KR20110072732A - A method for the diagnosis of colon cancer - Google Patents

Abstract

PURPOSE: A method for diagnosing colon cancer is provided to detect alpha 1-3/4 fucose level using AAL lectin and to enable simple diagnose of colon cancer. CONSTITUTION: A method for diagnosing colon cancer comprises: a step of pretreating alpha 1-6 fucosidase to test sample-derived blood; a step of treating the blood to an anti-beta-haptoglobin antibody-conjugated solid substrate and washing; a step of treating with AAL(Aleuria aurantia agglutinin) lectin on the substrate and washing; and a step of determining colon cancer in case that the AAL lectin attachment level is increased comparing the level of healthy person.

Description

A method for the diagnosis of colon cancer

The present invention relates to a colorectal cancer diagnostic kit using a sugar chain structure of blood-derived beta-haptoglobin, and a method for diagnosing colorectal cancer using the same.

Carcinoma-specific sugar chains are released into the blood, which can be used for diagnostic purposes using a variety of antibodies. Recently, plant-derived lectins have been used to recognize the altered sugar chain structure of haptoglobin. Such lectins are widely used for the purpose of detecting sugar chain structures because they are readily available, inexpensive, and have superior specificity and reactivity as compared to antibodies. The concentration of haptoglobin in the blood is increased in various types of carcinoma and inflammatory response, while the sugar chain structure has all the advantages of showing an unusual structure. However, until now, the scientific basis for the change in sugar chain structure in case of illness is unknown.

Meanwhile, as a method for measuring an increased fucose, a lectin blotting method using the Lotus tetragonoolobus lectin is used. Recently, in addition to the Lotus tetragonoolobus lectin, a method of viewing fucose of haptoglobin obtained from pancreatic cancer patients using Aleuria aurantia lectin (AAL) and Aspergillus oryzae lectin (AOL) has been used. It also became.

The present inventors compared the differences through sugar chain structure analysis of beta-haptoglobin purified from the blood of patients with Crohn's disease and colon ulcers, which are chronic diseases of colorectal cancer, and normal people, resulting in beta-hap. Alpha 1-3 / 4 fucose was found in colorectal cancer by identifying alpha 1-3 / 4 fucose in the sugar chain structure of toglobin. The present invention was completed by revealing that colorectal cancer can be diagnosed using fucosylation) as a marker.

An object of the present invention is a colon cancer diagnosis kit or immunochromatography using a marker using alpha 1-3 / 4 fucose in the sugar chain structure of beta-haptoglobin derived from the blood of a colon cancer patient To provide a strip.

Another object of the present invention is to provide a method for diagnosing colon cancer from blood using the kit or immunochromatography strip.

In order to achieve the above object, the present invention is a colon containing an anti-beta-haptoglobin antibody, AAL lectin (Aleuria aurantia agglutinin lectin) and alpha 1-6 fucosidase (α1-6-fucosidase) Provided are cancer diagnostic kits.

In addition,

1) an adhesive support;

2) a sample pad containing a test sample to be analyzed, which is attached to an upper surface of the adhesive plastic support;

3) a conjugate pad associated with the sample pad and containing a colorant-binding AAL lectin;

4) a test line linked to the sample pad, in which a beta-haptoglobin antibody is linearly fixed, and a glycoprotein that is located downstream of the signal detection region and binds to an anti AAL lectin antibody or AAL lectin A signal detection pad in association with said conjugate pad, said control line comprising said linearly fixed control line;

5) An immunochromatography strip for diagnosing colorectal cancer, comprising an absorption pad positioned downstream of the signal detection pad, for absorbing a test sample after the signal detection reaction is completed.

In addition,

1) pretreatment of alpha 1-6 fucosidase (α1-6-fucosidase) to the subject-derived blood;

2) treating the blood on a solid substrate to which an anti-beta-haptoglobin antibody is attached and then washing the blood;

3) treating the substrate with AAL lectin (Aleuria aurantia agglutinin lectin) and then washing; And

4) providing a protein detection method for providing information for diagnosing colorectal cancer, comprising determining a subject having an increased degree of adhesion of AAL lectin on the substrate as a subject having or at risk for colorectal cancer. do.

