KR20170010526A - Cancer diagnostic ELISA kit and method for detecting to cancer using the same - Google Patents

Abstract

The present invention relates to an ELISA kit for cancer diagnosis and a cancer diagnosis method using the same. The cancer diagnostic ELISA kit comprises a biotin-conjugated aptamer that is attached to a substrate and specifically binds to an ECPKA antigen.

Description

[0001] The present invention relates to an ELISA kit for cancer diagnosis and a cancer diagnosis method using the ELISA kit,

The present invention relates to an enzyme-linked immunosorbent assay (ELISA) kit for diagnosing cancer using a bio-marker in a sample such as blood, and a cancer diagnosis method using the same.

Cancer is a disease in which a part of the cells in the body develops in an abnormally proliferative state. When cancer occurs in the body, special proteins, enzymes, and hormones that are rarely healthy are increased in the blood or urine. It can be a clue to early detection, and this particular substance is called a tumor marker.

These markers are used to refer to tumor-associated antigens, which are substances that are formed in the tissues in which cancer cells are proliferating. They are not substances that are formed in the tumor itself, Also called a marker.

For example, antigens as markers for diagnosing the specific cancers can be diagnosed by assaying PSA (Prostate-specific antigen) specific to the prostate in the case of prostate cancer, and CEA (Carcino- Embryonic Antigen, CA15-3 for the diagnosis of breast cancer, CA19-9 for the diagnosis of gastric cancer, and CA125 for the diagnosis of ovarian cancer.

However, due to the lack of sensitivity and specificity, so far only one tumor marker has been able to isolate healthy and cancer patients.

In addition, since the types of cancer that can be diagnosed by specific tumor markers are determined, in order to diagnose the incidence of all cancers (for example, the National Cancer Information Center Classification: 96), a corresponding number of cancer diagnoses There was a problem that it was necessary.

It is an object of the present invention to provide an ELISA kit for cancer diagnosis which enables various cancer to be efficiently and more accurately diagnosed and a cancer diagnosis method using the same.

The ELISA kit for cancer diagnosis according to an embodiment of the present invention includes a biotin-conjugated aptamer which is attached to a plate and specifically binds to ECPKA (Extracellular Protein Kinase A) antigen.

The method of diagnosing cancer according to an embodiment of the present invention is characterized in that a first aptamer specifically binding to an ECPKA antigen and a second aptamer directly bound to a labeling substance and specifically binding to the ECPKA antigen are attached to a substrate, Treating the blood with the blood; And measuring the level of the labeled substance directly bound to the second aptamer to detect the amount of the ECPKA antigen bound to the first and second aptamers at different positions.

In addition, the method for diagnosing cancer according to another embodiment of the present invention comprises a first aptamer that specifically binds to an ECPKA antigen, and a second antibody that binds specifically to the Fc or Fab portion of the ECPKA autoantibody, Treating and reacting blood with an ELISA kit in which a human antibody is attached to a substrate; And measuring the level of the labeled substance directly bound to the anti-human antibody to detect the amount of the ECPKA autoantibody specifically bound to the anti-human antibody.

According to one embodiment of the present invention, cancer is diagnosed by using an ELISA kit containing an aptamer that specifically binds to an ECPKA antigen, thereby quantifying the possibility of developing a plurality of cancers.

FIGS. 1 and 2 are graphs for explaining the effect of cancer diagnosis using ECPKA (Extracellular Protein Kinase A) as a biomarker.
3 is a view showing an embodiment of the construction of an ELISA kit for cancer diagnosis for detecting the amount of ECPKA autoantibody.
4 is a flowchart showing a first embodiment of a method for diagnosing cancer using the ELISA kit for cancer diagnosis according to the present invention.
5 is a view showing an embodiment of the construction of an ELISA kit for cancer diagnosis for detecting the amount of ECPKA antigen.
6 is a flowchart showing a second embodiment of a method for diagnosing cancer using the ELISA kit for cancer diagnosis according to the present invention.
7 is a view showing another embodiment of the construction of an ELISA kit for cancer diagnosis for detecting the amount of ECPKA autoantibody.

Hereinafter, a cancer diagnosis ELISA kit according to an embodiment of the present invention and a cancer diagnosis method using the kit will be described in detail with reference to the accompanying drawings.

The foregoing objects, features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Like reference numerals designate like elements throughout the specification. In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail.

