KR100819122B1 - A Kit for Diagnosis of Pancreas Cancer - Google Patents

Abstract

The present invention relates to a pancreatic cancer diagnostic kit comprising a pancreatic cancer diagnostic substrate and an electric substrate capable of detecting pancreatic cancer specific antigen in a blood sample of a subject. The pancreatic cancer diagnostic kit of the present invention includes a pancreatic cancer diagnostic substrate to which an antibody capable of specifically binding to pancreatic cancer specific antigen is attached, and a detection means capable of detecting pancreatic cancer specific antigen bound to an antibody on an electrical substrate. The pancreatic cancer diagnostic kit of the present invention can determine whether pancreatic cancer develops even by blood samples of a subject, and thus, it may be widely used for early diagnosis of pancreatic cancer.

Matrilin 3, transthyretin (prealbumin, amyloidosis type I), lipocalin 2 and stratifin, protein chips, enzyme-linked immunosorbant assay (ELISA) , Immunochromatography, strip

Description

A kit for diagnosis of pancreas cancer

1 is a conceptual diagram of an immunochromatography strip for diagnosing pancreatic cancer of the present invention.

The present invention relates to a kit for diagnosing pancreatic cancer. More specifically, the present invention relates to a pancreatic cancer diagnostic kit comprising a pancreatic cancer diagnostic substrate and an electrical substrate capable of detecting pancreatic cancer specific antigen in a blood sample of a subject.

Among the major diseases of modern people, researches on the treatment and diagnosis of cancer are relatively active, mainly in lung cancer, liver cancer, gastric cancer, and the like. However, studies on esophageal cancer, colorectal cancer, and pancreatic cancer with low incidence are relatively low. On the other hand, pancreatic cancer initially does not feel much, and after the general metastasis, symptoms such as pain and weight loss usually occur, and the healing rate is lower, so regular diagnosis is very important. Most of the clinical symptoms are slow onset, loss of appetite and weakness, and weight loss is the most common symptom. Diagnosis has been performed by X-ray imaging of the stomach and duodenum, biliary tract imaging through the skin and liver, and retrograde endoscopic cholangiography, but recently ultrasonography and computed tomography are the most commonly used diagnostic methods. to be. By these methods, when a disease lesion is found, when a diagnosis is performed by performing a more accurate biopsy, a relatively accurate test result may be obtained. However, the method of diagnosis is very inconvenient because the patient suffers from pain. The subjects are reluctant to do this. Therefore, the development of a test method for diagnosing pancreatic cancer simply and quickly has been required.

On the other hand, a method using a gene has been developed to diagnose a genetic mutation such as cancer. Although significant results have been reported for lung cancer, liver cancer, and gastric cancer, which have high incidence, relatively low incidence is reported. In the case of pancreatic cancer, the research results are not much. In the diagnostic method using the gene, DNA is extracted from a tissue suspected of cancer by polymerase chain reaction (PCR), or RNA is extracted from the tissue and analyzed by gene expression analysis using a cDNA microarray. The former is effective only in certain cancers, such as chronic myelogenous leukemia (CML) or acute lymphocytic leukemia (ALL), mainly caused by chromosome translocation. There is a disadvantage that a conventional cancer diagnosis method such as examination or CT imaging should be preceded.

In order to overcome the drawbacks of the above-described cancer diagnostic method, a method for detecting and diagnosing a cancer-specific antigen has been developed. A representative example of the cancer-specific antigen may be a carcinoembryogenic antigen (CEA). CEA is known to increase in levels in patients with rectal-colon cancer, stomach cancer, breast chamber, lung cancer, ovarian cancer, prostate cancer, liver cancer and pancreatic cancer. A number of cancer diagnostic kits have also been developed through the method of detecting CEA. Korean Patent No. 151823 discloses monoclonal antibodies that can specifically bind to CEA, and Korean Patent No. 395206 discloses monoclonal antibodies against CEA. Ultrafast liver cancer, colorectal cancer, and prostate cancer diagnostic strips are disclosed. However, as described above, since CEA is expressed not only in pancreatic cancer but also in other cancer tissues, it cannot be referred to as pancreatic cancer specific antigen, and no pancreatic cancer specific antigen specifically expressed only in pancreatic cancer patients has been reported.

Therefore, there is a constant need to develop a method for screening pancreatic cancer specific antigens and rapidly and accurately diagnosing pancreatic cancer using antibodies thereto.

Accordingly, the present inventors have made efforts to develop pancreatic cancer-specific antigens and to develop a method for rapidly and accurately diagnosing pancreatic cancer using antibodies thereto. As a result, the present invention is directed to a protein that is specifically expressed in pancreatic cancer tissue and secreted into the blood. When the detection of the electrical protein in the blood sample of the subject, it was confirmed that the pancreatic cancer can be diagnosed quickly and accurately, and completed the present invention.

After all, the main object of the present invention is to provide a pancreatic cancer diagnostic substrate capable of detecting pancreatic cancer specific antigen in blood samples.

Another object of the present invention to provide a kit for diagnosing pancreatic cancer comprising an electrical substrate.

The substrate for diagnosing pancreatic cancer of the present invention includes (i) an anti-matrilin 3 antibody, an anti-transthyretin antibody, and an anti-lipocalin 2 antibody. And 1 to 4 antibodies selected from the group consisting of anti-stratifin antibodies; And, (ii) a pancreatic cancer diagnostic substrate comprising a solid phase support having an antibody attached thereto, wherein the solid phase support is not particularly limited thereto, but may be a nitrocellulose membrane, a polyvinylidene fluoride membrane, or a microplate. It is preferable that it is a glass substrate, a polystyrene board, a silicon board, or a metal board, and it is more preferable that it is an NC film, a microplate, or a glass board. In addition, the above-mentioned antibodies are not particularly limited, but it is preferable to use polyclonal antibodies or monoclonal antibodies, more preferably to use monoclonal antibodies, and the above-mentioned antibodies are hydrogen bonds, aldehyde bonds, poly-L- It is preferable to adhere to the solid-state support by a method such as lysine and a linkage by a linker.