In the present invention, alpha 1-3 / 4 fucose in the sugar chain structure of blood-derived beta-haptoglobin is significantly increased in the colorectal cancer patients compared to normal people. It provides a method for diagnosing colorectal cancer using the alpha 1-3 / 4 fucose as a marker. Therefore, by treating an enzyme that specifically cleaves alpha 1-6 fucose from blood from a subject, and confirming the degree of alpha 1-3 / 4 fucose using AAL lectin, colon cancer can be diagnosed easily and simply. Can be.

Hereinafter, the present invention will be described in detail.

The present invention provides a kit for diagnosing colorectal cancer containing an anti-beta-haptoglobin antibody, AAL lectin (Aleuria aurantia agglutinin lectin) and alpha 1-6 fucosidase (α1-6-fucosidase).

We collected blood from colorectal cancer patients and normal humans, and then isolated beta-haptoglobin using an anti-haptoglobin-affinity column (see FIG. 1). The beta-haptoglobin is then transferred to PVDF membranes after performing SDS-PAGE, followed by biotin-attached AAL lectin (Aleuria aurantia agglutinin lectin) and extra avidin-peroxidase (ExtrAvidin- After treatment with peroxidase), the band was identified through an ECL reaction, and a blasting was performed using an anti-haptoglobin antibody after undergoing a stripping step of exerting AAL lectin. By using this, the amount of fucose connected to haptoglobin can be easily measured, and it was confirmed that the intensity of the band detected in colorectal cancer was significantly higher than that of normal people, and it was normal in experiments using other lectins. It was confirmed that there is no significant difference between and colorectal cancer (see FIGS. 2 and 3).

In addition, the present inventors compared the results by performing AAL lectin blot after removing Fucα1-3 / 4 / 6GlcNAc using fucosidase, and then performing AAL after treatment with fucosidase. By confirming that the lectin blot showed the same signal as normal (see FIG. 5), it was found that the reaction of AAL was due to Fucα1-3 / 4 / 6GlcNAc.

In addition, the present inventors perform in-gel digestion of a band of beta-haptoglobin to cleave an N-linked glycan, and then GlyKo Signal to the cleaved N-linked glycan. The fluorescent material was bound using a 2-AB labeling kit, GlyKo GlycoClean S Cartridges were used to separate only glycans with 2-AB, and then analyzed using HPLC. Peak h, k, m It was confirmed that the colorectal cancer is higher than normal (see FIG. 6). In addition, the peak h, k, m was confirmed using a variety of fucosidase and enzymes in order to determine the relationship between the fucose, the peak h, k, m is associated with Fucα1-3 / 4GlcNAc rather than Fucα1-6GlcNAc It was confirmed that the peak with (see FIG. 6).

Therefore, the present inventors confirmed that alpha 1-3 / 4 fucose in the sugar chain structure of the blood-derived beta-haptoglobin in colorectal cancer patients was significantly increased compared to normal people, The alpha 1-3 / 4 fucose may be used as a diagnostic marker for colorectal cancer, and it may be a cleavage enzyme specific for alpha 1-6 fucose such as alpha 1-6 fucosidase and beta-haptoglobin. It can be seen that colorectal cancer can be easily diagnosed by detecting the signal from alpha 1-3 / 4 fucose by performing AAL after the treatment.

In the colorectal cancer diagnostic kit of the present invention, it is preferable to use the AAL lectin bound to a ligand which is a biotin or a biotin derivative having essentially the same binding action as biotin to avidin or streptavidin. It is preferable to use a binding conjugate bound to the ligand, such as a chromophore or a fluorescent conjugate bound to a binding molecule, but is not limited thereto.

The chromophore is preferably horseradish peroxidase (HRP) or basic phosphatase (alkaline phosphatase), and the fluorescent molecules are colloidal gold (coloid gold), poly L-lysine-fluorescein isothiocyanate (FITC), and rhodamine-B (RITC) -isothiocyanate) is preferably any one selected from the group consisting of, but is not limited thereto.

The colorectal cancer diagnostic kit of the present invention can diagnose colorectal cancer by quantitatively or qualitatively analyzing the binding reaction through an antigen-antibody-binding reaction, a sugar chain structure-lectin or a protein-ligand binding reaction, and the binding reaction is conventional. Enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, antigen- It can measure using methods, such as an antibody aggregation method.

The colorectal cancer diagnostic kit of the present invention may be a nitrocellulose membrane, a PVDF membrane, a well plate synthesized with polyvinyl resin or polystyrene resin, a slide glass made of glass, or the like. have.