Hereinafter, the term "diagnosis" refers to judging susceptibility to a disease of a test subject for a specific disease or disease, judging whether a specific disease or disease is presently present, Determining the prognosis of a cancer (e.g., identifying a transitional cancerous condition, determining the stage or progress of a cancer, or determining the response of a cancer to treatment), or determining therametrics (e.g., Monitoring the state of the object to provide it).

On the other hand, the term "biological sample" refers to a substance or mixture of substances, including one or more components capable of detecting a tumor marker, and refers to an organism, particularly a cell, tissue or body fluid derived from a human such as whole blood, urine, plasma, But is not limited thereto. It also includes cells or tissues cultured in Invitro as well as those directly derived from the organism.

Further, the term "detection" includes quantitative and / or qualitative analysis, including detection of presence and absence and detection of the expression level, and such methods are well known in the art, and those skilled in the art will appreciate that, .

The ELISA kit for cancer diagnosis according to an embodiment of the present invention quantitatively detects the possibility of developing cancer by quantitatively detecting ECPKA (auto-antibody) or antigen (ECPKA).

Extracellular protein kinase A (ECPKA) is secreted from cancer cells. ECPKA secretion leads to the formation of sero-autoantibodies, and the presence or concentration of anti-ECPKA autoantibodies is assayed to detect signs of cancer diagnosis and prediction . ≪ / RTI >

That is, the presence or absence of an anti-ECPKA autoantibody (particularly, an anti-ECPKA-IgG antibody) in a biological sample of a suspected cancer or confirmed cancer patient is tested to determine whether or not a specific cancer has occurred A comprehensive diagnosis of multiple, non-cancerous cancers may be possible.

Referring to the graphs shown in FIG. 1 and FIG. 2, the effect of cancer diagnosis using ECPKA autoantibodies is described. As a result of experiments with 295 cancer patients and 100 healthy individuals, the anti-ECPKA IgG antibody concentration (92%) than normal (14%) patients.

In addition, the ECPKA immunoassay method (measuring antigen concentration) shows statistically significant overlap between cancer patients (n = 66) and normal controls (n = 66). (Patient, frequency = 83%, mean value = 130 mU / ml, normal control, frequency = 21%, mean value = 60 mU / ml)

The ELISA kit for cancer diagnosis according to one embodiment of the present invention may be prepared by attaching an aptamer specifically binding to an ECPKA antigen and another aptamer or an antibody directly bonded to a labeling substance to a substrate have.

Aptamers are a special class of single-stranded nucleic acids (DNA, RNA or modified nucleic acids) that, by themselves, have a stable tertiary structure and are capable of binding to target molecules with high affinity and specificity.

Aptamers can be obtained through the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process, a random sequence nucleic acid library. The aptamer is thought to be a good alternative to antibodies, and many aptamers are known to bind specifically to metal ions and small chemical molecules, proteins, and even cells to have a dissociation constant at the nanomole level of picomoles .

In addition, aptamers have the following advantages over specific antibodies in comparison to antibodies:

The first feature is that the aptamer can be obtained from a nucleic acid library that is designed to target certain molecules (from small inorganic ions to cells). This feature makes it possible to overcome limitations of antibodies that must be obtained from cells or animals.

The second feature is that the selected aptamer can be amplified through a PCR (Polymerase chain reaction) process or can be transcribed to obtain a large amount of aptamers having high purity.

The third feature is that the aptamer has a relatively simple chemical structure and is therefore susceptible to change its functionalities when it is intended to be used for other purposes such as immobilization on a solid surface.

Finally, because aptamers are much more stable than antibodies, they can be used in chemical applications requiring tougher conditions. In addition, aptamers can be chemically synthesized in large quantities, so they are economical, have a target affinity comparable to that of antibodies, and are significantly smaller in size than antibodies.

On the other hand, the plate of the ELISA kit refers to a support capable of attaching an aptamer or an antibody, and may be glass, alumina, ceramic, carbon, gold, silver, copper, aluminum, compound semiconductor, , But the present invention is not limited thereto.

The substrate can be surface treated to bind an aptamer, which can be functionalized to include a functional group for immobilizing an aptamer, and can be modified, for example, with an aldehyde group, a carboxyl group, an amine group or a thiol group .

