KR101422700B1 - Synthetic antibody for detecting C-reactive protein - Google Patents

Abstract

The present invention provides a synthetic antibody for the detection of a C-reactive protein that is not an animal-derived antibody. The synthetic antibody does not require a complicated process including a fluorescent label, expensive equipment and the like required in conventional chromatography, So that the coupling amount can be easily analyzed.

Description

[0001] Synthetic antibodies for detecting C-reactive protein [0002]

The present invention relates to a synthetic antibody for the detection of C-reactive protein, wherein the synthetic antibody is excellent in sensitivity to C-reactive protein and capable of confirming the amount of binding of the synthesized antibody and C- RTI ID = 0.0 > antagonistic < / RTI >

Since gold nanoparticles are highly reactive with thiol groups of proteins due to their characteristics, they can be easily immobilized with proteins such as antibodies, and thus they have been widely used for immobilizing probe materials for target detection. In addition, since it absorbs light at a certain wavelength band (~ 530 nm) in the visible light region, it can discriminate the binding of the particles to the naked eye without using a probe such as a fluorescent substance, and thus can be used as a basic raw material for the rapid diagnosis kit using immunochromatography Has been used.

A synthetic antibody to a protein is an aptamer, and a technique for producing a synthetic antibody by a molecular imprint method has been developed. In the case of multiple subunits such as C-reactive protein (CRP), 2D molecular imprints are used using specific ligands, but most of them are applied to the surface of a chip, not a particle. Has come.

In recent years, immunochromatography (ICA) has been increasingly used because of its ability to measure various kinds of substances in various fields such as antigens and antibodies as well as hormones and drugs, Trend.

In addition, protein detection using immunochromatography has been used to immobilize an antibody of a target substance on the surface of gold nanoparticles by a non-specific binding force. An animal-derived monoclonal or polyclonal antibody is non-specifically immobilized on the surface of the gold nanoparticle to bind the target substance in the sample to the antibody on the gold nanoparticle surface, and another monoclonal or polyclonal antibody Is spotted on a strip to bind a target substance bound to the gold nanoparticles to the antibody immobilized on the strip, thereby determining the presence or absence of a target substance in the sample by forming a bright line or spot by gold nanoparticles.

However, this method requires two monoclonal or polyclonal antibodies, which are not able to bind two monoclonal antibodies to one target molecule at the same time, or when a polyclonal antibody is used, errors due to non-specific binding forces Has come.

In order to measure the binding amount of a target substance to an organic or inorganic polymer or a low-molecular-weight synthesized antibody immobilized on the surface of a nanoparticle when a protein is detected using a conventional immunochromatography method, Cleaning and so on. In order to finally measure the bonding amount, expensive equipment such as surface plasm resorption (SPR), spectrophotometer, and fluorescence meter is required, and many There were disadvantages of cost and time.

In order to solve the above-mentioned problems, the present invention provides a synthetic antibody having increased binding force with a target molecule including a molecule imprinting layer on the surface of gold nanoparticles. The present invention also provides a method for detecting C-reactive protein, which can detect low-level C-reactive protein using the synthetic antibody and can easily analyze the binding amount of a synthetic antibody bound to C-reactive protein.

In order to achieve the above object, the present invention provides a gold nanoparticle;

A pinning layer formed on the gold nanoparticle substrate and having an azide or acetylene terminal group R ₁ ; And

Formed on the fixed layer and formed of R ₂ -X-phosphocholine (wherein R ₂ represents an azide or acetylene terminal group and X represents a spacer group having a length of 0.1 nm to 5 nm) Molecular imprinting layer; / RTI >

Wherein the phosphocholine group contained in the molecular imprinting layer is oriented to correspond to a phosphocholine binding site of each molecule of a C-reactive protein, provides C- reactive protein synthesis hangcheeul for detecting which comprises coupled by a bond between the ring containing R ₂ in R ₂ -X- phosphocholine in a fixed bed end group R ₁ and the layer of molecular imprinting.

According to a preferred embodiment of the present invention, when the terminal groups R ₁ of a fixed bed is an azide group, if R ₂ is an acetylene group, and the end of the fixed bed short R ₁ is an acetylene group in the molecule imprinting layer, the molecular imprinting layer Lt; ₂ > may be an azide group.

According to another preferred embodiment of the present invention, the molecular imprinting layer is acetylene -C _{1 ~ 30} alkyl-phosphoryl choline, propargyl oxy -C _{1 ~ 30} alkyl-phosphoryl choline _{(propargyloxy-C 1 ~ 30 alkyl} -phosphorylcholine ) or azido -C _{1 ~ 30} alkyl- may be formed, including phosphoryl choline _{(azido-C 1 ~ 30 alkyl} -phosphoryl choline).