On the other hand, the pancreatic cancer diagnostic kit of the present invention includes a pancreatic cancer diagnostic substrate to which an antibody capable of specifically binding to pancreatic cancer specific antigen is attached, and a detection means capable of detecting pancreatic cancer specific antigen bound to the antibody on the electrical substrate: At this time, the detection means is not particularly limited thereto, but the primary antibody capable of specifically binding to the antigen bound to the antibody on the substrate or the primary antibody capable of specifically binding to the antigen bound to the antibody on the electrical substrate; It is preferable to use a secondary antibody-signal complex that specifically binds to the primary antibody. In particular, when the secondary antibody-signal complex is used as the detection means, the signal complex is not particularly limited thereto, but the fluorescent substance (for example, Cy-3, Cy-5, FITC, GFP (green fluorescent protein), RFP (red) fluorescent protein, Texas Red, etc.), luminescent materials, radioisotopes, enzymes (e.g. horseradish peroxidase (HRP), alkaline phosphatase, beta galactosidase (β-galactosidase), Luciferase and the like) may be preferably used, and when an enzyme is used as a signal complex, a kit for diagnosing pancreatic cancer may further include a coloring reagent causing a color reaction by an electric enzyme.

According to one embodiment of the present invention, the pancreatic cancer diagnostic kit of the present invention uses an NC (nitrocellulose) membrane or a polyvinylidene fluoride (PVDF) membrane with an antibody capable of specifically binding to the pancreatic cancer specific antigen as a pancreatic cancer diagnostic substrate. And an immunochromatography strip using a primary antibody-gold conjugate capable of specifically binding to an antigen bound to an antibody on an electrical substrate as a detection means for detecting pancreatic cancer specific antigen bound to an antibody on an electrical pancreatic cancer diagnostic substrate. (See FIG. 1).

1 is a conceptual diagram of an immunochromatography strip for diagnosing pancreatic cancer of the present invention. The electro immunochromatography strip is composed of a sample pad (1), an immune response member, and an anti-matriline 3 antibody, an anti-transtyretin antibody, an anti-lipocalin 2 antibody, and an anti-stratipine antibody bound to gold particles, respectively. Temporarily fixed glass fiber (GF) filters (2) and polyclonal antibody bands (determination lines, 3) specific for matriline 3, transtyretin, lipocalin 2 and stratipin, respectively, as result indicators The NC membrane 5 and the absorption pad 6 to which the crude secondary antibody band (control line 4) is fixed are mounted on the adhesive plastic backing 7.

According to another embodiment of the present invention, the pancreatic cancer diagnostic kit of the present invention uses a microplate attached with an antibody capable of specifically binding to pancreatic cancer specific antigens as a pancreatic cancer diagnostic substrate, and the antibody of the pancreatic cancer diagnostic substrate. Enzyme immunoassay using a primary antibody capable of specifically binding to pancreatic cancer specific antigen and a secondary antibody-signal complex that specifically binds to the primary primary antibody as a detection means capable of detecting pancreatic cancer specific antigen bound to (enzyme-linked immunosorbant assay: ELISA) kit: wherein the electric microplate is attached with an anti-matriline 3 antibody, an anti-transtyretin antibody, an anti-lipocalin 2 antibody and an anti-stratipine antibody, and is subjected to electrical detection Means include, but are not limited to, electric anti-matriline 3 antibodies, anti-transtyretin antibodies, and anti-lipocalin 2, each capable of specifically binding to an electrical protein. Thereof, or an anti-Strasse Tippin antibody, and contain the secondary antibody is coupled, an enzyme capable of specific binding to an electric antibody, a reagent causing a color reaction by the enzyme may be included additionally.

According to another embodiment of the present invention, the pancreatic cancer diagnostic kit of the present invention uses a protein chip with an antibody specifically binding to the pancreatic cancer specific antigen as a pancreatic cancer diagnostic substrate, and the antibody of the pancreatic cancer diagnostic substrate. Fluorescence immunity using a primary antibody capable of specifically binding to the pancreatic cancer specific antigen and a secondary antibody-signal complex that specifically binds to the primary primary antibody as detection means capable of detecting pancreatic cancer specific antigen bound to Assay kit: The protein chip used as the substrate is a glass substrate, a metal plate or a silicon substrate to which an anti-martryline 3 antibody, an anti-transtyretin antibody, an anti-lipocalin 2 antibody and an anti-stratipine antibody are attached. Fluorescence immunoassay kit including the electric protein chip is composed of matriline 3 and trans, respectively, bound to the electric protein chip and the electric antibody. Retinoic tea, lipocalin includes a detection means capable of detecting two or stripe Tippin. The electrical detection means is specific for an anti-matriline 3 antibody, an anti-transtyretin antibody, an anti-lipocalin 2 antibody, an anti-stratipin antibody, and an electrical antibody that can specifically bind to an electric pancreatic cancer specific antibody, respectively. It consists of a secondary antibody bound to a fluorescent substance that can be bound automatically. The electroluminescent material is not particularly limited thereto, but is preferably Cy-3, Cy-5, FITC, GFP (green fluorescent protein), red fluorescent protein (RFP), or Texas Red.