In addition, the labeling substance that is directly bound to the aptimer or the antibody means means for determining whether the target substance ECPKA autoantibody or antigen is bound to the aptamer or antibody and the level thereof.

The labeling substance may be a Q dot (quantum dot), an enzyme, a coloid gold, a fluorescent substance, a radioactive substance and a dye. The enzyme may be HRP (Horseradish peroxidase) The enzyme may be selected from the group consisting of Alkaline phosphatase, Glucose Oxidase, luciferase,? -D-galactosidase, malate dehydrogenase (MDH), acetylcholine Acetylcholinesterase, or analogs exhibiting similar activities thereto, but the present invention is not limited thereto.

Hereinafter, embodiments of a cancer diagnosis ELISA kit according to the present invention and a method of diagnosing cancer using the same will be described in detail with reference to FIGS. 3 to 6. FIG.

Figure 3 shows an embodiment of the construction of a cancer diagnostic ELISA kit for detecting the amount of ECPKA autoantibody.

Referring to FIG. 3, an ELISA kit for cancer diagnosis can be prepared with aptamer attached to a streptavidin coated plate.

The aptamer attached to the substrate is an aptamer that specifically binds to the ECPKA antigen and is a biotin-conjugated aptamer that specifically binds to the ECPKA catalytic alpha-subunit in blood. Lt; / RTI >

In addition, the substrate is bound with an antibody bound to a labeling substance. The antibody binds directly to HRP (Horseradish peroxidase), which is a labeling substance, and an anti-human Fc antibody that specifically binds to the Fc portion of the ECPKA autoantibody (HRP -conjugated anti-human Fc antibody).

The ECPKA catalytic alpha-subunit synthesized with a biotin-linked aptamer is attached to the substrate of the cancer diagnostic ELISA kit according to an embodiment of the present invention, and the synthesized ECPKA catalytic alpha-subunit is attached to the ECPKA catalytic alpha-subunit Can be specifically bound to the ECPKA catalytic alpha-subunit auto-antibody.

In addition, the HRP-conjugated anti-human Fc antibody attached to the substrate specifically binds to the ECPKA catalytic alpha-subunit autoantibody in the blood, and the level of HRP as the labeling substance Lt; RTI ID = 0.0 > ECPKA < / RTI > autoantibodies by detecting the amount of ECPKA catalytic alpha-subunit autoantibody.

On the other hand, the level of HRP as the labeling substance can be detected using the luminescence phenomenon by H ₂ O ₂ .

FIG. 4 is a flowchart illustrating a first embodiment of a method for diagnosing cancer using the ELISA kit for cancer diagnosis according to the present invention. Description about the same things as those described with reference to FIGS. 1 to 3 will be omitted below. .

4, the user can prepare an ELISA kit in which a first aptamer that specifically binds to an ECPKA antigen and an anti-human Fc antibody that is directly bound to a labeling substance and specifically binds to an ECPKA autoantibody are attached to a substrate (Step S400).

The substrate of the ELISA kit is further provided with a biotin-binding aptamer that specifically binds to the ECPKA catalytic alpha-subunit and HRP which is directly bound to HRP as a labeling substance and specifically binds to ECPKA catalytic alpha-subunit autoantibodies in blood Lt; RTI ID = 0.0 > anti-human < / RTI > Fc antibody.

Thereafter, the user treats the blood, which is a biological sample to diagnose cancer, with the prepared ELISA kit (step S410), measures the level of the labeled substance directly bound to the anti-human Fc antibody, The amount of the ECPKA autoantibody bound to the Fc antibody is detected (step S420).

That is, when the blood is treated and reacted with an ELISA kit to which a biotin-binding aptamer and an HRP-conjugated anti-human Fc antibody are attached on a substrate, an ECPKA catalytic alpha-subunit Quot; form of the first complex can be constructed.

Subsequently, the synthesized ECPKA catalytic alpha-subunit is combined with an ECPKA catalytic alpha-subunit autoantibody in blood to form a "substrate" - "biotin-linked aptamer" - "synthesized ECPKA catalytic alpha-subunit" A second complex in the form of an ECPKA catalytic alpha subunit autoantibody in blood "can be constructed.