The present invention also provides a method for preparing a gold nanoparticle comprising: forming a pinning layer having an azide or acetylene terminal group R ₁ on gold nanoparticles;

A molecular imprinting material comprising a C-reactive protein and an R ₂ -X-phosphocholine (wherein R ₂ represents an azide or acetylene end group and X represents a spacer group of 0.1 to 5 nm in length) Forming a molecular imprinting material / C-reactive protein mixed solution by mixing;

The molecular imprinted material / C- reactive protein mixture solution by immersing the gold nanoparticles are formed on the fixed bed, the end of the fixed bed short R ₁ and R ₂ -X- wherein the molecular imprinted material in the force between the fabric Colin R ₂ included in the Forming a gold nanoparticle / fixed layer / molecular imprint material / C-reactive protein complex layer by a ring addition reaction of And

Removing the C-reactive protein from the complex layer to form a molecular imprinting layer;

Reactive protein for the detection of C-reactive protein.

The present invention also relates to a method for detecting a C-reactive protein, comprising: a reaction membrane in which a test line and a control line containing a single or polyclonal antibody of C-reactive protein are treated;

A sample pad located on one side of the reaction membrane and absorbing the sample; And

An absorption pad located on the other side of the reaction film and absorbing a remaining amount of the sample;

Lt; RTI ID = 0.0 > immunochromatography < / RTI >

In addition, the present invention provides a method for detecting a C-reactive protein using the immunochromatographic strip and the synthetic antibody.

The term "biological sample " used in the present invention may include tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, urine and the like. Reactivity of the antibody or the same epitope of the present invention with an antibody having specificity can be measured without manipulating or manipulating these biological samples to measure the degree of expression of C-reactive protein.

The present invention provides a method for preparing a synthetic antibody that specifically binds to C-reactive protein (CRP) so as to function as nanomedicine that can be used in vivo and in vitro. These synthetic antibodies were prepared using the molecular imprinting method of CRP and applied to immunochromatography to be used as a detection probe for CRP concentration diagnosis in vitro. This is an improvement of the existing immunochromatography method which uses two antibodies to detect the target substance in the sample, and the detection limit is lowered to 0.1 ug / ml or less. In the case of a synthetic antibody, the chemical structure of the binding site to the target protein is well known, so that it is relatively easy to control the binding site of the other antibody.

In addition, as immunochromatography using a synthetic antibody other than an animal-derived antibody has not been successful in the past, various ligand substances based on chemical binding force can be utilized in immunochromatography and rapid diagnostic kits .

On the other hand, in order to determine the amount of the target substance bound to the surface of the nanoparticles, a complicated pretreatment process and an expensive apparatus were required, but procedures such as fluorescent substance labeling were required. The present invention provides a method for rapidly and simply measuring the protein binding on the nanoparticle surface without pretreatment and high-cost equipment by using an immunochromatography method. This method has the disadvantage that it can not discriminate the binding amount due to non-specific binding other than the target protein in the other non-labeled methods. However, since the final analysis is completed by the reaction of the anti-CRP antibody immobilized on the surface of the strip, .

1 is a cross-sectional view of a strip for immunochromatography according to an embodiment of the present invention.
FIG. 2 is a process diagram showing a process for producing a synthetic antibody according to another embodiment of the present invention.
FIG. 3 is a graph showing the binding amount of C-reactive protein tested in Experimental Example 1 (CM: using gold nanoparticles of Comparative Example 2, NIM: using gold nanoparticles of Comparative Example 1, MIM: Lt; / RTI > antibody).
Figure 4 is a strip of C-reactive protein detection chromatography using gold nanoparticles of Comparative Example 2 against 0.073 to 18 g / ml of pure C-reactive protein.
Figure 5 is a strip of C-reactive protein detection chromatography using gold nanoparticles of Comparative Example 1 against 0.073 to 18 g / ml of pure C-reactive protein.
Figure 6 is a strip of C-reactive protein detection chromatography using the synthetic antibody of Example 1 for 0.073 to 18 [mu] g / ml of pure C-reactive protein.
7 is a strip of C-reactive protein detection chromatography using gold nanoparticles of Comparative Example 2 for a mixed sample containing 1% human serum.
8 is a strip of C-reactive protein detection chromatography using gold nanoparticles of Comparative Example 1 for a mixed sample containing 1% human serum.
Figure 9 is a strip of C-reactive protein detection chromatography using the synthetic antibody of Example 1 for a mixed sample containing 1% human serum.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

1 is a cross-sectional view of a strip 100 for immunochromatography according to one embodiment of the present invention.

The immunochromatography comprises a reaction membrane 110 in which a test line 140 and a control line 150 containing a single or polyclonal antibody of C-reactive protein are treated;

A sample pad 120 located at one side of the reaction membrane and absorbing the sample; And

An absorption pad 130 located on the other side of the reaction film and absorbing a remaining amount of the sample; . ≪ / RTI >

The reaction membrane 110 includes a single or polyclonal antibody of a C-reactive protein. The reaction membrane 110 is not particularly limited as long as it is usually used in immunochromatography. More preferably, the nitrocellulose material can be used, but is not limited thereto . The thickness of the reaction film may be 0.01 to 1 mm, but is not limited thereto, and the size of the reaction film may be 0.5 to 5 cm ³ , but is not limited thereto.

The sample pad 120 is a material to which a sample can be absorbed and is not particularly limited as long as it is usually used for immunochromatography. More preferably, the sample pad 120 may be made of a cellulose material, but is not limited thereto. The sample pad may have a thickness of 0.01 to 2 mm, but the present invention is not limited thereto. The size of the sample pad may be 0.2 to 5 cm ³ , but is not limited thereto.