The pancreatic cancer diagnostic kit of the present invention can be widely used for the early diagnosis of pancreatic cancer because only the blood sample of the subject can confirm whether the pancreatic cancer develops.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Selection of Pancreatic Cancer Specific Genes

6 normal brain tissues (BN), 19 astrocytoma tissues (BA I) in the brain, 37 microsatellite stability (CMS) colorectal cancer tissues (CMSS), MSI (microsatellite instability) colon cancer tissues (CMSI) ) 13 cases, normal lung tissue (LN) 10 cases, stage 1 lung cancer tissue (LI) 71 cases, normal pancreas tissue (PN) 7 cases, pancreatic cancer tissue (AP) 8 cases and serous ovarian cancer tissue (ovarian cancer serous, OS) 27 samples were obtained respectively, RNA was extracted from the sample obtained previously using Trizol (Life Technologies, USA), purified using a column (RNesay spin column, Qiagen, USA), 18S and 28S rRNA Only RNAs with similar expression were selected.

5 μg of the RNA selected was applied to a cDNA synthesis kit (cDNA synthesis kit, Superscript Choice System; Life Technologies, USA) using oligo (dT) 24 as a primer, and then the synthesized cDNA was used as a template. CRNA was synthesized using a transcription system (T7 Megascript kit, Ambion, USA) under T7 polymerase laboratory conditions. At this time, the cRNA was labeled using biotin-11-CTP and biotin-16-UTP (Enzo, USA). Subsequently, 15 μg of the electrically labeled cRNA was treated with a fragmentation solution (40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and reacted at 94 ° C. for 30 minutes to randomize to a size of 30 to 50 bases. Sectioned. Then, hybridization cocktail (100 mmol / L MES, 1 mol / L NaCl, 20 mmol) containing 0.1 mg / ml of herring sperm DNA (Promega, USA) and 500 g / ml of acetylated BSA (Life Technologies, USA) 300 μl / L EDTA, 0.01% (v / v) Tween 20) was heated to a temperature of 94 ° C. for 5 minutes, cooled, and centrifuged at 16,000 × g at room temperature to obtain a supernatant. The hybridization solution obtained above was mixed with 10 µg of the electrically fragmented cDNA, applied to a microarray (HuGeneFL Array, Affymetrix, USA) capable of identifying 7129 genes, and hybridization was performed at 45 ° C. for 16 hours. .

After the reaction was completed, the electric microarray was washed, stained with streptavidin solution (streptavidin-phycoerythrin, Molecular Probes, USA) and then washed with 6x SSPE buffer (sodium chloride, sodium phosphate, EDTA). The electro-washed microarray was then treated with biotinylated anti-streptavidin IgG, stained again with electrostreptavidin solution, and washed again with 6X SSPE. The electrical microarrays were then scanned with a scanner (GeneArray Scanner, Affymetrix, USA), and then the results were measured using an image analyzer (GeneChip software, Affymetrix, USA) and analyzed via quantile-normalization. (See Table 1).

Result of measuring specific gene expression rate in various tissues gene AP MO R BN BA I CMSS CMSI L I LN PN OS L01406 1346 105 12.88 -18 -6 -39 105 -46 25 -75 12 HG2190 2241 203 11.05 9 -90 -256 -345 -459 -446 203 -336 J00124 7676 406 18.91 406 262 209 285 202 116 557 642 M60047 3180 426 7.46 7 -17 282 426 179 186 -27 57 X82693 2552 312 8.18 312 46 178 89 167 170 172 72 AJ001047 396 57 3.96 -166 -108 -123 -69 11 -31 57 -126 L14565 6264 1807 3.47 1318 941 661 714 935 997 1807 941 M31551 354 109 3.24 -60 -134 -141 -131 -144 -164 109 -155 Y07755 15681 5141 3.05 455 540 1006 785 1408 1021 320 5141 D12763 328 129 2.55 -15 -25 -43 -35 35 129 80 -34 AC002077 2423 956 2.53 367 299 186 279 956 812 188 525 X62891 311 130 2.39 -191 -266 -337 -320 -321 -286 130 -319 X52426 2022 950 2.13 67 29 140 95 448 305 950 108 D85527 3205 1528 2.10 1528 1236 917 933 962 764 1582 1102 U12139 2358 1171 2.01 224 101 155 196 147 -8 1171 106 AF000562 6738 3087 2.2 2867 1892 1779 1812 2221 2280 3087 2205 X83863 1407 735 1.91 270 351 86 113 271 158 735 135 M15517 10438 5464 1.91 190 352 177 133 450 197 5464 179 M76482 518 250 2.1 203 114 247 250 129 141 -106 99 J04177 5801 2049 2.8 769 2049 1588 1466 1477 314 856 1384 M28249 778 419 1.86 66 72 419 351 271 127 149 61 U07919 1506 817 1.84 170 817 462 283 441 469 496 216 U19796 4963 1972 2.5 2732 1939 1700 1888 1760 1543 1972 1664

gene AP MO R BN BA I CMSS CMSI L I LN PN OS X07696 1955 241 8.1 -87 -113 41 93 241 75 -86 -2 M11718 13921 2877 4.8 1085 1983 2877 2270 4127 2294 1740 2204 U52112 4170 2356 1.77 2356 1074 972 954 1203 1133 2348 1301 L42611 3326 928 3.6 557 499 867 835 850 583 928 884 M57506 181 107 1.68 82 79 17 25 51 7 107 39 J03779 1710 1019 1.68 490 589 856 527 674 1019 993 538 D30758 2530 1315 1.9 1238 762 553 621 1013 1277 1315 650 M64099 3628 2190 1.66 1021 2190 825 625 1321 1072 1123 755 M29610 228 126 1.8 49 71 67 78 46 0 126 38 L14754 1779 1083 1.64 1083 371 532 593 452 736 754 517 J00210 164 18 9.1 18 -8 -35 -49 -17 -43 -42 -69 K01396 22184 13712 1.62 390 1038 3262 9218 11095 13712 3019 1908 U58331 162 58 2.8 58 -27 -38 -22 -24 -48 40 -13 U21051 216 129 1.7 48 100 107 87 90 63 129 66 U17760 3308 1793 1.8 165 144 868 833 1793 661 37 1050 U40763 400 222 1.8 134 185 182 217 180 195 145 222 X99133 10786 6257 1.7 180 223 4492 6257 2085 757 1967 2923 AB000467 454 288 1.58 -67 124 63 123 2 38 288 37 X16707 157 13 1.57 -167 -169 -100 -244 -334 -371 13 -280 X52425 2147 1373 1.6 510 568 1177 1274 1373 1372 528 703 X57348 4564 2922 1.6 264 255 2214 2922 2660 1075 614 870 L12350 3264 1489 2.2 478 743 1489 1106 1427 655 297 776 X75546 597 395 1.51 40 77 55 212 142 135 352 395

