KR101186411B1 - A kit for detecting target comprising thrombin marker - Google Patents

Abstract

The present invention relates to a kit for detecting a target substance using a thrombin-labeled blood coagulation reaction. The present invention is an antibody bound to thrombin; And it provides a kit for detecting a target substance comprising a biochip fixed to the surface of the antibody capable of binding to the antigen of the antibody. The present invention also provides a method for producing a target material detection kit comprising a method for producing a biochip comprising an antibody and a method for producing a thrombin-binding antibody. In addition, the present invention comprises the steps of reacting a target material on the surface of the biochip; Reacting the thrombin bound antibody to the surface of the biochip; And it provides a method for detecting a target material comprising the step of reacting fibrinogen (fibrinogen) on the surface of the biochip. Since the kit for detecting a target substance containing thrombin according to the present invention may indicate the presence or absence of a target substance by reacting with fibrinogen, which is a substrate, not only immunoassay but also interaction between a target such as genes, proteins, and heavy metals with a probe It is expected that it can be used for the detection of all possible substances.

Description

A kit for detecting target comprising thrombin marker

The present invention relates to a kit for detecting a target substance comprising a thrombin label, and a method for manufacturing the same. The present invention also relates to a method for detecting a target substance through a blood coagulation reaction using the kit.

Generally, the coagulation process causes blood platelets in the blood to break down to form thromboplastin, and thromboplastin works with calcium ions (Ca ²⁺ ) in the blood to form thrombin, one of the plasma proteins. To change. This thrombin catalyzes the reaction of hydrolyzing soluble fibrinogen in the blood and converting it into insoluble fibrin.

Fibrin is a thread-like substance, entangled like a net and trapping blood cells in it, and it becomes smaller over time. Thrombin has a very high substrate specificity for fibrinogen, and four peptide bonds between fibrinogen's arginine and glycine residues. Cut In addition, the optimum pH of this reaction is near neutral or weakly alkaline, and the main component is a protein with a minimum molecular weight of 8,000 and easy to polymerize.

On the other hand, aptamers are oligonucleotides or peptides that bind to specific target molecules, and are usually made by selecting from a large random sequence pool, but natural aptamers also exist in riboswitches. . Aptamers are used not only for basic research but also for clinical purposes as macromolecular drugs. Aptamers may be combined with ribozymes for self cleavage in the presence of their target molecules. Usually short aptamers are classified into peptide aptamers consisting of DNA or RNA aptamers consisting of oligonucleotide chains and various short peptide domains attached to both ends of a protein scaffold.

In addition, DNA or RNA aptamers isolate oligomers that have a high affinity and selectivity for a specific chemical or biological molecule by an evolutionary method using an oligonucleotide library called systemic evolution of ligands by exponential enrichment (SELEX). (C. Tuerand L. Gold, Science 249, 505-510, 2005; AD Ellington and JW Szostak, Nature 346, 818-822, 1990; M. Famulok, et. Al., Acc. Chem. Res. 33, 591-599, 2000; DS Wilson and Szostak, Annu. Rev. Biochem. 68, 611-647, 1999).

In addition, aptamers recognized as substitutes for antibodies can be used as elements of biosensors that can recognize molecules in detection and analysis systems, as well as antibodies. Oligonucleotide-based aptamers can be used in comparison to protein-based antibodies. There are several advantages (R. Nutiu, et. Al., Pure Appl. Chem. 76, 1547-1561, 2004).

First, aptamers are obtained in vitro, and second, various organic and inorganic substances including toxins can be used as target molecules of aptamers. When specific aptamers that specifically bind are identified and obtained, they can be reproduced with low cost and high consistency by automated oligomer synthesis methods. In addition, aptamers are relatively easy to introduce useful functional groups such as fluorescent molecules or photoreactive groups, and the structure of aptamers is reversible due to the denaturation-renaturation process by heat. It can have a long time of activity.

It is an object of the present invention to provide a kit capable of detecting a target substance using a blood coagulation reaction of thrombin.

In addition, another object of the present invention is to provide a method for producing a kit containing the thrombin.

In addition, another object of the present invention to provide a method for detecting a target material using a kit containing the thrombin.

In order to achieve the object of the present invention as described above, the present invention provides a biochip and fibrinogen in which a thrombin-bound antibody, an antibody or a probe to a target substance capable of binding to the antibody, is immobilized on a surface thereof. It provides a kit for detecting a target substance comprising.

