KR20110019953A - Method for sensing a specific protein using a molecular probe and binding pattern - Google Patents

Abstract

PURPOSE: A method for analyzing a material using difference of binding affinity is provided to determine presence of a specific protein and minute changer of the specific protein. CONSTITUTION: A method for analyzing a specific protein comprises: a step of patterning a binding affinity of the specific viral protein of a substrate and plural probes containing an amino acid dimer or derivatives thereof and making database; a step of measuring binding affinity by contacting the plural probes with a sample; a step of comparing the measured binding affinity with the database; and a step of confirming the specific protein. The amino acid dimer or derivative thereof is a compound of chemical formula 1.

Description

Method for detecting specific protein using molecular probe and binding pattern {METHOD FOR SENSING A SPECIFIC PROTEIN USING A MOLECULAR PROBE AND BINDING PATTERN}

The present invention relates to a method for detecting a specific protein using a molecular probe, particularly a binding pattern using various kinds of amino acid dimers as molecular probes.

Measuring and analyzing specific proteins or peptides (hereinafter, proteins) is a very important field throughout the bio / medical / chemical industry. The most widely used methods to measure specific proteins to date are 1) using an immune response (eg ELISA), 2) analytical chemistry such as MALDI-TOF, NMR, liquid chromatography, and the like. Among them, the method of applying the immune response is most commonly used for screening. However, the biggest problem so far known when using immunological methods is the error of the results due to non-selective binding. The problem of non-selective binding arises not only from the binding between the antigen and the antibody but also from the antigen or antibody binding to the reaction vessel or other foreign body.

Until now, the methods developed or proposed to solve these problems are mostly dependent on the experimenter's experience or the choice of materials, such as developing more selective antibodies or improving the pretreatment of the sample. However, the problem of recognizing a specific protein by this type of approach is that different results may occur depending on the situation, and if the analysis of a specific protein is difficult with only one antibody, the analysis result may be different depending on the subjective judgment of the analyst. Is the point. In addition, the analysis method using immunology had a disadvantage that the antibody itself is very expensive when the antibody is used as a capturing agent.

Therefore, research on the development of a method for precisely analyzing a specific protein without using an antibody in analyzing a specific protein has been continued. In particular, a low-molecular organic compound that selectively binds to a specific protein is used as a capture material for detection. Research has been conducted to detect the presence of specific proteins.

U.S. Patent Application Publication No. US20090074663 has developed a peptide that binds to IL-4R of atherosclerotic flakes to diagnose / treat atherosclerosis, and International Application Publication No. WO06 / 128294 binds the virus to protect the activity of the virus. Inhibiting peptides have been developed, and Korean Patent Application Publication No. 2007-0062467 discloses a method for detecting AI virus using a gene probe.

Tannish Nandi, on the other hand, theoretically suggested substances that bind to viruses such as AI using computational chemistry (Tannish Nandi, Bioinformation, Vol. 2 (6), pp 240-244, 2008.01), and James Stevens et al. A system that can be used to determine the presence or absence of AI virus was proposed (James Stevens et al., Glycan microarray technologies: tools to survey host specificity of influenza viruses, Nature Reviews, Vol. 4, pp. 857-864, 2006.12 ).

However, up to now, no system has been developed that can synthesize amino acid combinations into actual capture materials to detect viruses and determine minute changes such as genetic variation of specific proteins.

The present invention is to develop a method for analyzing a specific protein, without using an antibody.

Another object of the present invention is to develop a method for identifying details by recognizing patterns for various substances of a specific protein as well as simple binding as in ELISA.

Another object of the present invention is to develop a method that can be easily used for the detection of viruses with high strains such as AI virus, SARS virus and the like.

Another object of the present invention is to develop a method for measuring microstructural variations (eg, secondary or tertiary structural changes in amino acid sequences) of a protein or peptide.

It is an object of the present invention to develop a method that can ultimately analyze specific viruses by profiling patterns in which various organic substances (a kind of molecular probe) bind to specific proteins.

The present invention provides a method of patterning and database-binding affinity data with a plurality of probes composed of amino acid dimers or derivatives thereof and a specific protein of a known virus, and a plurality of probes composed of the amino acid dimers or derivatives thereof. Determining the binding affinity by contacting the sample to be detected, comparing the measured binding affinity with the database, and confirming the presence or absence of a specific protein by the comparison. Provides a method for analyzing proteins.