gene AP MO R BN BA I CMSS CMSI L I LN PN OS D17525 2004 1330 1.51 616 586 471 489 593 598 1330 522 Y08766 1388 861 1.6 502 587 330 292 705 483 861 616 AC000066 186 115 1.6 108 45 26 9 35 -21 115 7 Z19574 4813 1851 2.6 318 60 351 399 1851 273 50 1584 J02906 3666 2486 1.47 1068 846 653 713 771 806 2486 688 U21689 1458 989 1.47 592 331 277 206 197 311 989 336 L37033 10203 6964 1.47 6927 3972 1503 1223 2113 2224 6964 2168 M26041 5673 3886 1.46 610 997 1696 1376 2610 3886 2451 1542 X86779 3194 2106 1.5 2082 1509 1353 1452 1510 1098 2106 1523 D42055 420 241 1.7 67 277 197 168 164 167 241 92 U58096 344 204 1.7 204 96 103 116 134 105 189 115 M90657 5268 2859 1.8 -115 521 1075 461 2859 2486 809 3257 M20203 236 167 1.41 -39 -51 -51 -136 -3 19 167 -74 L37036 1429 663 2.2 280 179 421 201 663 238 205 260 L34155 3319 2360 1.41 26 12 781 869 875 608 377 2360 X57766 4783 3402 1.41 918 775 3402 1765 2322 542 1066 2436 U07424 2660 1770 1.5 1770 831 1357 1357 800 868 408 1304

gene AP MO R BN BA I CMSS CMSI L I LN PN OS U62800 2135 1163 1.8 -79 -37 4 41 1154 1163 -58 320 D86479 4904 3616 1.36 1054 3616 1336 1200 2287 2067 944 1201 L38500 183 135 1.35 135 106 99 57 109 17 122 105 D50840 4626 2895 1.6 1484 1050 1197 842 2895 2701 1031 1887 U45983 140 66 2.1 66 -13 -20 -19 11 -24 59 -11 M55682 247 183 1.35 -48 -47 -62 -78 -50 -84 183 -10 M60094 198 90 2.2 90 72 67 45 70 25 90 57 M59371 1051 729 1.4 306 392 729 654 641 637 186 611 D50923 787 572 1.4 237 372 524 493 361 172 572 481 V00536 222 162 1.4 162 107 109 141 120 95 140 96 X70218 3877 2531 1.5 623 1260 2200 2186 2229 1382 1697 2531 M13452 3530 2656 1.33 1169 1089 1494 1790 2026 2656 2071 1588 X60382 376 184 2.0 91 33 116 126 184 -4 22 108 S66896 332 106 3.1 -9 52 27 41 96 97 106 38 M30607 300 229 1.31 -22 26 45 89 12 17 229 -3 X14830 573 352 1.6 352 334 235 218 316 222 254 205 U08191 457 250 1.30 126 135 124 90 170 178 350 197 U11863 8393 6444 1.30 3560 2432 2221 2389 2502 1690 6444 2267 X66610 164 110 1.5 42 44 41 82 36 -31 110 8 L40379 1809 1391 1.30 12 534 1047 1116 1041 1391 -43 1245

In Table 1, AP represents the average value of the specific gene expression ratio in pancreatic cancer tissue, MO represents the highest value of the expression ratio of the gene in other tissues, R means the ratio of the electrical AP divided by MO.

