition Cocktail Set 1 [Calbiochem]), and in buffer fragment 1 (weight) in buffer 3 (volume). Homogenize at the rate of. The homogenized is centrifuged at 10,000 rpm for 10 minutes and the supernatant collected. The extract is incubated with Protein-A Sepharose (3: 1) while rotating at 4 ° C. for 1 hour and then centrifuged for 2 minutes at 6,000 rpm. The supernatant is collected and dialyzed by contacting PBS overnight at 4 ° C. After dialysis, the extracts are prepared for coating on microtiter plates of ELISA. Figure 4 shows the titers of anticancer antigen antibodies in serum from cancer patients (n = 36) and healthy people (n = 25). An average value of 1.5 or greater is positive. FIG. 5 shows titers of antigenic antibodies in serum from cancer patients with various forms of cancer (lung, kidney, melanoma, pancreas, ovary, colon, liver, stomach, bladder, cervical carcinoma, sarcoma, leiomyoma). The serum (n = 36) and normal serum (n = 25) of the patient were as in FIG.

From the above experimental results, it was confirmed that the autoantibody immunoassay method using the immunocomplex or kit of the present invention for detecting cancer antigens of humans and animals is excellent in sensitivity, specificity, and reliability. It can also be seen that ELISAs for detecting autoantibodies using the present invention, rather than antigens against ECPKA and other cancer antigens, offer an excellent new approach for diagnosing cancer in humans and animals.

Claims (21)

An immunoassay comprising the steps of assaying for the presence or concentration of anti-ECPKA autoantibodies in a biological sample from a mammal: (a) contacting a biological sample with an antigen specific to an anti-ECPKA autoantibody: in the above step, the anti-ECPKA autoantibody is present in the biological sample to bind the antigen to the antigen-anti-ECPKA Autoantibody complexes are formed; (b) contacting the anti-ECPKA autoantibody binding molecule to the antigen-anti-ECPKA autoantibody complex formed above, wherein the anti-ECPKA autoantibody binding molecule is formed of the antigen-anti- Combine with the anti-ECPKA autoantibodies of the ECPKA autoantibody complex to form an expanded complex; And, (c) determining the presence and concentration of anti-ECPKA autoantibodies in the biological sample by assaying the presence and concentration of the expanded complex formed above. The immunoassay according to claim 1, wherein the specific antigen for the anti-ECPKA autoantibody is an extracellular PKA protein. The immunoassay according to claim 1, wherein said anti-ECPKA autoantibody is an antibody of a mammalian species different from said mammal, and is specific for the antibody produced in said mammal. The immunoassay according to claim 3, wherein the mammal is a human, and the anti-ECPKA autoantibody is a human IgG antibody. The immunoassay according to claim 1, wherein the anti-ECPKA autoantibody binding molecule is labeled so as to be detected. The immunoassay according to claim 5, wherein the detectable label is a chemical label. 2. The immunoassay according to claim 1, wherein said antigen specific for anti-ECPKA autoantibody in step (a) is immobilized on a solid support prior to contact with said biological sample. The immunoassay according to claim 1, wherein said antigen specific for anti-ECPKA autoantibody in step (a) is immobilized on a solid support after contact with said biological sample. The method of claim 1, wherein the immunoassay is an immunochromatographic immunoassay. In step (a) above, the biological sample is placed in contact with the first porous carrier, wherein the first porous carrier contains an unfixed, labeled antigen specific for the anti-ECPKA autoantibody; In step (b) above, the formed antigen-anti-ECPKA autoantibody complex is placed in contact with a second porous carrier, and the second porous carrier is connected to and fixed with the first porous carrier. Containing anti-ECPKA autoantibody binding molecule; And, In step (c) above, the presence or concentration of anti-ECPKA autoantibody in the biological sample detects the presence of the labeled antigen specific for the anti-ECPKA autoantibody in the second porous carrier. Immunoassay characterized in that the assay by. An immunocomplex containing an antigen specific for an anti-ECPKA autoantibody bound to an anti-ECPKA autoantibody, wherein the anti-ECPKA autoantibody is additionally bound to an anti-ECPKA autoantibody binding molecule. . The immunocomplex of claim 10, wherein the antigen specific for the anti-ECPKA autoantibody is an extracellular PKA protein. The immunocomplex of claim 10, wherein the anti-ECPKA autoantibody binding molecule is labeled so as to be detected. The immunocomplex of claim 12, wherein the detectable label is a chemical label. The immunocomplex of claim 10, wherein the anti-ECPKA autoantibody binding molecule is an immunological molecule. The immunocomplex of claim 14, wherein the anti-ECPKA autoantibody is a human autoantibody, and the anti-ECPKA autoantibody binding molecule is an anti-human IgG antibody. Kits consisting of the following constructs for assaying the presence and concentration of anti-ECPKA autoantibodies in a biological sample from a mammal: The kit consists of an empty case consisting of a multi-filter system, a first porous carrier and a second porous carrier, the second porous carrier being connected to a first porous carrier, and the first porous carrier being a multiple filter. Is connected to the system, a portion of which can be accessed from the case; The first porous carrier contains an unfixed, labeled PKA Cα or Cα fragment; And The second porous carrier is immobilized and contains antibodies bound to unlabeled human IgG. The kit of claim 16, wherein said labeled PKA Cα is ECPKA. The kit of claim 16 wherein said label of labeled PKA Cα is a chemical label. The kit of claim 16, wherein the kit detects human anti-ECPKA autoantibodies, and wherein the antibody binding to human IgG is an antibody of a mammal other than human. A diagnostic method for diagnosing cancer in mammals other than humans using the immunocomplex according to any one of claims 10 to 15. 20. A diagnostic method for diagnosing cancer in mammals other than humans using the kit according to claim 16.

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