The present invention 1) can determine the presence or absence of a specific protein even in the absence of a specific antibody development 2) to determine the minute changes (specific changes in composition of one or two amino acids) of a specific protein due to genetic variations 3) The profiled data can be easily databased.

The present invention is a method for analyzing a protein using a difference in binding affinity for various substances of the protein,

Patterning and database binding affinity data with a plurality of probes composed of amino acid dimers or derivatives thereof and specific proteins of known viruses,

Measuring a binding affinity by contacting a plurality of probes composed of the amino acid dimer or a derivative thereof with a sample to be detected;

Comparing the measured binding affinity with the database;

Confirming the presence or absence of a specific protein by the comparison,

It provides a method for analyzing a specific protein comprising a.

A schematic overview of the method of analyzing a substance of the present invention is shown in FIG. 1.

Known viruses used in the present invention may be avian influenza virus (AI virus) or SARS virus.

As used herein, the term "probe" refers to "various types of materials used for the detection of specific proteins of interest".

The probe consisting of the amino acid dimer or derivative thereof may be a compound of Formula 1:

[Formula 1]

Wherein X and X 'are each independently hydrogen; Biotin; Streptavidin; Avidin; Or a functional group comprising a biotin, streptavidin or avidin and a linker which binds the biotin, streptavidin or avidin to the main structure of the compound of Formula 1;

n is 0 or 1;

R and R 'are each independently selected from the groups listed below:

* When R or R 'is -CH ₂ -CH ₂ -CH ₂ -and -CH ₂ -SS-CH _2- , hydrogen of the carbon to which the substituent is attached is eliminated.

Examples of the linker include polyethylene glycol (PEG), DNA, C1-C20 alkylene, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), carbonyldiimidazole (CDI), Sulfosuccinimidyl (Sulfosuccinimidyl, Sulfo-NHS), isocyanate derivatives, acyl azide derivatives, N-hydroxysuccinimides (NHS), sulfonyl chloride derivatives, aldehyde derivatives, epoxy derivatives, and the like.

Preferably, X 'may include 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), carbonyldiimidazole (CDI), sulfosuccinimidyl (Sulfo-NHS) and the like. Being; X isocyanate derivative which forms an isocyanate compound to form isothiourea bond, acyl azide derivative which forms an amide bond, N-hydroxysuccinimide (NHS), sulfonyl chloride derivative which forms sulfonamide bond, secondary Aldehyde derivatives and epoxy derivatives that form amide bonds may be included.

Preferred examples of the amino acid dimer or its derivative represented by Formula 1 include Phe-Tyr, Tyr-Trp, Phe-Phe, Phe-Trp, Phe-Leu, Phe-Arg, Gln-Trp, Trp-Phe, Phe -Ala, Trp-Gln, Ala-Ile, Phe-Met, Cys-Phe, Val-Met, Tyr-Gln, Leu-Val, Arg-Phe, Ile-Met, Val-Ala, Lys-Phe, Ser-Cys , Ser-Asp, Asp-Ser, Ser-Glu, Glu-Ser, Asp-Cys, Asp-Glu, Cys-Glu, Cys-Asp, Glu-Asp may be preferably used, but is not limited thereto.

Determining binding affinity by contacting a plurality of probes consisting of the amino acid dimer or derivatives thereof with a sample to be detected is:

Immobilizing the plurality of probes or the sample to be detected, consisting of the amino acid dimers or derivatives thereof, on the substrate surface, and the plurality of probes or the sample to be detected, consisting of the amino acid dimers or derivatives thereof, the fixed probe or sample And contacting it. At this time, when a plurality of probes composed of the amino acid dimer or derivative thereof is fixed to the surface of the substrate, and the sample to be detected is contacted with the immobilized material, the second capture conjugated with the labeling substance after the contact The method may further include contacting the material.

As the substrate, silicon wafers, metals, glasses, crystals and the like used in the art can be used without limitation. In the present invention, the secondary trapping material is a polyclonal compound that can bind to the protein captured by the compound of Formula 1 Me means an antibody-like substance.

The step of contacting the plurality of probes consisting of the amino acid dimers or derivatives thereof with the sample to be detected may be performed after the step of fixing the probes consisting of the amino acid dimers or derivatives thereof to the surface of the nano- or micro-sized particles. . As the nano or micro sized particles, those used in the art may be used without limitation.