The absorbent pad 130 is a material to which the sample can be absorbed and is not particularly limited as long as it is usually used for immunochromatography. More preferably, the absorbent pad 130 is made of a cellulose material, but is not limited thereto. The thickness of the absorbent pad may be 0.01 to 2 mm, but is not limited thereto. The absorbent pad may have a size of 0.2 to 5 cm ³ , but the present invention is not limited thereto.

The present invention also relates to gold nanoparticles; A pinning layer formed on the gold nanoparticle substrate and having an azide or acetylene terminal group R ₁ ; And a -R ₂ -X-phosphocholine (wherein R ₂ represents an azide or acetylene terminal group and X represents a spacer group having a length of 0.1 nm to 5 nm) formed on the fixed layer A molecular imprinting layer formed by the method; Wherein the phosphocholine group contained in the molecular imprinting layer is oriented to correspond to a phosphocholine binding site of each molecule of C-reactive protein, imprinting layer is C- reactive protein detection, comprising coupled by click (click) a chemical reaction between the R ₂ included in the R ₂ -X- phosphocholine the end group R ₁ and the molecular imprinting layer of said fixed bed Thereby providing a synthetic antibody.

The gold nanoparticles are not particularly limited so long as they are used as a basic raw material of immunoassay kit, but those having diameters of 20 to 80 nm are more preferable.

The target substance to be detected herein is a C-reactive protein, and the C-reactive protein is one of proteins observed in the blood. In addition, the C-reactive protein is a protein that precipitates in association with the C-polysccharide of Diplococcus pneumoniae and exists in human serum at an average concentration of 580 ng / ml. Since the C-reactive protein rapidly increases upon inflammatory diseases or necrosis of tissues, it can be mainly used as a marker of inflammation. In addition, the detection of C-reactive protein concentration in the blood is clinically important and is used to diagnose diseases related to rheumatism, bacterial infection, viral infection, tuberculosis, inflammation at the onset of nephritis, or tissue necrosis. The physiological function of the C-reactive protein is to bind the posococholine appearing on the surface of dead or dying cells (and some types of bacteria) to activate the complement system through the C1Q complex (antigen + antibody) Protein enhances the phagocytosis of macrophages expressing the receptor of C-reactive protein. Each molecule of the C-reactive protein has a binding site capable of specifically binding to five phosphocholine groups.

The fixing layer can be used without particular limitation as long as it is fixed on the gold or nano particle base material through a functional group such as a thiol group and has an azide or acetylene group on its surface. For example, the polymer layer can be formed of a polymeric material such as polyacrylate, polymethacrylate, ethylene glycol amine, ethylene glycol thiol, N-hydroxysuccinimide-ethylene glycol, But are not limited to, those formed from N-hydroxysuccinimide-polyethylene glycol, maleimide-polyethylene glycol, polymers based on carbohydrate unit linkages, and combinations thereof.

According to a preferred embodiment of the present invention, when the terminal group R ₁ of the fixing layer is an azide group, R ₂ of the molecular imprinting layer is an acetylene group, and when the terminal group R ₁ of the fixing layer is an acetylene group, R ₂ May be an azide group.

According to another preferred embodiment of the present invention, the pinning layer can be a self-assembled monolayer having an azide or acetylene terminal group R ₁ on its surface.

Also, the self-assembled monolayer may be formed of HS-YR ₁ wherein R ₁ represents an azide or acetylene terminal group, and Y represents a spacer group having a length of 0.1 nm to 5 nm.

According to another preferred embodiment of the present invention, the spacer group Y may be a hydrocarbon group or an oxyhydrocarbon group having 1 to 30 carbon atoms which may include at least one of a double bond, a triple bond and an aromatic ring, but is not limited thereto . For example, the spacer group Y may be a hydrocarbon group having 1 to 30 carbon atoms, or 1 to 25 carbon atoms, or 1 to 20 carbon atoms, or 1 to 15 carbon atoms, which may contain at least one of a double bond, a triple bond and an aromatic ring Or an oxyhydrocarbon group, but is not limited thereto.

According to another preferred embodiment of the present invention, the spacer group Y has 1 to 30 carbon atoms, or 1 to 25 carbon atoms, or 1 to 20 carbon atoms, which may contain at least one of a double bond, a triple bond and an aromatic ring, An alkyl group having 1 to 15 carbon atoms or an alkyl oxide (e.g., a polyethyloxide, a polypropoxide, etc.), but is not limited thereto.

In addition, the self-assembling monolayer is azido -C _{1 ~ 30-alkyl-1-thiol (azido-C 1 ~ 30 alkyl} -1-thiol), acetylenic -C _{1 ~ 30-alkyl-1-thiol} or propargyl oxy -C _{1 to 30} may be formed, including alkyl-1-thiol.

The molecular imprinting layer may be formed comprising R ₂ -X-phosphocholine (wherein R ₂ represents an azide or acetylene terminal group and X represents a spacer group of 0.1 nm to 5 nm in length) But is not limited thereto.