Subsequently, when the HRP-conjugated anti-human Fc antibody is added to the second complex and reacted, the "substrate" - "biotin-linked aptamer" - "synthesized ECPKA catalytic alpha-subunit" Quot; HRP-conjugated anti-human Fc antibody ", wherein the level of HRP, the labeling substance bound to the third complex, is measured to determine the amount of the ECPKA catalytic alpha subunit autoantibody in the blood Can be detected.

The quantitative value of the ECPKA autoantibody in the blood detected as described above can indicate the invention probability of cancer and the ECPKA autoantibody value measured in the blood as measured by the method described with reference to Figure 4 is provided to the user, And / or to compare the average value of the ECPKA autoantibody levels of the cancer generator.

According to another embodiment of the present invention, it is possible to diagnose whether or not cancer has occurred by quantitatively detecting ECPKA antigen in blood.

FIG. 5 shows an embodiment of the configuration of an ELISA kit for cancer diagnosis for detecting the amount of ECPKA antigen, and a description of the same components as those described with reference to FIG. 1 to FIG. 4 will be omitted below do.

Referring to FIG. 5, the cancer diagnostic ELISA kit according to another embodiment of the present invention can be manufactured in a state where a plurality of app tamers are attached to a streptavidin coated plate (Streptavidin coated plate).

The first aptamer attached to the substrate is an aptamer that specifically binds to the ECPKA antigen and may be a biotin-conjugated aptamer that specifically binds to the ECPKA catalytic alpha-subunit in blood.

Also, the second aptamer attached to the substrate is an aptamer directly bound to the labeling substance and specifically binding to the ECPKA antigen, and is an HRP-conjugated aptamer (HRP-binding domain) specifically binding to the ECPKA catalytic alpha-subunit in blood, conjugated aptamer).

The biotin-linked aptamer and the HRP-bound aptamer attached to the substrate of the cancer diagnostic ELISA kit according to another embodiment of the present invention can recognize different sites by being bonded to different positions of the ECPKA catalytic alpha subunit, respectively.

In addition, the HRP-conjugated aptamer specifically binds to the ECPKA catalytic alpha-subunit, thereby detecting the amount of the ECPKA catalytic alpha-subunit by detecting the level of the HRP as the labeling substance, quantitatively indicating the ECPKA antigen in the blood have.

On the other hand, the level of HRP as the labeling substance can be detected using the luminescence phenomenon by H ₂ O ₂ .

FIG. 6 is a flowchart showing a second embodiment of a method for diagnosing cancer using the ELISA kit for cancer diagnosis according to the present invention.

6, a user prepares an ELISA kit in which a first aptamer that specifically binds to an ECPKA antigen and a second aptamer that is directly bound to a labeling substance and specifically binds to the ECPKA antigen are attached to a substrate (Step S600).

The substrate of the ELISA kit is further provided with a biotin-binding aptamer that specifically binds to the ECPKA catalytic alpha-subunit and an HRP-binding aptamer that binds directly to the marker substance HRP and specifically binds to the ECPKA catalytic alpha-subunit in blood Or may be attached.

Thereafter, the user treats and reacts the blood, which is a biological sample to be diagnosed with cancer, with the prepared ELISA kit (step S610), measures the level of the labeled substance directly bound to the second aptamer, The amount of ECPKA antigen bound to different positions of the aptamers is detected (step S620).

That is, when blood is treated and reacted with an ELISA kit in which a biotin-binding aptamer and an HRP-binding aptamer are attached to a substrate, first, a "substrate" - "biotin-conjugated aptamer" - "an ECPKA catalytic alpha- 1 complex can be constructed.

Subsequently, when the HRP-bound aptamer is added to the first complex and reacted, a second complex in the form of "substrate" - "biotin-linked aptamer" - "ECPKA catalytic alpha-subunit in blood" - "HRP binding aptamer" And the amount of the ECPKA catalytic alpha subunit in the blood can be detected by measuring the level of HRP, which is a labeling substance bound to the second complex.

The quantitative value of the detected ECPKA antigen in the blood as described above can indicate the inventive probability of cancer and the ECPKA antigen level measured by the method described with reference to Fig. 6 is provided to the user, Can be compared with the mean value of the ECPKA antigen level of the subject.

According to another embodiment of the present invention, in a cancer diagnostic ELISA kit for detecting the amount of an ECPKA autoantibody, a synthesized ECPKA antigen that specifically binds to the ECPKA autoantibody in the blood is fusion with an Fc protein, Lt; RTI ID = 0.0 > ELISA < / RTI > kit.