In one embodiment of the present invention, the thrombin-coupled antibody may be a thrombin bound to the antibody through a thrombin aptamer (aptamer).

In one embodiment of the present invention, the thrombin-coupled antibody is streptavidin-antibody complex (streptavidin-antibody complex), streptavidin bound to the streptavidin of the streptavidin-antibody complex and the thrombin app And thrombin bound to the timer.

In one embodiment of the present invention, the target material may be selected from the group consisting of antigens, nucleic acids, metal ions and glucose (glucose).

In one embodiment of the present invention, the biochip comprises a substrate to which maleimide-biotin is attached; And a streptavidin-antibody complex bound to the maleimide-biotin.

Further, according to the present invention,

Forming a monomolecular film to which a thiol group is connected through silanization;

The step of dropping the biotin (maleimide-PEO ₂ -biotin) solution secure the biotin-maleimide -PEO ₂ on the monomolecular film surface;

Binding an avidin-antibody complex or a streptavidin-antibody complex to the biotin;

Binding a biotin linked thrombin aptamer to the avidin of the avidin-antibody complex or streptavidin-antibody complex; And

It provides a method for producing a target material detection kit comprising the step of fixing the thrombin to the thrombin aptamer.

Further, according to the present invention,

Reacting a target material on a surface of the biochip according to the present invention;

Reacting the thrombin-bound antibody according to the present invention to the surface of the biochip; And

It provides a method of detecting a target material comprising the step of reacting fibrinogen (fibrinogen) on the surface of the biochip.

Further, according to the present invention,

Reacting a target with a surface of a biochip according to the present invention;

Reacting the streptavidin-antibody complex with the biochip to which the target material is reacted;

Binding a thrombin aptamer to the streptavidin-antibody complex;

Fixing thrombin to the thrombin aptamer; And

It provides a method for detecting a target material comprising the step of reacting fibrinogen on the surface of the biochip to which the thrombin is fixed.

In one embodiment of the present invention, in the step of reacting fibrinogen on the surface of the thrombin-fixed biochip may be mixed with a material for increasing the signal for fibrin production in the reaction solution.

In one embodiment of the present invention, the material is a color bead (color bead), fluorescent particles, quantum dots, gold particles, electrochemically active particles (silver, copper, lead, cadmium, iron , Zinc), carbon nanotubes, graphene, fullerene, fluorescent proteins, GOX (Glucose oxidase), Alkaline Phosphatase (ALP) and Horse-Radish Peroxidase (HRP).

As a result of reacting the kit for detecting a thrombin-containing target substance according to the present invention with fibrinogen, the substrate was able to confirm the progress of the blood coagulation process even with the naked eye. Therefore, the kit containing the thrombin according to the present invention can be usefully used as a simple and accurate measurement method than the conventional method for detecting substances using the blood coagulation mechanism. In addition, this method is expected to be used not only for immunoassay but also for detecting genes and other proteins, heavy metals and the like by changing only a target substance detection portion.

Figure 1 shows the manufacturing process of the kit for detecting a target material containing thrombin according to an embodiment of the present invention.
Figure 2 shows the result of the reaction of the kit for detecting the target material containing thrombin and the fibrinogen solution as a substrate according to an embodiment of the present invention.

The present invention is characterized in providing a kit for detecting a target substance comprising a thrombin label.

Kits of the present invention include a thrombin-bound antibody; A biochip immobilized on a surface of an antibody or a probe for a target substance capable of binding to the antibody; And fibrinogen.

In the present invention, the thrombin-coupled antibody may be one in which thrombin is bound to the antibody through a thrombin aptamer, and the thrombin-coupled antibody is a streptavidin-antibody complex, the strep Thrombin aptamer bound to streptavidin of the tavidin-antibody complex and thrombin bound to the thrombin aptamer.

In general, “aptamer” refers to an aptamer capable of specifically binding to a target substance to be detected. In the present invention, thrombin aptamer was used.

In addition, the target material may include all of the materials detectable using the kit according to the present invention, and the target material may include, but is not limited to, antigens, nucleic acids, metal ions, and glucose (glucose). have.

The biochip comprises a substrate to which maleimide-biotin is attached; And a streptavidin-antibody complex bound to the maleimide-biotin.