According to a further preferred embodiment of the present invention, the molecular imprinting layer is acetylene -C _{1 ~ 30} alkyl-phosphoryl choline, propargyl oxy -C _{1 ~ 30} alkyl-phosphoryl choline _{(propargyloxy-C 1 ~ 30 alkyl} -phosphorylcholine ) or azido -C _{1 ~ 30} alkyl- may be formed, including phosphoryl choline _{(azido-C 1 ~ 30 alkyl} -phosphoryl choline).

As described above, an azide or acetylene-terminal self-assembled monolayer fixed to the gold nanoparticle substrate is formed through a functional group capable of binding to gold nanoparticles such as thiol groups, and the azide or acetylene- By forming the molecular imprinting layer on the self-assembled monolayer by click chemistry, it is easy to immobilize the molecule imprinting layer on the gold nanoparticle substrate and the binding constant to the C-reactive protein is improved, A synthetic antibody for the detection of an improved C-reactive protein can be produced. Therefore, the synthetic antibody for the detection of the C-reactive protein of the present invention exhibits a high binding constant to the C-reactive protein, so that a low concentration of C-reactive protein can be detected. Thus, The concentration of reactive protein is also measurable. In addition, the click chemistry reaction is pH-independent and is a very fast and efficient reaction so that the molecular imprinting layer can be easily and efficiently immobilized on the gold nanoparticle substrate. Thus, in the present application, a synthetic antibody for the detection of C-reactive protein can be easily produced without using an antibody.

The present invention also provides a method for preparing a gold nanoparticle comprising: forming a pinning layer having an azide or acetylene terminal group R ₁ on gold nanoparticles;

A molecular imprinting material comprising a C-reactive protein and an R ₂ -X-phosphocholine (wherein R ₂ represents an azide or acetylene end group and X represents a spacer group of 0.1 to 5 nm in length) Forming a molecular imprinting material / C-reactive protein mixed solution by mixing;

The molecular imprinted material / C- reactive protein mixture solution by immersing the gold nanoparticles are formed on the fixed bed, the end of the fixed bed short R ₁ and R ₂ -X- wherein the molecular imprinted material in the force between the fabric Colin R ₂ included in the Forming a gold nanoparticle / fixed layer / molecular imprinting material / C-reactive protein complex layer by a click chemistry reaction; And

Removing the C-reactive protein from the complex layer to form a molecular imprinting layer;

Reactive protein for the detection of C-reactive protein.

According to another preferred embodiment of the present invention, when the terminal group R ₁ of the fixing layer is an azide group, R ₂ of the molecular imprinting material is an acetylene group, and when the terminal group R ₁ of the fixing layer is an acetylene group, R ₂ May be an azide group.

According to another preferred embodiment of the present invention, the fixation layer may be a self-assembled monolayer having an azide or acetylene terminal group R ₁ on the surface.

According to another preferred embodiment of the present invention, after formation of the complex layer, the method may further comprise reacting the propargyl alcohol to the fixed bed portion which is not bound to the molecular imprinting material.

According to another preferred embodiment of the present invention, before immersing the immobilized gold nanoparticles, the molecular imprinting material / C-reactive protein mixed solution is preliminarily cultured to form five phosphocholine groups To correspond to the phosphocholine binding site of each molecule of the C-reactive protein.

As shown in FIG. 2, a method for producing a synthetic antibody for detecting C-reactive protein according to an embodiment of the present invention will be described. A glass substrate is treated with a pyranose solution (piranha soln) (3-aminopropyl trimethoxysilane (APTMS) solution). Thereafter, the glass substrate processed in step 1 may be immersed in distilled water containing gold nanoparticles having a size of 50 nm to immobilize the gold nanoparticles on the glass substrate. Step 3 can treat the glass substrate on which the gold nanoparticles are immobilized by a click chemical reaction and a pyramidal-alcohol blocking reaction. Then, the glass substrate may be subjected to ultrasonic treatment to produce a synthetic antibody according to the present invention.

The present invention also relates to a method for preparing a compound of the present invention comprising the steps of: adding a synthetic antibody prepared according to the present invention to a buffer solution;

Adding a sample to the buffer solution; And

Immersing the sample pad end of the strip prepared for immunochromatography according to the present invention in a buffer solution to which the sample is added;

Lt; RTI ID = 0.0 > C-reactive < / RTI >

The buffer solution facilitates the binding of the C-reactive protein contained in the sample to the synthetic antibody by well dispersing the synthetic antibody of the present invention, and is not particularly limited as long as it is usually used in immunochromatography, A buffer solution applicable to neutral, weakly acidic, weakly alkaline pH can be used. And more preferably at least one selected from the group consisting of a borate buffer solution, a phosphate buffer solution, a tris buffer solution and a MOPS buffer solution.

The concentration of the synthesized antibody added to the buffer solution is not particularly limited as long as it is an amount normally used in immunochromatography. More preferably, the synthetic antibody is added to the buffer solution at a concentration of 1 쨉 g / ml to 100 ㎎ / ml based on gold nanoparticles But the present invention is not limited thereto.