The colorectal cancer diagnostic kit of the present invention is a label, and a color developing enzyme or a fluorescent molecule is a conventional color developing agent that is a color developing reaction, horseradish peroxidase (HRP), alkaline phosphatase (colloid gold), colloid gold (coloid gold), FITC (poly L-lysine-fluorescein isothiocyanate), fluorescent materials such as rhodamine-B-isothiocyanate (RITC) and dyes (dye) and the like can be used.

Colorectal cancer diagnostic kit of the present invention preferably comprises a phosphate buffer, NaCl and Tween 20 as a wash solution, but is not limited thereto.

The present invention also provides an immunochromatography strip for diagnosing colorectal cancer comprising an AAL lectin, an anti-beta-haptoglobin antibody, and a protein that specifically binds to the AAL lectin.

The immunochromatographic strip according to the present invention preferably comprises an adhesive plastic support, and a sample pad, a conjugate pad, a signal detection pad, and an absorption pad attached to the adhesive plastic support, but are not limited thereto.

Specifically, the immunochromatography strip preferably has the following configuration, but is not limited thereto.

1) an adhesive support;

2) a sample pad containing a test sample to be analyzed, which is attached to an upper surface of the adhesive plastic support;

3) a conjugate pad associated with the sample pad and containing a colorant-binding AAL lectin;

4) a test line linked to the sample pad, in which a beta-haptoglobin antibody is linearly fixed, and a glycoprotein that is located downstream of the signal detection region and binds to an anti AAL lectin antibody or AAL lectin A signal detection pad in association with said conjugate pad, said control line comprising said linearly fixed control line;

5) an absorption pad positioned downstream of the signal detection pad, the sample being absorbed after the signal detection reaction is completed.

In the above configuration, the color developer of step 3) is preferably colloidal gold particles, but is not limited thereto.

In the above configuration, the signal detection pad of step 4) is preferably made of a nitrocellulose film, but is not limited thereto.

In the above configuration, the absorbent pad of step 5) preferably includes a porous support, and an absorbent dispersed in a pores of the porous support or adsorbed or coated on the fiber yarn of the porous support. It is preferable to further include a porous film layer attached to the upper surface, but is not limited thereto. The absorbent is preferably selected from the group consisting of calcium chloride, magnesium chloride, diatomaceous earth, bentonite, dolomite, gypsum, silica gel and mixtures thereof, but is not limited thereto.

The immunochromatography strip according to the present invention is characterized in that a test sample is determined as a positive sample of colorectal cancer when colored lines appear in the control line and the detection line on the immunochromatography strip.

In the immunochromatography strip according to the present invention, blood, which is a test sample, is first supplied to the immunochromatography strip through the sample pad. The sample pad may further have a function of filtering in order to further improve the selectivity for the analyte or to minimize the influence of the interference material which may be included in the test sample. If necessary, an auxiliary pad may be further provided upstream of the sample pad containing a substance that can increase the reaction between the analyte and the conjugate or eliminate the influence by the interference. Blood introduced through the sample pad is transferred to a conjugate pad located upstream of the sample pad through chromatographic movement. The conjugate pad contains an anti-beta-haptoglobin antibody conjugate that specifically binds to beta-haptoglobin contained in blood. The conjugate is labeled with gold particles, latex particles, fluorescent materials, enzymes and the like. The test sample passing through the conjugate pad moves to the signal detection pad. The signal detection pad includes a detection line for detecting whether an analyte is present in the sample to be tested, and a control line for confirming whether the analytical kit is normally operated regardless of the presence or absence of the analyte. To this end, the detection line is coated with a material (or signal detection material) that selectively and specifically binds to the binding product between the analyte and the conjugate contained in the conjugate pad, and the control line is coated with the conjugate line. It is preferable to coat a material that specifically binds to the conjugate contained in the pad. The signal detection pad is composed of a porous membrane pad, it may be made of nitrocellulose, cellulose, polyethylene, polyethersulfone, nylon and the like.

In addition, the present invention is a composition or colon for diagnosing colon cancer comprising an anti-beta-haptoglobin antibody, AAL lectin (Aleuria aurantia agglutinin lectin) and alpha 1-6 fucosidase (α1-6-fucosidase) Provided are methods for diagnosing colorectal cancer using a cancer diagnostic kit.