As described above, the method described above is schematically illustrated in FIG. The assay shown in FIG. 2 consists of:

i) coating the protein or fragment thereof on the surface of the 96 well plate;

ii) reacting a plurality of probes consisting of amino acid dimers conjugated with biotin with an immobilized protein or fragment thereof;

iii) reacting streptavidin-HRP conjugated with biotin with a secondary capture agent;

iv) Perform HRP-TMB analysis.

At this time, HRP-TMP analysis uses the following reaction. Horse radish peroxidase (HRP) and TMB (3,3 ', 5,5'-tetramethylbenzidine) are the most widely used reaction assays for ELISA reactions. When TMB reacts with HRP, it forms a blue product. However, if the reaction continues to be maintained (by enzyme), the reaction intensity is indistinguishable, so usually a strong acid (the same amount of 2N HCL in this patent) is added to stop the reaction and the color turns yellow and is analyzed at a wavelength of 450 nm. The reference wavelength is usually 650 nm.

In the present invention, the greatest effect is that the specific protein can be detected without using a specific antibody. In particular, the infection rate is so fast that it can be used for screening when there is not enough time for antibody production. In addition, the same kind of virus can be used as additional data when it is difficult to detect or difficult to detect due to variation of amino acid sequence. In addition, it can be used as a comparative data by analyzing simultaneously with the antibody.

Industrial applications are also possible in the bio- or pharmaceutical industry, particularly in quality control in the production of peptide formulations.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited to the following examples and may be variously modified and changed within the scope of the invention.

Example  1: Selection of the measurement target and preparation of the antigen solution

In this example, peptide fragments (14-15 peptides) of the following four viruses were used as targets.

Avian Influenza A (H5N1) Hemagglutinin (N-terminus) (Amino Acid Configuration: NH ₂ -CYPGDFNDYEELKHL-COOH)-ProSCI Catalog No: 3427P

-Avian Influenza A (H5N1) hemagglutinin (middle domain) (amino acid sequence: NH ₂ -RNSPQRERRRKKRG-COOH)-ProSCI Catalog No: 3425P

-SARS envelope peptide (C-terminus)-ProSCI Catalog No: 3533P

-SARS Matrix Peptide (C-terminal) ProSCI Catalog No: 3529P.

All used antigens were purchased in solution from ProSCI (12170 Flint Place, Poway, CA 92064).

Example 2 : amino acid Dimer  Preparation of Level Peptides

Amino acid dimers (thermosynthetic company) were purchased and the purity was over 95%. The types of amino acid dimers used are as follows: Phe-Tyr, Tyr-Trp, Phe-Phe, Phe-Trp, Phe-Leu, Phe-Arg, Gln-Trp, Trp-Phe, Phe-Ala, Trp- Gln, Ala-Ile, Phe-Met, Cys-Phe, Val-Met, Tyr-Gln, Leu-Val, Arg-Phe, Ile-Met, Val-Ala, Lys-Phe, Ser-Cys, Ser-Asp, Asp-Ser, Ser-Glu, Glu-Ser, Asp-Cys, Asp-Glu, Cys-Glu, Cys-Asp, Glu-Asp.

Example  3: Of probe  Produce

In the present invention, a probe conjugated with a sulfo-NHS-biotin (Product #: 21217) manufactured by Pierce was used as an amino acid dimer.

In this example, the probe was obtained by the following procedure. In order to conjugate the biotin, 10 μM of the amino acid dimer solution prepared in Example 2 and 10 μM of Sulfo-NHS-biotin were mixed in the same amount and reacted for 10 minutes at room temperature to conjugate the biotin to the amino acid dimer.

Example  4: coating of antigen ( Coating of antigen )

1) The antigen solution described in Example 1 was diluted 100-fold using carbonate buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , 3 mM NaN ₃ ).

2) 110 μl of diluted antigen solution was dispensed into NUNC's high binding 96 wells (Maxi-sorp F series).

3) After dispensing, the cells were incubated at room temperature (about 20 ° C.) for about 24 hours to fix the antigen in 96-well plates.

4) After washing three times with PBS buffer, 200 μl of blocking buffer was dispensed (blocking buffer: 1 × PBS, pH 7.2, 0.1% BSA, 0.02% thimersol) for 2 hours at room temperature. Store at

5) After the incubation, wash 5 times using washing buffer (1 × PBS, pH 7.2, 0.05% Tween-20, 0.02% thimerosal) and PBS buffer.