Figure 7 shows another embodiment of the construction of a cancer diagnostic ELISA kit for detecting the amount of ECPKA autoantibody.

Referring to FIG. 7, an ECPKA catalytic alpha-subunit attached to a substrate of an ELISA kit can be produced in association with an Fc protein.

This is because a lot of production cost and time are required to synthesize and purify the ECPKA catalytic alpha-subunit protein. When the Fc protein is bound to the ECPKA catalytic alpha-subunit, mass production of protein It is possible to remarkably reduce the production cost and time of the ELISA kit according to the present invention.

On the other hand, when the ECPKA catalytic alpha-subunit is attached to the substrate in the state that the ECPKA catalytic alpha-subunit is bound to the substrate, the anti-ECPKA autoantibody, which binds directly to the substrate of the ELISA kit and specifically binds to the Fab portion of the ECPKA autoantibody, An anti-human Fab antibody may be attached.

For example, as shown in FIG. 7, an anti-human Fab antibody (HRP-conjugated anti-human Fab antibody), which is directly bound to HRP as a labeling substance and specifically binds to a Fab portion of an ECPKA autoantibody, , And the labeling substance can quantitatively indicate ECPKA autoantibodies in the blood.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention.

Claims (18)

An ELISA kit for cancer diagnosis comprising a biotin-conjugated aptamer attached to a plate and specifically binding to an ECPKA (Extracellular Protein Kinase A) antigen. The method of claim 1, wherein the biotin-conjugated aptamer
A cancer diagnostic ELISA kit that specifically binds to ECPKA catalytic alpha-subunit in blood. The method according to claim 1,
A cancer diagnostic ELISA kit quantitatively showing the ECPKA antigen in blood. The method of claim 3,
Wherein a second aptamer directly bound to the labeling substance and specifically binding to the ECPKA antigen is attached to the substrate. 5. The method of claim 4,
Wherein the labeling substance is HRP (Horseradish peroxidase). 5. The method of claim 4,
Wherein the biotin-linked aptamer and the second aptamer bind to different sites of the ECPKA antigen, respectively. The method according to claim 1,
An ELISA kit for cancer diagnosis quantitatively showing ECPKA autoantibodies in blood. 8. The method of claim 7,
Wherein the synthesized ECPKA antigen that specifically binds to the ECPKA autoantibody is attached to the substrate. 9. The method of claim 8, wherein the synthesized ECPKA antigen is
An ELISA kit for cancer diagnosis in which the Fc protein is bound to an ECPKA catalytic alpha-subunit. 10. The method of claim 9,
Wherein an anti-human Fab antibody, which is directly bound to a labeling substance and specifically binds to a Fab portion of the ECPKA autoantibody, is attached to the substrate. 8. The method of claim 7,
Wherein an anti-human Fc antibody, which is directly bound to a labeling substance and specifically binds to an Fc portion of the ECPKA autoantibody, is attached to the substrate. 12. The method according to any one of claims 10 and 11,
Wherein the labeling substance is HRP (Horseradish peroxidase). A first aptamer which specifically binds to an ECPKA antigen and a second aptamer which is directly bound to the labeling substance and specifically binds to the ECPKA antigen, And
Measuring the level of the labeled substance directly bound to the second aptamer to detect the amount of the ECPKA antigen bound to the first and second aptamers at different positions. A first aptamer that specifically binds to an ECPKA antigen, and an ELISA kit in which an anti-human antibody directly bound to a labeling substance and specifically binding to an Fc or Fab portion of an ECPKA autoantibody is attached to a substrate, ; And
Determining the level of the labeled substance directly bound to the anti-human antibody to detect the amount of the ECPKA autoantibody specifically bound to the anti-human antibody. 15. The method of claim 14,
The synthesized ECPKA antigen was attached to the substrate of the ELISA kit,
Wherein said ECPKA autoantibodies in the blood specifically bind to said synthesized ECPKA antigens. 16. The method of claim 15, wherein the synthesized ECPKA antigen is
Wherein the Fc protein is bound to the ECPKA catalytic alpha-subunit. 15. The method according to any one of claims 13 to 14,
Wherein the first aptamer is a biotin-linked aptamer. 15. The method according to any one of claims 13 to 14,
Wherein the labeling substance is HRP (Horseradish peroxidase).

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