The method for diagnosing colorectal cancer is specifically, but not limited to, the following steps:

1) pretreatment of alpha 1-6 fucosidase (α1-6-fucosidase) to the subject-derived blood;

2) treating the blood on a solid substrate to which an anti-beta-haptoglobin antibody is attached and then washing the blood;

3) treating the substrate with AAL lectin (Aleuria aurantia agglutinin lectin); And

4) Determining that the subject with increased AAL lectin adhesion on the substrate compared to the normal person as an individual with or at risk of colorectal cancer.

In the method, the subject of step 1) is a vertebrate, including human, preferably a mammal, more preferably human, ape, bovine, pig, rat, rabbit, guinea peak, hamster, dog or Cats can be used, but are not limited to these.

In the above method, the solid substrate of step 2) may be any one selected from the group consisting of a nitrocellulose (NC) film, a polyvinylidene fluoride (PVDF) film, a microplate, a glass substrate, a polystyrene, a silicon substrate, and a metal plate. It doesn't work.

In the method, the AAL lectin of step 3) is bound to a ligand, the ligand is bound to a specific binding molecule, it is preferable to use a binding enzyme or a chromophore or fluorescent molecules bound to the binding molecule, but is not limited thereto. .

In the method, the binding degree of step 4) is Western blotting method, Enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, immunoprecipitation And immunofluorescence (immunofluorescence) is preferably measured by any one selected from the group consisting of, but not limited to.

Hereinafter, the embodiment of the present invention will be described in detail.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Clinical Analysis of Colorectal Cancer Patients

In the case of colorectal cancer, colonoscopy or S colonoscopy was used to determine the location of colorectal cancer, and pathologists diagnosed it through biopsy. Seventeen patients with colorectal cancer were classified as follows: "rectal cancer" (n = 6, mean age: 60 yr), "ascending colon cancer" (n = 5, Mean age: 75 yr), "descending colon cancer" (n = 2, mean age: 78 yr), "sigmoid colon cancer" (n = 3, mean age: 71 yr) ), And blood of "cecal cancer" (n = 1, age: 63 yr) were collected at Chungnam National University Hospital. The number of biopsy sites, diagnosis system (AJCC-TNM system), and classification of patients are summarized in Table 1 below. Three cases of rectal cancer (Case: # S1-S3) were summed according to the purification method (anti-haptoglobin-affinity chromatography or hemoglobin-affinity chromatography). It was used to compare the glycosylation changes of toglobin.

In the case of chronic colorectal disease, the blood of colorectal ulcer patients (UC) (n = 4, mean age: 49 yr) and the Crohn's disease (CD) blood (n = 5, mean age: 34 yr) were received at Chungnam National University Hospital And Mount Sinai School of Medicine.

In normal cases, blood from healthy volunteers (n = 14, mean age: 28 yr) was collected from Korea Advanced Institute of Science and Technology (Daejeon, Korea).

All blood (colon cancer patients, patients with chronic colon disease, normal people) were collected and allowed to stand at room temperature for 30 to 60 minutes, then centrifuged at 3,000 rpm for 10 minutes to pass the supernatant only through a 0.45 um filter before use at -80 ° C. Stored.

Diagnosis and Information on Colorectal Cancer Using Biospy Case number age gender Biopsy AJCC / TNM ¹ category score Diagnosis S1 67 M 3 Rectal cancer T3N1M0 ⅢB S2 40 F 5 Rectal cancer T3N1M0 ⅢB S3 56 M 4 Rectal cancer T2N0M0 I CP1 50 M 7 Rectal cancer T3N0M0 ⅡA CP2 79 M 5 Ascending Colon Cancer T3N0M0 ⅡA CP3 85 F 5 Rectal cancer T3N1M0 ⅢB CP4 80 F 5 Ascending Colon Cancer T3N0M0 ⅡA CP5 82 F 6 Descending Colorectal Cancer T3N1M0 ⅢB CP6 74 F 5 Descending Colorectal Cancer T3N1M0 ⅢB CP7 74 M 2 Ascending Colon Cancer T3N0M0 ⅡA CP8 69 F 4 Ascending Colon Cancer T3N1M0 ⅢB CP9 82 F 2 S phase colon cancer T2N0M0 I CP10 66 F 3 S phase colon cancer T2N0M0 I CP11 65 F 4 S phase colon cancer T3N1M0 ⅢB CP12 67 M One Appendix cancer T3N1M0 ⅢB CP13 72 M 3 Ascending Colon Cancer T3N0M0 ⅡA CP14 63 F 4 Rectal cancer T2N0M0 I

¹ American Joint Communittee on Cancer (AJCC), known as the TNM system.