The kit according to the invention may in particular comprise an antibody or probe capable of specifically binding to a target material, wherein the antibody capable of specifically binding to a target material is a polyclonal antibody, a monoclonal antibody and a recombinant antibody. And the like, as commonly referred to in the art.

Methods for generating antibodies to a target material can be readily prepared by those skilled in the art using known techniques. For example, in the case of polyclonal antibodies, antigens can be injected into the animals and collected from the animals to obtain serum containing the antibodies, which can be produced by methods well known in the art such as goats, rabbits, sheep, It can be produced from any animal species host such as monkey, horse, pig, cow, dog and the like. In the case of monoclonal antibodies, the hybridoma method (Kohler et al., European Jounral of Immunology , 6, 511-519, 1976) or phage antibody libraries (Clackson et al, Nature , 352, 624-628, 1991, Marks et al, J. Mol. Biol ., 222: 58 , 1-597, 1991). In addition, the antibodies according to the invention may comprise functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In addition, in the case of the probe capable of specifically binding to a target substance, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, and includes deoxyribonucleotides and ribonucleotides and targets. Refers to one that hybridizes specifically to a nucleotide (nucleic acid) sequence and that is naturally present or artificially synthesized The probe according to the invention may be a single chain, preferably an oligodioxyribonucleotide. Probes of the invention may include natural dNMPs (ie, dAMP, dGMP, dCMP, and dTMP), nucleotide analogues or derivatives, and probes of the invention may also comprise ribonucleotides. Backbone modified nucleotides such as peptide nucleic acids (PNA) (M. Egholm et al., Nature, 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2′-O-methyl RNA, alpha-DNA And methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-0-allyl DNA, 2′-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, Chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-)), C-7 7-deazapurine with substituents (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl- , Imidazolyl-, pyridyl-), inosine and diaminopurine It may include.

In addition, the kit according to the present invention may include a biochip, and an antibody or a probe may be immobilized on a substrate. Preferred substrates may include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. have. Immobilization can be carried out by chemical bonding methods or by covalent binding methods such as UV. It can also be bound to a substrate via the linker (eg ethylene glycol oligomer and diamine).

Meanwhile, when the sample applied to the biochip of the present invention is a nucleic acid, it may be labeled and hybridized with the array element on the chip. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may vary depending on the labeling substance.

In addition, the present invention may provide a method for producing a kit capable of detecting a target substance, preferably, the method for producing a kit according to the present invention forms a monomolecular film to which a thiol group is connected through silanization. Making a step; The step of dropping the biotin (maleimide-PEO ₂ -biotin) solution secure the biotin-maleimide -PEO ₂ on the monomolecular film surface; Binding an avidin-antibody complex or a streptavidin-antibody complex to the biotin; Binding a biotin linked thrombin aptamer to the avidin of the avidin-antibody complex or streptavidin-antibody complex; And fixing thrombin to the thrombin aptamer.

In addition, the present invention comprises the steps of reacting the target material on the surface of the biochip according to the present invention; Reacting a thrombin bound antibody to the surface of the biochip; And it may provide a method for detecting a target material comprising the step of reacting fibrinogen (fibrinogen) on the surface of the biochip.

Furthermore, another method for detecting a target material, the method comprising: reacting the target material on the surface of the biochip according to the present invention; Reacting the streptavidin-antibody complex with the biochip to which the target material is reacted; Binding a thrombin aptamer to the streptavidin-antibody complex; Fixing thrombin to the thrombin aptamer; And it provides a method for detecting a target material comprising the step of reacting fibrinogen (Fibrinogen) on the surface of the thrombin fixed biochip.

In the method, the step of reacting fibrinogen on the surface of the thrombin-immobilized biochip may mix a material for increasing the signal for fibrin generation in the reaction solution, and the material may be colored particles or fluorescent particles. Particles, quantum dots, gold particles, electrochemically active particles, carbon nanotubes, graphene, fullerenes, fluorescent proteins, GOX (Glucose oxidase), Alkaline Phosphatase (ALP) and HRP (Horse- Radish Peroxidase), and the electrochemically active particles may be silver, copper, lead, cadmium, iron or zinc.

As described above, the method according to the present invention for detecting a target substance using a kit for detecting a target substance containing thrombin is involved in a thrombin that can amplify a detection signal for more accurate and rapid detection of a target substance. It is characterized by the fact that it is used as a label.