In addition, in Table 1, the gene LO1406 is a growth hormone releasing hormone receptor, HG2190 is crystallin beta 3 (crystallin, beta 3), J00124 is keratin 14 (epidermolysis bullosa simplex (Dowling-Meara, Koebner)), M60047 is a heparin-binding growth factor binding protein, X82693 is a lymphocyte antigen 6 complex, locus D, AJ001047 is matrilin 3, L14565 is peripherin, M31551 is serine or cysteine proteinase inhibitor, clade B (ovalbumin), member 2), Y07755 is S100 Calcium binding protein A2, D12763 for interleukin 1 receptor-like 1, AC002077 for KIAA0677 gene product, X62891 for arginine vasopressin (neurophysin II, antidiuretic hor mone, diabetes insipidus, neurohypophyseal), X52426 for keratin 13, D85527 for LIM domain kinase 2, U12139 for collagen type 11 alpha 1, AF000562 for europlakin 2, X83863 is prostaglandin E receptor 3 (subtype EP3), M15517 is transthyretin (prealbumin (amyloidosis type I)), M76482 is desmoglein 3 (pemphigus vulgaris antigen) , J04177 is collagen type 11 alpha 1, M28249 is integrin, aplha 2 (CD49B, alpha 2 subunit of VLA-2 receptor), U07919 is aldehyde dehydrogenase 1 family member A3 (aldehyde dehydrogenase 1 family, member A3), U19796 is mitochondrial ribosomal protein L28, X07696 is keratin 15, M11718 is collagen type 5 alpha 2, U52112 is L1 cell adhesion protein, L42611 is keratin 6E, M57506 is small inducible Cytokine A1 (small inducible cytokine A1), J03779 is a membrane metal Endopeptidase (membrane metallo endopeptidase), D30758 for centaurin beta 1, M64099 for gamma-glutamyltransferase-like activity 1, M29610 for glycophorin E, L14754 is immunoglobulin mu binding protein 2, J00210 is interferon alpha 1, K01396 is a serine (or cysteine) protease inhibitor, U58331 is sarcoglycan (delta), and U21051 is a G protein binding receptor 4, U17760 for laminin beta 3, U40763 for peptidyl-prolyl isomerase G, X99133 for lipocalin 2, AB000467 for chromosome ORF 9, X16707 is FOS-like antigen 1, X52425 is interleukin 4 receptor, X57348 is stratifin, L12350 is thrombospondin 2, X75546 is fibromodulin, D17525 Manon-bound Lectin Serine 1 (mannan-binding lectin serine protease 1), Y08766 for splicing factor 1, AC000066 for kinase anchor protein, Z19574 for keratin 17, J02906 for cytochrome P450 Group 2 protein subfamily F polypeptide 1, U21689 for glutathione S-transferase pi, L37033 for FK506 binding protein 8, M26041 for major histocompatibility complex, X86779 for FAST kinase, D42055 for neural precursor cell expressed, developmentally downregulated 4, U58096 for testis specific protein (Y-linked), M90657 for transmembrane 4 superfamily member 1 ), M20203 is elastase 2 (neutrophil), L37036 is small inducible cytokine subfamily B (Cys-X-Cys) member 5 (epithelial derived neutrophil-a) ctivating peptide 78), L34155 is laminin alpha 3, X57766 is matrix metalloproteinase 11 (stromelysin 3), and U07424 is phenylalanine-tRNA synthetase-like protein (phenylalanine-tRNA synthetase-). like protein), U62800 for cystatin E / M, U31201 for laminin gamma 2 kalinin BM600, D86479 for AE binding protein 1, L38500 for solute carrier 5 (inositol carrier) (inositol transporter)) member 3, D50840 for UDP glucose ceramide glucosyltransferase, U45983 for chemokine (CC motif) receptor 8, M55682 Is the triglyceride 1 cartilage matrix protein, M60094 is the H1 histone family, member T (testis-specific), M59371 is EpHA2, and D50923 is the KIAA0133 gene product, V00536 is interferon gamma rferon, gamma), X70218 is protein phosphatase 4, catalytic subunit, M13452 is lamin A / C, X60382 is collagen X type alpha 1, S66896 is serine (or cysteine) protein Hydrolase inhibitor clade B member 3 (claine B (ovalbumin), member 3), M30607 is zinc finger protein (Y-linked), X14830 is choline receptor nicotine Beta polypeptide 1 (cholinergic receptor, nicotinic, beta polypeptide 1 (muscle)), U08191 is a nuclear factor related to kappa B binding protein, U11863 is an amyloide binding protein 1 (Copper containing amine oxidant), X66610 means lung specific enolase alpha (lung-specific), and L40379 means thyroid hormone receptor interactor 10, respectively.

As shown in Table 1, the genes were confirmed that the expression level in pancreatic cancer tissue is much higher than the expression level in normal pancreatic tissue as well as other cancer tissues.

Example 2 Western Blot Analysis

Among the above 95 pancreatic cancer specific expression genes, genes encoding six proteins secreted extracellularly are selected and antibodies that specifically bind to the proteins expressed by these genes are used to detect pancreatic cancer patients, normal people and other cancer patients. Western blot analysis was performed on the obtained blood samples. The electrical proteins are matryline 3, kalinin, transthyretin, UDP glucose ceramide glucosyltransferase, lipocalin 2 and stratipin.

First, serum was separated from the blood of a patient who provided the normal tissue and the cancer tissue of Example 1, and then electrophoretically separated electrophoresed on 6 10% SDS-polyacrylamide gels, respectively, followed by an electrical method. To PVDF (poly vinylidene flouride, Millipore, USA) membrane. In order to reduce the nonspecific reaction of the protein transferred to the PVDF membrane, the electric PVDF membrane was blocked for 12-14 hours with blocking buffer (3% bovine serum albumin, 0.05% Tween 20 in PBS). Then, anti-matriline 3 rabbit polyclonal antibody (Biocat, Germany), anti-calilin rabbit polyclonal antibody (Abcam, UK), anti-transtyretin rabbit polyclonal antibody (Abcam, UK) ), Anti-UDP glucose ceramide glucosyltransferase rabbit polyclonal antibody (Abcam, UK), anti-lipocalin 2 rabbit polyclonal antibody (Abcam, UK) and anti-stratipine rabbit polyclonal antibody (Alpha Diagnostic International, USA ) Were diluted in an electric blocking buffer at a concentration of 1: 200 (v / v), respectively, and reacted by stirring at room temperature for 2 hours. Then, washed three times for 10 minutes with PBST (0.05% Tween 20 in PBS), and anti-rabbit goat IgG (Santa Cruz Biotechnology, USA) conjugated with horseradish peroxidase (HRP) 1: 200 (v / v) Blocking buffer diluted to the treated with an electro-washed PDVF membrane, and stirred for 45 minutes at room temperature to react. Subsequently, the plate was washed three times with PBST for 10 minutes, and then chemiluminescent reaction was generated using an ECLTM kit (Amersham, UK), which was analyzed by PhosphaImager (Fuji, Japan).

As a result, matriline 3, transthyretin, lipocalin 2 and stratipin were clearly detected in the blood of pancreatic cancer patients. On the other hand, kalinin, UDP glucose ceramide glucosyltransferase was most clearly detected in pancreatic cancer, but was detected in the blood of some brain, pancreatic and ovarian cancer patients.

Thus, it was confirmed that matriline 3, transthyretin, lipocalin 2 and stratipin may be useful markers for pancreatic cancer diagnosis as proteins specifically expressed in pancreatic cancer.