Example 2 Purification of Beta-haptoglobin from Blood

In order to separate haptoglobin from the blood isolated in Example 1, a rabbit anti-human haptoglobin polyclonal antibody was used as CNBr-activated Sepharose 4B (cyanogens bromide). After preparing an anti-haptoglobin-affinity column bound using -activated sepharose 4B], beta-haptoglobin was purified from blood.

Specifically, 0.3 g of CNBr-Sepharose-4B was placed in a 100 ml beaker, activated at room temperature for 1 hour in 1 mM HCl, and then transferred to a poly-prep column. Then, washed with 60 ml of 1 mM HCl and 4 ml of coupling buffer (0.1 M NaHCO 3 / 0.5 M NaCl, pH 8.3), and then transferred to a 15 ml tube (500 ug). The reaction was carried out at 4 ° C. for 17 hours in an anti-haptoglobin antibody / 4 ml coupling buffer. Then, after transferring to a new poly-prep column, washed with 4 ml coupling buffer (blocking buffer) and blocking buffer (blocking buffer) (0.2 M glycine, pH 8.0). After blocking for 2 hours at room temperature with a blocking buffer, 6.5 ml of blocking buffer, acetate buffer (0.1 M acetic acid / 0.5 M NaCl, pH 4.0) and binding buffer (coupling buffer), and then finally washed with PBS to prepare a tablet. Then, 500 ul of blood was diluted with 4 ml PBS (10 mM phosphate buffer / 2.7 mM Potassium chloride / 137 mM sodium chloride, pH 7.4) and mixed with the affinity column. After shaking for 2 hours at room temperature, the binding of the antibody and haptoglobin in the blood occurs, and then rinsed thoroughly with PBS. The bound haptoglobin was eluted with elution buffer (0.1 M glycine / 0.5 M NaCl, pH 2.8), then concentrated and quantified using a centrifugal filter (MWCO 10,000). Afterwards, it was confirmed using SDS-PAGE and Coomassie Brilliant Blue staining method.

As a result, as shown in Figure 1, it was confirmed that the beta-haptoglobin (β-haptoglobin) in the blood was purified. An alpha chain subunit is also shown, and since no N-linked sugar chain is present in alpha-haptoglobin, no separate separation was performed.

Example 2 Analysis of Sugar Chain Structure of Beta-haptoglobin

<2-1> Lectin blotting analysis

In order to analyze the sugar chain of the beta-haptoglobin isolated in Example 2, lectin blotting was performed.

Specifically, 1 ug of haptoglobin was transferred to PVDF membranes after 12.5% SDS-PAGE. The transferred PVDF membrane was blocked for 2 hours at room temperature with 3% BSA / T-TBS (TBS (140 mM NaCl, 10 mM Tris-HCl, pH 8.0) / 0.05% Tween 20)]. After washing three times with T-TBS for 5 minutes, it was reacted with AAL lectin (Aleuria aurantia agglutinin lectin) diluted with biotin at 1: 1,000 for 4 hours at 4 ° C. After washing three times with T-TBS for 5 minutes, the extra avidin-peroxidase diluted to 1: 2,000 was treated for 1 hour at room temperature, and then washed three times for 5 minutes with T-TBS. The band was confirmed by the ECL reaction.

Also, SNA lectins (Sambucus nigra lectin, purchased from EY Laboratories) with HRP (Horse Radish Protein) linked to AAL lectins (targeting NeuAc alpha2-6 / 3Gal / GalNAc), PHA-E lectins (Phaseolus vulgaris-E lectin) ,, Buy EY Laboratories) (targeting GlcNAc beta4 Man beta4 GlcNAc), or PHA-L with biotin linked (Phaseolus vulgaris-Llectin, purchased from Vector Laboratories) (targeting GlcNAc beta6 Man beta6 Man side chain) Lectin blotting was performed as described above.

As a result, as shown in Figure 2, when using the AAL lectin that can detect Fucose (Fucose), it was confirmed that the intensity of the band detected in colorectal cancer is significantly higher than that of normal people (Fig. 2). In addition, as shown in Figure 3, the experiment using another lectin (lectin) was confirmed that the difference between the normal and colorectal cancer did not appear significantly (Fig. 3).