The sample to be added to the buffer solution may be at least one selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, urine, and manipulated or purified form of the sample.

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

[ Example ]

Preparation Example  1. 2- (2- (2- azidoethoxy ) ethoxy ) ethyl  11- ( tritylthio ) undecanoate Synthesis of

1.00 g (4.58 mmol) of 11-mercaptoundecanoic acid, 1.40 g (5.02 mmol) of trityl chloride and 0.95 g (6.87 mmol) of potassium carbonate were added to 20 ml of acetonitrile and stirred while heating to 100 ° C. After 7 hours of reaction, acetonitrile was removed by distillation under reduced pressure, dissolved in chloroform, and washed several times with 0.1N hydrochloride aqueous solution. Chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate. The chloroform was removed by distillation under reduced pressure. The hexane: ethyl acetate = 9: 1 was added to remove the trityl chloride. (yield = 83%) ( ¹ H NMR (CDCl ₃ 400 MHz):? (ppm) 7.42-7.17 (m, ( _{C 6 H 5) 3 -CS-} CH 2 -, 15H), 2.35 (t, -CH 2 -CH 2 -CH 2 -C (O) OH, J = 7.6, 2H), 2.14 (t, -CS- _{CH 2 -CH 2 -, J =} 7.6, 2H), 1.65 (p, -CH 2 -CH 2 -CH 2 -C (O) OH, J = 7.2, 2H), 1.41-1.12 (m, -S- CH ₂ -CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -CH ₂ -CH ₂ -, 14H)).

5.00 g (33.3 mmol) of triethylene glycol and 2.70 mL (19.40 mmol) of triethyl amine were added to 50 mL of chloromethane, cooled to 0 ° C using an ice bath, and then 1.00 mL (12.90 mmol) of methanesulfonyl chloride was slowly added thereto. After the addition, the ice-bath was removed and stirred at room temperature. After about 20 hours of reaction, the reaction mixture was distilled off under reduced pressure, and 2.55 g of product was obtained by removing impurities as much as possible using dichloromethane: methanol = 95: 5. 1.10 g (16.90 mmol) of sodium azide and 1.40 g (8.43 mmol) of potassium iodide were added to 50 ml of ethanol and the mixture was stirred at 110 ° C. After about 14 hours of reaction, the reaction mixture was distilled off under reduced pressure, dissolved in water and extracted with chloroform several times. The extracted chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate, and the chloroform was distilled off under reduced pressure to obtain 0.91 g of compound 2 (yield = 40%) ( ¹ H NMR _{3 400MHz): δ (ppm)} 3.75-3.59 (m, -O-CH 2 -CH 2 -O-, 10H), 3.40 (t, -O-CH 2 -CH 2 -N 3, J = 4.8, 2H )).

1.73 g (4.17 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1.73 g 5.74 mmol) and N, N'-dimethylamino pyridine (0.70 g, 5.73 mmol) in chloroform (20 mL), and the mixture was stirred at room temperature. After about 17 hours of reaction, the reaction mixture was diluted with chloroform and washed several times with 0.1 N hydrochloride aqueous solution and brine. Chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate, and the chloroform was distilled off under reduced pressure. The residue was purified by column chromatography using hexane: ethyl acetate = 3: (yield = 79%) ( ¹ H NMR (CDCl ₃ 400 MHz):? (ppm) 7.40-7.17 (m, (C ₆ H ₅ ) ₃ -CS-CH ₂ -, 15H), 4.24 _{m, -CH 2 -C (O)} O-CH 2 -CH 2 -, 2H), 3.71-3.64 (m, -O-CH 2 -CH 2 -O-, 8H), 3.39 (t, -O- CH ₂ -CH ₂ -N ₃ J = 4.8, 2H), 2.33 (t, -CH 2 -CH 2 -CH 2 -C (O) OH, J = 7.6, 2H), 2.14 (t, -CS-CH 2 -CH 2 -, J = 7.6, 2H), 1.64 (m , -CH 2 -CH 2 -CH 2 -C (O) OH, 2H), 1.41-1.12 (m, -S-CH 2 -CH 2 -CH 2 -, -CH 2 _{-CH 2 -CH 2 -CH 2 -,} 14H)).

1.80 g (2.92 mmol) of 2- (2- (2-azidoethoxy) ethoxy) ethyl 11- (tritylthio) undecanoate and 0.56 mL (3.51 mmol) of triethylsilane and 2.00 mL of trifluoroacetic acid were added to 8 mL of chloroform and stirred at room temperature. After about 4 hours, the reaction solution was diluted with chloroform and washed several times with aqueous sodium bicarbonate solution. Chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate, and the chloroform was distilled off under reduced pressure. The residue was purified by column chromatography using hexane: ethyl acetate = 3: It was obtained for g) (yield = 43%) (1 H NMR (CDCl 3 400MHz): δ (ppm) 4.24 (m, -CH 2 -C (O) O-CH 2 -CH 2 -, 2H), 3.71 _{-3.64 (m, -O-CH 2} -CH 2 -O-, 8H), 3.40 (t, -O-CH 2 -CH 2 -N 3 J = 4.8, 2H), 2.54 (q, --CS-CH 2 -CH 2 -, J = 7.6, 2H), 2.34 (t, CH 2 -CH 2 -CH 2 -C (O) OH, J = 7.6, 2H), 1.63 (m , -CH 2 -CH 2 -CH 2 -C (O) OH, -S-CH 2 -CH 2 -CH 2 -, 2H), 1.38-1.27 (m, -CH 2 _{-CH 2 -CH 2 -CH 2 -,} 12H)).