Example 3 Fabrication and Analysis of Strips

Through the following process, an immunochromatography strip for diagnosing pancreatic cancer, including anti-matriline 3, anti-transtyretin, anti-lipocalin 2, and anti-stratipin polyclonal antibody, was prepared as shown in FIG. 1.

Example 3-1 Preparation of Sample Pad

A cellulose filter (Millipore, USA) was cut into 0.8 cm x 1.2 cm size and used as a sample pad (1).

Example 3-2 Preparation of Anti-Matrilin 3, Anti-Transtyretin, Anti-Lipocalin 2, and Anti-Stratipin Polyclonal Antibodies, Gold Conjugates, and Glass Fiber (GF) Filters

Immune response between the pancreatic cancer specific antigen in the blood sample and the electric anti-matriline 3, anti-transtyretin, anti-lipocalin 2, and anti-stratipine polyclonal antibody included in the present invention occurs. GF) The conjugate of the electric polyclonal antibody and the gold particles was temporarily fixed on the surface of the filter 2, and was prepared in the following manner.

Gold chloride was reduced to sodium citrate solution to prepare 40 nm-sized gold particles with an absorbance of 10 ± 1 at 532 nm. 10 μg / ml of anti-growth hormone releasing hormone receptor, anti-crystallin beta B3, anti-keratin 14, and anti-heparin binding growth factor binding protein polyclonal antibody were attached to electro-gold chloride solution and polyethylene glycol (PEG) solution To stabilize the gold particles. Meanwhile, a glass fiber filter (1.0 cm x 0.8 cm, Millpore, USA) was divided into quarters with a width of 0.2 cm, and each of the polyclonal antibody-gold conjugates previously prepared was wetted and dried at 37 ° C.

Example 3-3 Preparation of NC Membranes

The NC membrane (nitrocellulose membrane, Millipore, USA) was cut to the appropriate size (0.8 cm x 5 cm), and then divided into quarters of 0.2 cm in width, and then placed as a control line (3) at a point about 1.6 cm from the bottom of each membrane. Goat anti-rabbit IgG (Goat anti-rabbit IgG, Santa-Cruz, USA) was diluted 1:50 concentration in PBST and treated in a straight line, the pancreatic cancer at the point 0.8 cm in the lower direction in the control line (3) As the detection result determination line (4) of specific antigen, anti-growth hormone releasing hormone receptor, anti-crystallin beta B3, anti-keratin 14, and anti-heparin binding growth factor binding protein polyclonal antibody included in the present invention were used as PBST. Was diluted 1:50 (v / v), treated in a straight line, and then dried at 37 ° C. to prepare an NC film 5 having a control line and a detection result judgment line on the film surface.

Example 3-4 Preparation of Absorbent Pads

Absorption pad 6 absorbs unreacted substances in the sample after the immune reaction, thereby moving the sample solution containing the analyte by capillary action, the cellulose filter (Millipore, USA) 0.8cm × 3cm Cut to size was used.

Example 3-5 Adhesive Plastic Backing

As a part for supporting each pad, a commercial plastic plate (Millipore, USA) was used. As shown in FIG. 1, the sample pad 1 prepared in Example 3-1 was placed on the adhesive plastic backing 7, and the anti-growth hormone releasing hormone prepared in Example 3-2 was placed thereon. A glass fiber filter (2) having a receptor, an anti-crystallin beta B3, an anti-keratin 14, and an anti-heparin binding growth factor binding protein polyclonal antibody-gold conjugate, respectively, was connected in parallel in order from the left to the right. Next, the NC membrane 5 prepared in Example 3-3 and the absorbent pad 6 prepared in Example 3-4 were sequentially mounted, but 0.1 was placed so that the material was continuously moved by capillary action. An immunochromatographic strip for diagnosing pancreatic cancer was prepared by arranging and assembling the parts partially overlapping, and fixing them by adhesion, and attaching an absorbent paper (Millipore, USA) under the membrane.

Example 3-6 Analysis of Blood Specimens

Blood was collected from five normal patients, seven colon cancer patients, five pancreatic cancer patients, four ovarian cancer patients, and three brain cancer patients, and then each dropped 3 ml into the absorbent pad (6) of the electrochromatically produced immunochromatography strip. Then, after 5 minutes, color development was observed.

As a result, matriline 3, transthyretin, lipocalin 2 and stratipine were specifically detected only in pancreatic cancer patients.

Example 4 Preparation and Analysis of Enzyme-linked Immunosorbant Assay (ELISA) Kit for Pancreatic Cancer Diagnosis

Example 4-1 : Preparation of enzyme immunoassay plate for pancreatic cancer diagnosis

Coating buffers (Na ₂ CO ₃ 0.188) for adsorption of anti-matriline 3, anti-transtyretin, anti-lipocalin 2 and anti-stratipine polyclonal antibodies at a concentration of 1:50 (v / v), respectively. % (w / v), NaHCO ₃ 0.271% (w / v), NaCl 0.731% (w / v), pH 9.6) and 50 μl / well each on an enzyme immunoassay plate (Nunc, USA) Aliquoted and coated overnight at 4 ° C. The electrocoated plates were washed three times with PBS and then treated at room temperature for 1 hour with blocking buffer (3% (w / v) BSA, 0.05% (v / v) Tween 20 in PBS) to block nonspecific binding. It was then dried, sealed and stored at 4 ° C. until used in the dark.