<2-2> Reblotting Assay Using Anti-haptoglobin Antibody

To check if there is the same amount of haptoglobin, remove the reacted PVDF membrane with stripping buffer (2% SDS, 62.6 mM Tris-HCl, pH 6.7, 0.78% 2-ME) at 60 ° C for 30 minutes. Shake reaction. Then, after rinsing with distilled water, TBS and T-TBS for 5 minutes each 5 minutes, it was blocked with 5% skim milk / T-TBS at room temperature for 2 hours. After washing three times with T-TBS for 5 minutes, the reaction was carried out at room temperature for 2 hours using an anti-haptoglobin antibody diluted 1: 50,000. Then, washed three times with T-TBS for 5 minutes, and then reacted at room temperature for 1 hour using anti-rabbit (anti-rabbit) IgG-HRP diluted 1: 3,000, and then bands through the ECL reaction. It was confirmed. Blotting index (Blotting index) was calculated as the numerical value obtained by dividing the band obtained by reblotting the numerical value of the band from the lectin blot.

As a result, as shown in Table 2 and Table 3, 50% (7/14) of colorectal cancer patients showed more than 1.5 in the blotting index, 64% (9/14) had more than 1.0, And 100% (14/14) showed more than 0.5, while 21% (3/14) of normal people had more than 1.5, 36% (5/14) had more than 1.0, and 43% (6/14) It can be seen that shows a value of 0.5 or more (Table 2 and Table 3).

sample Case number Blotting Indicator <0.5 0.5-1.0 1.0-1.5 > 1.5 Normal N1 + N2 + N3 + N4 + N5 + N6 + N7 + N8 + N9 + N10 + N11 + N12 + N13 + N14 + cancer Ascending Colon Cancer CP2 + CP4 + CP7 + CP8 + CP13 + Appendix cancer CP12 + Descending Colorectal Cancer CP5 + CP6 + Rectal cancer CP1 + CP3 CP14 + S phase colon cancer CP9 + CP10 + + CP11 +

(Blotting Metrics, Corresponding / Total) ratio Positive Cases for Normal People (> 1.5, 3/14) 21% (> 1.0, 5/14) 36% (> 0.5, 6/14) 43% Positive case for cancer (> 1.5, 7/14) 50% (> 1.0, 9/14) 64% (> 0.5, 14/14) 100% Ascending Colon Cancer (> 1.5, 2/5) 40% (> 1.0, 3/5) 60% Appendix cancer (> 1.5, 1/2) 100% (> 1.0, 1/2) 100% Descending Colorectal Cancer (> 1.5, 1/3) 50% (> 1.0, 2/3) 50% Rectal cancer (> 1.5, 1/3) 33% (> 1.0, 2/3) 67% S phase colon cancer (> 1.5, 2/3) 67% (> 1.0, 2/3) 67%

Example 3 AAL Reaction of Beta-haptoglobin Purified from Serum

To determine how AAL reacts specifically to colorectal cancer, we compared the beta-haptoglobin sugar chain structure of patients with colorectal ulcer (UC) and Crohn's disease (CD), the most common chronic diseases of colorectal cancer. The experiment was conducted.

Specifically, beta-haptoglobin was purified by anti-haptoglobin affinity column chromatography, followed by western blot on PVDF membrane with biotinylated AAL, followed by reblotting with anti-haptoglobin antibody. The relative intensities of the AAL bands were then compared to the haptoglobin band, a blotting index. Three normal (n) and three colorectal cancer (CP) were compared to nine chronic diseases (5 UC and 4 CD).

As a result, as shown in FIG. 4, all of them were similar to normal except for colorectal ulcer (CD) # 1 of lane 4, and showed lower signals than colorectal cancer (FIG. 4).

Example 4 N-linked glycan structure analysis of beta-haptoglobin purified from serum

<4-1> Structural analysis of N-linked glycans of beta-haptoglobin using lectin blotting

The reason why beta-haptoglobin responds largely to AAL is because it responds to Fucα1-3 / 4 / 6GlcNAc. Thus, the fucosidase was used to remove Fucα1-3 / 4 / 6GlcNAc, and then AAL lectin blots were performed to compare the results. Specifically, 2 ug of haptoglobin was reacted daily in 50 mM sodium acetate buffer (pH 5.5, 37 ° C.) containing 1 mU bovine kidney α-fucosidase. Then, SDS-PAGE and lectin blots were performed.

As a result, as shown in FIG. 5, the AAL lectin blot performed after the treatment with fucosidase showed a normal signal (FIG. 5). Through this, it was found that AAL reacted due to Fucα1-3 / 4 / 6GlcNAc.