Preparation Example  2. 6-Propargylhexylphosphorylcholine (propargyl- Phosphorylcholine ) Synthesis of

A solution of 1,6-hexanediol (3 g, 25.4 mmol) in 20 mL of dimethylformamide (DMF) was added to a solution of sodium hydride (1.52 g, 38.1 mmol) in DMF (20 mL) Was added dropwise to the suspension and stirred for 30 min under ice-bath conditions. Propargyl bromide (5.7 g, 38.1 mmol) in DMF (20 mL) was added to the mixture, and then stirred at room temperature for 20 hours. The solvent was removed under reduced pressure, and the crude product was dissolved in diethyl ether. The mixture was rinsed with water, dried over anhydrous magnesium sulfate and purified by column chromatography on silica gel (Rf = 0.5, hexane / EtOAc = 1: 1) to give compound 5 (1.80 g, 45.4%) to give (IR: 3373, 3292, 2933 , 2858, 1093; 1 H NMR (400MHz, CDCl 3) δ4.13 (s, 2H, HCCCH 2 O), 3.59 (t, 2H, CH 2 OH), 3.52 ( _{t, 2H, CH 2 OCH 2} ), 3.03 (s, 1H, CH 2 OH), 2.48 (s, 1H, HCCCH 2 O), 1.58 (m, 4H, OCH 2 CH 2 CH 2 CH 2 CH 2), _{1.38 (m, 4H, OCH 2} CH 2 CH 2 CH 2); 13 C NMR (100MHz, CDCl 3) δ 79.8, 74.3, 70.0, 62.3, 57.9, 32.5, 29.3, 25.8, 25.5; exact mass calcd for C 9 H ₁₆ O ₂ : 156.12, found: 179 [M + Na] < ^{+ &} gt ^; ).

Chloro-l, 3,2-dioxaphosphorane 2-oxide (1.09 g, 7.68 mmol) and triethylamine (777 mg, 7.68 mmol) were dissolved in 30 mL dichloromethane And dissolved in dichloromethane (DCM). The reaction mixture was stirred at room temperature under a dark room condition for 72 hours. The solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (Rf = 0.3, hexane / EtOAc = 1: 4) to give compound 6 (700 mg, 69.5% Respectively.

Compound 6 (700 mg, 2.66 mmol) and trimethylamine (1.57 g, 26.6 mmol) were dissolved in 8 mL acetonitrile. The reaction mixture was stirred at 60 < 0 > C for 20 hours. The solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (chloroform: methanol 2: 1 and chloroform: methanol: water 50: 50: 4, Rf = 0.2). The solvent was removed under a reduced pressure to the residue is filtered to dissolved in dry chloroform, compound 7 (411 ㎎, 48.1%) to give the (IR: 3350, 3296, 2936 , 2860, 1086, 1059; 1 H NMR (400MHz, CD3OD) δ 4.28 (m, 2H, POCH 2 CH 2 N +), 4.15 (d, 2H, HCCCH 2 O), 3.90 (q, 2H, CH 2 CH 2 CH 2 OP), 3.69 (m _{, 2H, POCH 2 CH 2 N} +), 3.54 (t, 2H, HCCCH 2 OCH 2), 3.27 (s, 9H, N + (CH 3) 3), 2.85 (t, 1H, HCCCH 2 O), 1.64 _{(m, 4H, OCH 2 CH} 2 CH 2 CH 2 CH 2), 1.44 (m, 4H, OCH 2 CH 2 CH 2 CH 2); 13 C NMR (100MHz, CDCl 3) δ79.7, 74.8, 69.6, 65.5, 59.0, 57.4, 53.5, 30.4, 29.2, 25.6, 25.3; exact mass calcd for C 14 H 28 NO 5 P: 321.17, found: 322 [M + H] +).

Example  1. Synthesis of antibody

A glass plate treated with a pyran solution (3: 7 = H ₂ O ₂ : Conc. H ₂ SO ₄ ) was immersed in a 1% APTMS (3-aminopropyl trimethoxysilane) solution and reacted at room temperature for 8 hours. The glass plate was rinsed well with toluene, immersed in distilled water containing 50 nm gold nanoparticles dispersed therein, and reacted at room temperature for 12 hours. The glass plate on which the gold nanoparticles are immobilized is washed well with ethanol and water and immersed in a solution of 2 mM 2- (2- (2-azidoethoxy) ethoxy) ethyl 11- (tritylthio) undecanoate, The fixed layer was immobilized on the gold nanoparticle surface.