On the other hand, blood and tissues have been found to be involved in the coagulation of more than 50 kinds of blood, the involvement in the coagulation of blood in the blood is called procoagulants (procoagulants) to prevent blood coagulation (anticoagulants) It is called). In the blood and tissue, these two opposing groups of substances are always in equilibrium, but usually anticoagulants predominate to prevent coagulation. However, when blood vessels are damaged, procoagulants are activated at this site, which prevails over the action of anticoagulants, causing blood to coagulate. There are three basic processes involved in the coagulation of blood: first, when a blood vessel or blood is damaged, a complex called a prothrombin activator is formed, and second, a prothrombin activator is called thrombin. Third, thrombin converts fibrinogen into fibrin thread, which surrounds platelets, blood cells, and plasma to form blood clots. Through this mechanism, the mechanism of blood coagulation is achieved.

Therefore, in the present invention, when a target substance is detected by using a coagulation reaction that is accompanied by a reaction mechanism even in a very systematic and fine reaction in vivo, it is expected that the target substance can be detected more quickly and accurately. When thrombin involved in the coagulation reaction was used as a label, it was possible to amplify the detection signal easily and effectively, so that it was possible to purify the labeling substance. Furthermore, the optical or electrochemical equipment used in the past was used. There is a feature that is easy to detect because it does not have to, and has a very high sensitivity that can detect up to aM level even when visually observed.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

Fabrication of biochips according to the present invention

Through silanization, a monomolecular film with a thiol group was formed, a solution containing Maleimide-PEO ₂ -biotin was dropped to fix the biotin on the surface, and the avidin-antibody was bound to the biotin (see FIG. 1). . The antigen solution was dropped on the surface to bind the antibody and the avidin-antibody was immobilized to the antigen. Next, Biotin linked thrombin aptamers were bound and thrombin fixed with aptamers.

< Example  2>

Biochip of the present invention and Thrombin  Target substance detection using blood coagulation

The prepared biochip and thrombin containing antibody were reacted with fibrinogen. The fibrinogen solution containing the microparticles was placed on the surface of the electrode, and after a predetermined time, the presence or absence of the antigen was determined according to whether the polymer was formed (FIG. 2).

< Example  3>

Mouse according to the detection method of the present invention IgG (Target material) detection

One) Maleimide - PEG ₂ - Biotin Fabrication of glass substrates

Indium-tin-oxide (ITO) coated glass substrates (hereinafter referred to as ITO) were washed with acetone, ethanol and water, and then dried with nitrogen gas. Thereafter, ITO was dipped in a mixed solution of about 1: 3 H ₂ O ₂ / H ₂ SO ₄ for several seconds, washed thoroughly with a large amount of tertiary distilled water, and dried with nitrogen gas. ITO was placed in a toluene solution (v / v) containing 5% MPTS (3-Mercaptopropyltrimethoxysilane) at 40 ° C. for 4 hours, washed with toluene, toluene / ethanol 1: 1, ethanol and dried with nitrogen gas.

After that, the PBS solution containing 5 mM Maleimide-PEG ₂ -Biotin was dropped on the surface of ITO and left for 1 hour and 20 minutes, followed by washing with water.

2) anti-mouse- IgG Biotin (antibodies) and Streptavidin  Fabrication of the composite

An equal amount of 20 mg / ml anti-mouse-IgG-biotin and streptavidin were added to PBS (0.1 M, pH 7.2) and left for 3 hours before centrifugal membrane filter Excess streptavidin molecules that did not form a complex (complex) were removed, and diluted in a 1.5 ml PBSTB ( PBST : PBS 0.1M pH 7.2 + 0.05% Tween20) + 0.1% BSA) solution.

3) anti-mouse- IgG Biotin (antibodies) and Streptavidin  Adhesion to Composite Glass Substrates

Subsequently, a PBSTB ( PBST (PBS 0.1M pH7.2 + 0.05% Tween20) + 0.1% BSA) solution containing a complex of anti-mouse-IgG-biotin and streptavidin was dropped on the ITO surface for 2 hours. Leave on for a while and then rinse thoroughly with water. PBS (0.1 M, pH 7.2) solution containing 10 mM amine-PEG ₂ -biotin was dropped on the surface of ITO in order to remove the remaining pockets of biotin of streptavidin and washed with water for 1 hour.