<4-2> Structural Analysis of N-Linked Glycans by HPLC

After confirming beta-haptoglobin using the SDS-PAGE, the band of beta-haptoglobin was subjected to in gel digestion. Specifically, the gel pieces were rinsed twice with acetonitrile and PNGaseF buffer solution (20 mM NaHCO ₃ , pH 7.0), and then treated with PNGase F at 37 ° C. for 17 hours to obtain N-linked glycan ( N-linked glycan) was cleaved. The fluorescent substance was bound to the cleaved N-linked glycans using a GlyKo Signal 2-AB labeling kit, and only glycans with 2-AB were separated using GlyKo GlycoClean S Cartridges. Glycans with 2-AB were then analyzed using a TSK gel Amide-80 4.6 × 250 mm column and HPLC. All reactions were at 30 ° C. and the mobile phase started with Sol A [20%, v / v, 50 mM ammonium formate, pH 4.4] and Sol B (80%, v / v, acetonitrile). , SolA was changed to 58% by 152 minutes. The flow rate is 0.4 ml / min, and the sugar chain structure is compared by comparing the retention time of N-glycan with glucose unit derived from 2-AB glucose homopolymer standard. Predicted.

As a result, as shown in Figure 6, it was found that the colorectal cancer is larger than normal in peak h, k, m.

In addition, to determine whether the peaks h, k, m are related to fucose, various fucosidases (Almond meal alpha-fucosidase, prozyme) (Fucα1-3, Specific cleavage 4), Bovine kidney α-fucosidase (sigma) (specifically cleaving Fucα1-6> 2,3,4)] and other enzymes Bacter ureapaciene sialidase (specifically cut sialic acid α2-6> 3> 8,9), S. pneumonia N-acetylhexamidase (S. pneumonia N -acetylhexosamidase (prozyme) (specifically cleaving GlcNAcβ1-2> 3,4,6) and beta-galactosidase (β-galactosidase, sigma) (Galβ1-3,4> 6)] Confirmed by.

As a result, as shown in FIG. 6, peaks h, k, and m were confirmed to be peaks associated with Fucα1-3 / 4GlcNAc rather than Fucα1-6GlcNAc (FIG. 6).

Therefore, by treating beta-haptoglobin with an enzyme specific for Fucα1-6GlcNAc, and then performing AAL, only signals from Fucα1-3 / 4GlcNAc can be easily identified, thereby diagnosing colorectal cancer. Able to know.

As can be seen from the above, the present invention can be usefully used to develop a composition and diagnostic kit for diagnosing colorectal cancer, and to develop a method for diagnosing colorectal cancer and a therapeutic agent for colorectal cancer.

1 shows the purification of beta-haptoglobin using an anti-haptoglobin-affinity column, SDS-PAGE, and Coomassie Brilliant Blue staining method. to be:

Lane 1: size marker;

Lane 2: blood diluted to 1/100; And

Lane 3: eluent derived from anti-haptoglobin affinity chromatography.

Figure 2 is a graph showing the results of comparing the sugar chain structure of beta-haptoglobin by using a blotting of beta-haptoglobin in the colorectal cancer patients and normal blood derived HRP-bound AAL lectin.

Figure 3 is a beta-haptoggle using blotting of beta-haptoglobin from colorectal cancer patients and normal blood with other lectins (SNA lectin, PHA-L lectin, PHA-E lectin) other than HRP-linked AAL lectin. This graph shows the result of comparing the sugar chain structure of robin.

Figure 4 shows the beta-haptoglobin purified from patients with colorectal ulcer (UC) and Crohn's disease (CD) chronic colon cancer, Western blot on the PVDF membrane with biotinylated AAL, followed by anti-haptoglobin antibody After reblotting, the graph shows the relative intensity of the AAL bands compared to the haptoglobin band, an blotting index:

N: from normal subjects (3 cases);

UC: from patients with colorectal ulcers (4 cases);

CD: from Crohn's disease patients (5 cases); And

CP: 3 cases from colorectal cancer patients.

FIG. 5 is a diagram showing the results of comparing SDS-PAGE and lectin blots using AAL lectins when the beta-haptoglobin was treated with or without fucosidase. .