To form a molecular imprinted monolayer (MIM) on the gold nanoparticles, 3.32 nmol propargyl proline was dissolved in 1 ml of binding buffer (0.1 M Tris / HCl, _{150 mM NaCl, 5 mM CaCl 2} , pH 8.0) 0.66 nmol of C- reactive protein (c-reactive protein; CRP) ( propargyl phosphocholine: CRP = 5: pre-culture was at a molar ratio) of 1. The 1,3-dipolar cycloaddition reaction, which is one of the click chemistries, involves 1.66 nmol of copper sulfate (II) pentahydrate, 3.32 nmol of sodium ascorbate, and a reserve of 16 hours at 4 ° C Gold nanoparticles coated with a self-assembled monolayer of 2- (2- (2-azidoethoxy) ethoxy) ethyl 11- (tritylthio) undecanoate were added to a 20 ml PBS solution containing the cultured propargylphosphocholine- And placing the immobilized glass plate. The molar ratio of propargylphosphoryl choline: copper sulfate: sodium ascorbate was 1: 0.5: 1.

Propargyl-alcohol was added to the gold nanoparticles for another click reaction in order to block the azide end that was not involved in the click reaction. Gold nanoparticles were immersed at room temperature for 5 hours in a 20 ml PBS furber solution containing 20 μmol propargyl-alcohol, 10 μmol copper sulfate (II) · hexahydrate, and 20 μmol sodium ascorbate.

In order to remove CRP from the molecular imprinting layer, it was immersed in 2% sodium dodecyl sulphate (SDS) solution at room temperature for 30 minutes. Finally, the gold nanoparticles were thoroughly washed with deionized water, acetone and ethanol, dried under nitrogen gas, and the glass plate immersed in distilled water was ultrasonicated in an ultrasonic cleaner for 30 minutes in order to obtain a synthetic antibody from the glass plate.

Comparative Example  One.

To prepare a non-imprinted monolayer (NIM), all steps are the same as in Example 1 except that no CRP is added to the reaction mixture.

Comparative Example  2.

To prepare a control monolayer (CM), the gold nanoparticles coated with the azide (N ₃ ) -terminal self-assembled monolayer prepared in Example 1 above were reacted with propargyl-alcohol . This resulted in a surface uncoated by propargylphosphorylcholine

Experimental Example  1. Analysis of C-reactive protein binding of synthetic antibodies

To analyze the amount of CRP bound to gold nanoparticles having a monomolecular layer (CM) for control, gold nanoparticles having a molecular unprinted monolayer (NIM) and gold nanoparticles having a molecularly imprinted monolayer (MIM) Respectively. The glass substrate immobilized with gold nanoparticles was immersed in a 10 nM CRP solution to bind CRP to the molecularly phosphorylated phosphocholine binding site and to form anti-rabbit (anti-rabbit) conjugated with rabbit (IgG fraction) and horse radish peroxidase IgG antibody was serially treated and developed with 3,3 ', 5,5'-tetramethylbenzidine (TMB) solution, and the absorbance at 450 nm was measured. NIM showed a basic binding level similar to that of CM, while MIM showed an increased amount of CRP binding (FIG. 3)

Experimental Example  2. Immunochromatography

As shown in Fig. 1, a cellulose-based membrane and a nitrocellulose membrane were attached to produce a strip for immunochromatography.

The synthetic antibody according to the present invention was mixed with a sample containing CRP such as human plasma and dispensed into a 96-well plate. One end of the immunochromatographic strip immersed in the anti-CRP monoclonal antibody was immersed in the sample, Respectively. After all the samples were developed, the intensity of the spot was measured according to the concentration of each sample.

Figs. 4 to 6 show the results of immunochromatography performed at 0.073 , 0.22, 0.66, 2, 6, 9, and 18 占 퐂 / ml of pure CRP at various concentrations ranging from 0.073 占 퐂 / ml to 18 占 퐂 / ml. As can be seen in FIG. 6, the strip containing the synthetic antibody (MIM) of Example 1 exhibited a concentration-dependent binding amount from a concentration of 0.073 mu g / ml.

On the other hand, as can be seen from FIG. 4, the gold nanoparticles of Comparative Example 2 (gold nanoparticles having a monolayer (CM)) exhibited binding amounts at only 18 μg / Showed a nonspecific response.

Further, as shown in FIG. 5, the gold nanoparticles of Comparative Example 1 (gold nanoparticles having a non-molecule-imprinted monolayer (NIM)) showed similar tendency to gold nanoparticles having a monolayer, Of CRP binding capacity.

7 to 9 show that when a sample in which CRP was mixed with a concentration of 0.073 to 18 μg / ml (0.073 , 0.22, 0.66, 2, 6, 9, 18 μg / ml) and human serum was used in the buffer solution . As shown in Figures 7 to 9, using a synthetic antibody (MIM) of Example 1 (see Figure 9), which is the same result as the chromatography using pure CRP, for samples containing 1% human serum and CRP 7) or gold nanoparticles of Comparative Example 1 (gold nanoparticles having a non-molecularly-imprinted monolayer (NIM)) (Comparative Example 2) 8), the concentration-dependent binding pattern was exhibited even at a low concentration of 5 μg / ml or less.