Example 4-2 Treatment and Analysis of Blood Samples

An electrocoated plate was treated with an electric cancer patient and a blood sample of a normal person 200μl per well, the reaction was slowly shaken at room temperature for 1 hour, and then washed five times with PBST. Subsequently, the electric anti-matriline 3, anti-transtyretin, anti-lipocalin 2, and anti-stratipin polyclonal antibodies diluted 1: 200 (v / v) in PBST were added to the anti-matriline 3, Dispense 50 μl into wells coated with anti-transtyretin, anti-lipocalin 2 and anti-stratipine polyclonal antibodies, treat at room temperature for 2 hours, wash three times with PBST, and use blocking buffer. Treatment at room temperature for 1 hour blocked nonspecific binding. Then, the washing was performed twice more, and anti-rabbit goat IgG (Santa Cruz Biotechnology, USA) conjugated with horseradish peroxidase (HRP) was diluted in blocking buffer at a concentration of 1: 400 (v / v), Each well was dispensed into the antibody-treated wells and allowed to react slowly by shaking for 2 hours at room temperature. Then, it was washed four times with PBST, and color was developed by adding the coloring agent TMB (tetramethyl benzidine, Sigma, USA) according to the manufacturer's concentration, and then stopped by adding 1.25M sulfuric acid solution. Subsequently, the degree of color development of the plate on which color was stopped was measured by absorbance at 450 nm using a microplate reader (Model 680 Microplate Reader, Biorad, USA) to determine the result (see Table 2).

Results of Enzyme Immunoassay on Blood Samples of Normal Patients Cancer specific antigen Normal people Pancreatic cancer Colorectal cancer Ovarian Cancer Brain cancer Matryline 3 0.081 ± 0.016 0.254 ± 0.039 0.076 ± 0.021 0.077 ± 0.024 0.092 ± 0.031 Transthyretin 0.025 ± 0.008 0.102 ± 0.007 0.028 ± 0.010 0.031 ± 0.008 0.029 ± 0.009 Lipocalin 2 0.067 ± 0.017 0.156 ± 0.034 0.071 ± 0.028 0.065 ± 0.013 0.074 ± 0.029 Stratipine 0.055 ± 0.011 0.136 ± 0.032 0.061 ± 0.019 0.054 ± 0.012 0.061 ± 0.013

As shown in Table 2, the absorbance in serum obtained from patients with pancreatic cancer is more than twice higher than that of serum obtained from normal and other cancer patients for matriline 3, transthyretin, lipocalin 2, and stratipine. As it turned out, it was confirmed that the onset of pancreatic cancer through enzyme immunoassay.

Example 5 Fabrication and Analysis of Protein Chips

Example 5-1 Preparation of Protein Chips

Electrical anti-matriline 3, anti-transtyretin, anti-lipocalin 2 and anti-stratipine polyclonal antibodies were arranged on a slide glass for microscopy coated with poly-L-lysine.

First, prior to attaching the antibodies, the crosslinker EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) was added to the microscope slide glass coated with electric poly-L-lysine at a concentration of 1 mg / ml. It was pretreated for a minute and washed three times with PBS solution. The antibodies were then diluted in PBST at concentrations of 1:50, 1: 100, 1: 200 and 1: 400 (v / v), and dispensed into 96-well microplates, followed by arraying of the antibodies (MicroGrid II). , BioRobotics, USA) was arranged dropwise at 150 μm in size and 400 μm in thickness on a microscope slide glass coated with electric poly-L-lysine. The electric slide glass was divided into 16 sections according to the antibody type and dilution concentration, and each compartment was composed of 48 dots in an area of 1 mm ² . Then, the electric slide glass in which the electric antibodies were arranged was left in a humid chamber for 1 hour at 37 ° C., combined with the electric antibodies, and then taken out and dried. Subsequently, the slide glass was immersed in 100% cold acetone for 1 minute so that the antigen was firmly bound to the slide glass, and then taken out again and dried.

Example 5-2 Serum Analysis of Normal and Various Cancer Patients Using Protein Chips

The protein chip prepared in Example 5-1 was immersed in the blocking buffer used in Example 2, left to stand for 10 minutes, and washed three times with PBST. Subsequently, blood samples of normal persons and various cancer patients obtained in the above Examples 3-6 were dropped by 1 ml on each of the protein chips, which were washed electrically, and allowed to stand at room temperature for 15 minutes and washed three times with PBST. The washed protein chip was immersed in the electric blocking buffer and allowed to stand for 15 minutes, and then washed three times with PBST. Then, after diluting anti-matriline 3, anti-transtyretin, anti-lipocalin 2 and anti-stratipine polyclonal antibody at a concentration of 1: 200 in the electric blocking buffer, respectively, 1 ml of the mixed antibody mixture was mixed. The reaction was carried out for 15 minutes at room temperature by dropping the protein chip was washed, and washed three times with PBST. Subsequently, anti-rabbit IgG goat immunoglobulin G (An + -rabbit IgG (H + L)) conjugated with fluorescein isothiocyanate (FITC) diluted 1: 400 in an electric blocking buffer on a protein chip treated with an electropolyclonal antibody. , F (ab ') 2, FITC, Goat, Roche Applied Science, USA) 1ml was added and allowed to stand at room temperature for 15 minutes, and then washed three times with PBST. Then, the protein chip treated with the anti-rabbit IgG goat immunoglobulin G conjugated with the electric FITC was dried at room temperature, and scanned with a flatbed scanner (GenePix Scanner 4000A, Axon Inc, USA) to fluoresce the electric protein chip. The extent was measured and analyzed via quantile-normalization (Table 3).