6 shows bands of beta-haptoglobin in various fucosidases [sialidases; Sialidase and alpha-fucosidase; Alpha-fucosidase and beta-galactosidase; Sialidase, alpha-fucosidase (Bovine), alpha-fucosidase (Almond) and beta-galactosidase; In-gel digestion was performed using sialidase, alpha-fucosidase (Bovine), alpha-fucosidase (Almond), beta-galactosidase and N-acetylhexosamidase]. After labeling with 2-AB, only glycans with 2-AB were isolated, and then glycans with 2-AB were analyzed using a TSK gel Amide-80 4.6 X 250 mm column and HPLC. This figure shows one result.

Claims (19)

A kit for diagnosing colorectal cancer containing an anti-beta-haptoglobin antibody, AAL lectin (Aleuria aurantia agglutinin lectin), and alpha 1-6 fucosidase (α1-6-fucosidase). The kit of claim 1, wherein the AAL lectin is bound to a ligand. 3. The kit of claim 2, wherein the ligand is a biotin or a biotin derivative having essentially the same binding action as biotin to avidin or streptavidin. The kit according to claim 2, further comprising a visualization conjugate in which chromophores or fluorescent molecules bound to ligand specific binding molecules are bound. The kit according to claim 4, wherein the chromophore is horseradish peroxidase (HRP) or alkaline phosphatase. The kit of claim 4, wherein the fluorescent molecule is any one selected from the group consisting of colloidal gold, poly L-lysine-fluorescein isothiocyanate (FITC), and rhodamine-B-isothiocyanate (RITC). 1) an adhesive support; 2) a sample pad containing a test sample to be analyzed, which is attached to an upper surface of the adhesive plastic support; 3) a conjugate pad associated with the sample pad and containing a colorant-binding AAL lectin; 4) a test line linked to the sample pad, in which a beta-haptoglobin antibody is linearly fixed, and a glycoprotein that is located downstream of the signal detection region and binds to an anti AAL lectin antibody or AAL lectin A signal detection pad in association with said conjugate pad, said control line comprising said linearly fixed control line; 5) An immunochromatography strip for diagnosing colorectal cancer, comprising an absorption pad located downstream of the signal detection pad, the sample absorbing the test sample after the signal detection reaction is completed. 8. The immunochromatographic strip of claim 7, wherein the colorant of step 3) is a colloidal gold particle. The immunochromatography strip according to claim 7, wherein the signal detection pad of step 4) is made of any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone and nylon. 8. The immunochromatography according to claim 7, wherein the absorbent pad of step 5) comprises a porous support and an absorbent dispersed in a pore of the porous support or adsorbed or coated on the fiber yarn of the porous support. strip. 8. The immunochromatographic strip according to claim 7, wherein if a colored line appears in the control line and the detection line on the immunochromatography strip, the test sample is determined as a positive sample of colorectal cancer. 1) pretreatment of alpha 1-6 fucosidase (α1-6-fucosidase) to the subject-derived blood; 2) treating the blood on a solid substrate to which an anti-beta-haptoglobin antibody is attached and then washing the blood; 3) treating the substrate with AAL lectin (Aleuria aurantia agglutinin lectin) and then washing; And 4) A method for detecting a protein for providing information for diagnosing colorectal cancer, comprising determining a subject having an increased degree of adhesion of AAL lectin on the substrate as a subject having or at risk of developing colorectal cancer. The method of claim 12, wherein the solid substrate of step 2) is any one selected from the group consisting of NC (nitrocellulose) membrane, PVDF (polyvinylidene fluoride) membrane, microplate, glass substrate, polystyrene, silicon substrate and metal plate Way. 13. The method of claim 12, wherein the AAL lectin of step 3) is bound to a ligand. 15. The method of claim 14, wherein the ligand is biotin or a biotin derivative having essentially the same binding action as biotin to avidin or streptavidin. The method of claim 12, wherein step 3) comprises attaching a ligand-bound AAL lectin, followed by binding to a chromophore or fluorescent molecule to which a binding molecule specifically binding to the ligand is attached. 17. The method of claim 16, wherein the chromatase is horseradish peroxidase (HRP) or alkaline phosphatase. The method of claim 16, wherein the fluorescent molecule is any one selected from the group consisting of colloidal gold, poly L-lysine-fluorescein isothiocyanate (FITC), and rhodamine-B-isothiocyanate (RITC). The method of claim 12, wherein the binding degree of step 4) is composed of Western blotting, Enzyme-linked immunosorbent assay (ELISA), immunoprecipitation and immunofluorescence. Measuring by any one selected from the group.

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