4) Target substance (mouse- IgG ) To glass substrate

Mouse after that PBST (PBS 0.1M pH7.2 + 0.05% Tween20) solution containing IgG was dropped on the surface of the ITO, left for 1 hour, and washed with water. Again, PBSTB ( PBST (PBS 0.1M pH7.2 + 0.05% Tween20) + 0.1% BSA) solution containing a complex of anti-mouse-IgG-biotin and streptavidin was placed for 40 minutes, and then cleaned with water. Washed.

5) Thrombin  Binding to Target Material

Subsequently, a solution of Tris buffer (25 mM Tris, 150 mM NaCl, pH 7.2) containing 1 uM of thrombin aptamer-biotin (5'-biotin-TTTTTTTTTTTTTTTTTTTTGGTTGGTGTGGTTGG-3 ') was dropped on the surface of the ITO. Leave for 40 minutes and wash with water. Subsequently, binding buffer (50 mM Tris-HCl, 140 mM NaCl, 1 mM MgCl ₂ , 0.05% Tween20 and 0.1% bovine serum albumin pH 7.4) containing 10 mg / ml of Bovine thrombin was dropped on the surface of the ITO and at 25 ° C. Leave for 1 hour and wash with water.

6) Detection using fibrinogen

Subsequently, a cleavage buffer solution (20 mM Tris-HCl, 150 mM NaCl, 2.5 mM CaCl ₂ , pH 8.4) containing 2.5 mg / ml Fibrinogen and 20 ml of polystyrene microparticles (diameter: 3 um) was dropped on the surface of the ITO. Set to ℃. After about 50 minutes, the ITO was washed with water to visually confirm the formation of polymer on the ITO surface. As a result, the presence or absence of Mouse-IgG, a target substance, could be determined by the generation of the polymer.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (10)

Thrombin bound antibody; A biochip immobilized on a surface of an antibody or a probe for a target substance capable of binding to the antibody; And a target substance detection kit comprising fibrinogen. The method of claim 1,
The thrombin coupled antibody is a target material detection kit, characterized in that the thrombin is bound to the antibody through a thrombin aptamer (aptamer). The method of claim 2,
The thrombin-coupled antibody consists of a streptavidin-antibody complex, a thrombin aptamer bound to streptavidin of the streptavidin-antibody complex, and a thrombin bound to the thrombin aptamer. Target material detection kit characterized in that. The method of claim 1,
The target material is a kit for detecting a target material, characterized in that selected from the group consisting of antigens, nucleic acids, metal ions and glucose (glucose). The method of claim 1,
The biochip comprises a substrate to which maleimide-biotin is attached; And a streptavidin-antibody complex bound to the maleimide-biotin. Forming a monomolecular film to which a thiol group is connected through silanization;
The step of dropping the biotin (maleimide-PEO ₂ -biotin) solution secure the biotin-maleimide -PEO ₂ on the monomolecular film surface;
Binding an avidin-antibody complex or a streptavidin-antibody complex to the biotin;
Binding a biotin linked thrombin aptamer to the avidin of the avidin-antibody complex or streptavidin-antibody complex; And
Fixing a thrombin to the thrombin aptamer, a method for producing a kit for detecting a target substance. Reacting the target material on the surface of the biochip of any one of claims 1 to 5;
Reacting the thrombin-bound antibody of any one of claims 1 to 5 to the surface of the biochip; And
A method of detecting a target material comprising the step of reacting fibrinogen on the surface of the biochip. Reacting the target material on the surface of the biochip of any one of claims 1 to 5;
Reacting the streptavidin-antibody complex with the biochip to which the target material is reacted;
Binding a thrombin aptamer to the streptavidin-antibody complex;
Fixing thrombin to the thrombin aptamer; And
And a method of reacting fibrinogen with the surface of the biochip to which the thrombin is immobilized. 9. The method of claim 8,
The method of detecting a target substance, characterized in that the reaction solution is mixed with a substance for increasing the signal for fibrin production in the reaction solution fibrinogen on the surface of the thrombin fixed biochip. 10. The method of claim 9,
The material for increasing the signal may be colored particles, fluorescent particles, quantum dots, gold particles, electrochemically active particles, carbon nanotubes, graphene, fullerenes, fluorescent proteins , GOX (Glucose oxidase), ALP (Alkaline Phosphatase) and HRP (Horse-Radish Peroxidase) detection method of the target material, characterized in that selected from the group consisting of.

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