As can be seen from the above Examples, Comparative Examples and Examples, the synthetic antibodies of the present invention can be detected by binding to low-level C-reactive proteins and can be detected by using expensive equipments And the binding amount could be analyzed without complicated procedures.

Claims (17)

Gold nanoparticles;
The gold is formed on the nano-particles [2 - {(azido-C _{1 ~ 3} alkoxy) C _{1 ~ 3} alkoxy} C _{1 ~ 3} alkyl (trityl Please) C _{9 ~ 13} alkanoic acid (2 - {( _{azidoC 1 ~ 3 alkoxy) C 1} ~ 3 alkoxy] C 1 ~ 3 alkyl} (tritylthio) C 9 ~ fixed bed containing ₁₃ alkanoic acid); And
Formed on the fixed bed, and NC _{2 ~ 5} alkynyl-layer phosphoryl choline imprinted molecule comprising a _{(NC 2 ~ 5 alkynyl-phosphorylcholine} ); includes,
The phosphocholine group contained in the molecular imprinting layer is oriented to correspond to a phosphocholine binding site of each molecule of C-reactive protein,
Wherein the fixed layer and the molecular imprinting layer are bound to each other by a ring bond between an azido group and an acetylene group of the fixed layer. According to claim 1, C- reactive protein detection according to claim 1, further comprising a C _{2 ~ 5} alkynyl, (C _{1 ~ 3} alkynyl) layer in combination with azido groups that are not bonded acetyl groups in the molecule imprinting layer fixed bed Soluble synthetic antibody. The synthetic antibody for detecting C-reactive protein according to claim 1, wherein the gold nanoparticles have an average diameter of 20 to 80 nm. The synthetic antibody for the detection of C-reactive protein according to claim 1, wherein the synthetic antibody detects a C-reactive protein at a concentration of 0.07 ㎍ / ml or more. delete delete On the gold nanoparticles [2 - {(azido-C _{1 ~ 3} alkoxy) C _{1 ~ 3} alkoxy} C _{1 ~ 3} alkyl (trityl Please) C _{9 ~ 13} alkanoic acid (2 - {(azidoC _{1 ~ 3 alkoxy) C 1 ~ 3 alkoxy} ] to form a fixed layer including a _{C 1 ~ 3 alkyl} (tritylthio} ) C 9 ~ 13 alkanoic acid);
Forming a phosphoryl choline molecular imprinted material / C- reactive protein mixture solution by mixing the molecular imprinted material comprising a _{(NC 2 ~ 5 alkynyl-phosphorylcholine} ) - C- reactive protein and NC _{2 ~ 5} alkynyl;
Immobilized gold nanoparticles on the molecular imprinting material / C-reactive protein mixed solution to immobilize gold nanoparticles / fixed layer / molecular imprint material / C - forming a reactive protein complex layer; And
Removing the C-reactive protein from the complex layer to form a molecular imprinting layer; Reactive protein. ≪ / RTI > delete delete The method of claim 7, further comprising, after forming the complex layer, further comprising reacting the propargyl alcohol with the immobilization layer portion that is not bound to the molecular imprinting material. 8. The method of claim 7, wherein before immersing the immobilized gold nanoparticles, the molecular imprinting material / C-reactive protein mixed solution is pre-incubated so that five phosphocholine groups derived from the molecular imprinting material are immobilized on the C- Reactive protein to correspond to the phosphocholine binding site of each molecule of the reactive protein. delete Adding a synthetic antibody of any one of claims 1 to 4 to a buffer solution;
Adding a sample to the buffer solution; And
Immersing the sample pad end of the strip for immunochromatography in a buffer solution to which the sample is added,
The immunochromatographic strip includes a reaction membrane on which a test line and a control line containing a single or polyclonal antibody of C-reactive protein is treated, a membrane on one side of the reaction membrane, A sample pad on which a sample is absorbed and an absorption pad on the other side of the reaction membrane and absorbing a remaining amount of the sample. 14. The method of claim 13, wherein the buffer solution is at least one selected from the group consisting of a borate buffer solution, a phosphate buffer solution, a tris buffer solution and a MOPS buffer solution. - Reactive protein diagnostic method. 14. The method for diagnosing C-reactive protein according to claim 13, wherein a synthetic antibody is added to the buffer solution at a concentration of 1 mu g / ml to 100 mg / ml.
8. The method of claim 7, wherein the step of forming the immobilization layer is performed by adding 0.5 to 5 mM of [2 - {(C ₁ -C ₃ alkoxy) C _1-3 alkoxy} C _1-3 alkyl] (trityl cyano) C _{9 To} C ₁₃ alkanoic acid), which is characterized in that a solution containing an alkanoic acid ([2- (azidoC _1-3 alkoxy) C _1-3 alkoxy] C _1-3 alkyl} (tritylthio) C 9-13 alkanoic acid is used. A method for producing a synthetic antibody for the detection of a reactive protein. According to claim 7, wherein forming the molecular imprinted material / C- reactive protein mixture is NC _{2 ~ 5} alkynyl-phosphoryl choline molecular imprinted material comprising a _{(NC 2 ~ 5 alkynyl-phosphorylcholine} ) and the C- Reactive protein is in a molar ratio of 3 to 7: 1.

Images