Measurement of Fluorescence Responses to Blood Samples of Normal and Various Cancer Patients Cancer specific antigen Concentration of antibody (v / v) Normal people Pancreatic cancer Colorectal cancer Ovarian Cancer Brain cancer Matryline 3   1:50 112.2 352.1 105.1 122.1  98.1   1: 100 92.1 311.4  91.3 103.2  83.4   1: 200 72.4 234.2  71.3  82.4  68.4   1: 400 52.3 142.1  42.1  56.3  56.5 Transthyretin   1:50 31.1  68.1  29.6  33.7  33.1   1: 100 27.1  58.1  22.1  26.5  27.2   1: 200 18.2  32.3  16.5  18.2  19.1   1: 400 10.2  18.3  13.5  12.5  13.1 Lipocalin 2   1:50 102.4 234.5 106.5  98.3  86.1   1: 100 84.1 201.5  86.3  83.1  74.1   1: 200 62.3 163.3  64.2  74.1  62.3   1: 400 48.1  91.3  52.1  56.3  46.3 Stratipine   1:50 93.5 212.4 104.2 100.5  97.3   1: 100 83.5 195.3  90.6  93.2  85.2   1: 200 64.3 154.4  71.3  75.3  68.2   1: 400 52.1 104.5  62.2  61.2  53.4

As shown in Table 3, in the case of matriline 3, transthyretin, lipocalin 2, and stratipine, when the concentration of antibodies that can specifically bind is diluted 1: 200 (v / v) or more, In addition, the fluorescence coloration of blood samples from pancreatic cancer patients was more than twice that of normal or other cancer patients. However, in case of transthyretin, when the concentration of antibody was diluted to 1: 100 (v / v) or more, the fluorescent color of blood samples of pancreatic cancer patients or other cancer patients was normal. By recording more than twice the degree, it was confirmed that when the concentration of the antibody used is increased, it can function as a pancreatic cancer specific label.

Example 6 Analysis of Random Specimens Using Protein Chips

Based on the results of Example 5-2, the concentrations of anti-matriline 3, anti-lipocalin 2 and anti-stratipine polyclonal antibodies were diluted 1: 200 (v / v) and anti-transtyretin Dilute the concentration of polyclonal antibody to 1: 100 (v / v) and drop the poly-L-lysine-coated slide glass to make protein chips, respectively. Except for dropping blood samples of 12 subjects whose opacity was unclear, the fluorescence coloration of the electric protein chip was measured in the same manner as in Example 5-2, and analyzed by quantile-standardization. 4). In this case, a blood sample of a subject who was found to be a normal person was used as a control.

Diagnosis of Pancreatic Cancer in Subjects Using Protein Chips Experimental group Matryline 3 Lipocalin 2 Stratipine Transthyretin Control     72.5     65.1   59.3   28.1 One     74.2     63.1   63.3   26.2 2     78.5     67.4   57.3   25.1 3     73.1     64.2   64.5   26.3 4     82.4     71.5   67.3   29.1 5     84.1     72.4   63.2   30.2 6    313.1    165.3  156.3   59.1 7     81.4     69.2   57.3   26.3 8     78.5     64.1   65.3   25.4 9     73.1     68.2   62.3   29.1 10     75.3     70.4   71.2   22.6 11    295.5    172.5  161.3   45.1 12     74.1     67.3   66.3   25.3

As shown in Table 4, two blood samples (Experimental Group 6 and 11) more than doubled the degree of fluorescence development for matriline 3, transthyretin, lipocalin 2, and stratipine more than twice that of normal blood samples. As a result of performing a close examination such as a CT scan for the two subjects, both patients were found to be pancreatic cancer.

As described and demonstrated in detail above, the present invention provides a pancreatic cancer diagnostic kit comprising a pancreatic cancer diagnostic substrate and an electrical substrate capable of detecting pancreatic cancer specific antigen in a blood sample of a subject. The pancreatic cancer diagnostic kit of the present invention can determine whether pancreatic cancer develops even by blood samples of a subject, and thus, it may be widely used for early diagnosis of pancreatic cancer.

Claims (11)

(Iii) 1 to 3 selected from the group consisting of an anti-matrilin 3 antibody, an anti-transthyretin antibody and an anti-stratifin antibody Dog antibodies; And, (Ii) A pancreatic cancer diagnostic substrate comprising a solid support having an antibody attached thereto. The method of claim 1, Solid phase support is characterized in that the NC (nitrocellulose), PVDF (polyvinylidene fluoride) membrane, microplate, glass substrate, polystyrene, silicon substrate or metal plate Pancreatic cancer diagnostic substrate. The method of claim 1, The antibody is characterized in that the polyclonal antibody or monoclonal antibody A substrate for diagnosing pancreatic cancer. The method of claim 1, Antibodies are attached to a solid phase support by hydrogen bonding, by aldehyde or by poly-L-lysine and linkage. A substrate for diagnosing pancreatic cancer. A pancreatic cancer diagnostic kit comprising detection means capable of detecting pancreatic cancer specific antigens bound to the antibody of the pancreatic cancer diagnostic substrate of claim 1 and an electric pancreatic cancer diagnostic substrate. The method of claim 5, The detecting means is a primary antibody-gold conjugate capable of specifically binding to an antigen bound to an antibody on a substrate. Pancreatic cancer diagnostic kit. The method of claim 5, The detecting means is a primary antibody capable of specifically binding to an antigen bound to an antibody on a substrate, and a secondary antibody-signal complex that specifically binds to an electrical primary antibody. Pancreatic cancer diagnostic kit. The method of claim 7, wherein The signal complex is characterized in that the fluorescent material, luminescent material, radioisotope or enzyme Pancreatic cancer diagnostic kit. The method of claim 8, The fluorescent material is Cy-3, Cy-5, FITC, GFP (green fluorescent protein), RFP (red fluorescent protein) or Texas Red (Texas Red) characterized in that Pancreatic cancer diagnostic kit. The method of claim 8, Enzyme is characterized in that the horseradish peroxidase (HRP), alkaline phosphatase (beta), beta galactosidase (luciferase) or luciferase (luciferase) Pancreatic cancer diagnostic kit. The method of claim 10, It further comprises a coloring reagent causing a color reaction by the enzyme Pancreatic cancer diagnostic kit.

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