Example  5: measuring object Probe  Analysis of binding and bonding strength of materials

1) Dispense 10 μl of the probe solution prepared in Example 3 onto the antigen-coated plate obtained in Example 4, incubate at room temperature for 1 hour, and then wash buffer (1 × PBS, pH 7.2, 0.05% Tween-20, 0.02%). Thimerosal) and PBS buffer.

2) The step 1) was repeated five times.

3) 50 μl of streptavidin-HRP (Streptavidin-HRP) (Sigma-Aldrich, Cat. No. S2438) diluted to 1/10000 using PBS solution was dispensed.

9) After 1 hour of incubation at room temperature, the cells were washed five times using washing buffer (1 × PBS, pH 7.2, 0.05% Tween-20, 0.02% thimerosal) and PBS buffer.

10) 50 μl of tetramethylbenzidine (TMB) solution was added thereto, followed by reaction for 15 minutes. The reaction was stopped using 2N HCl, and analyzed using a micro-well plate. The instrument used a microwell plate reader (absorbance at 450.0 nm) from Molecular Device.

The experimental results were as follows.

* AI virus IN represents H5N1 hemagglutinin intermediate domain, AI virus NT represents H5N1 hemagglutinin N-terminus, SARS M represents the SARS matrix, and SARS E represents the SARS envelope. )

As shown in Table 1 above, experiments with probes composed of capture compounds prepared from 30 selected amino acid dimers and two commercialized antigens revealed that the probes of the capture compounds prepared with the respective amino acid dimers Different binding properties were shown for the antigens, and the patterning thereof was shown in FIGS. 3 and 4, respectively. As can be seen in Figures 3 and 4, each specific protein showed a distinct pattern.

Example  6: antigen in the plasma of poultry spiking After  Analysis

1) NUNC's maxisorp type microplate was used as the microplate used in this experiment.

2) As the antigen, the avian influenza A (H5N1) hemagglutinin N-terminal antigen of Example 1 was used and diluted at a ratio of about 1/1000 (10 µl of the antigen solution stock and 10 ml of the coating buffer were mixed. Carbonate buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , 3 mM NaN ₃ ) was used as the coating buffer.

3) Chicken blood (hereinafter: CRS-> Chicken Red Serum) was mixed with the coating buffer at a rate of 1/10. CRS collected the blood of chickens grazing in the mountains using a 50ml conical tube.

3) The blood of the collected chicken was removed after 3 hours by centrifugation to remove the gore (gore) and only the blood plasma was collected.

4) In the case of CRS, diluted 1/10 with the coating buffer. The reason for this is that the CRS is used as a negative control and the total protein concentration may be higher than the antigen, but the concentration of each protein is low because it is low to be used as a negative control group, so it is maintained at about 1/10.

5) In the case of Spiked CRS, 50 μl of antigen stock and 950 μl of CRS were mixed and coated with a coating buffer in a ratio of 1/10.

6) Thereafter, the same procedure as in Example 2 and Step 2) and Example 5 were performed.

The experimental results were as follows.

Dimer Experimental average Dimer Experimental average Ile-Thr 0.97 Thr-gln 0.33 Asn-tyr 0.89 Gln-his 0.32 Tyr-ala 0.59 Thr-glu 0.28 Thr-lys 0.59 Met-ala 0.25 Trp-Arg 0.57 Glu-his 0.24 Glu-lys 0.53 Cys-val 0.22 Ala-ala 0.53 Gly-glu 0.21 Phe-Asn 0.52 Glu-his 0.21 Gln-ile 0.51 Arg-Arg 0.19 Lys-lys 0.51 Ser-Val 0.18 Ser-cys 0.50 Val-His 0.17 Ser-lys 0.42 Pro-Ala 0.13 Leu-ala 0.37 Arg-Asn 0.12 Ser-arg 0.37 Val-Ala 0.09 Met-aps 0.35

The experimental results are compared with the results in PBS obtained in Example 5 as follows.

Dimer PBS Chicken blood Ile-Thr One One Asn-tyr 2 2 Tyr-ala 3 4 Thr-lys 4 5 Trp-Arg 5 7 Glu-lys 6 6 Ala-ala 7 3 Phe-Asn 8 8 Gln-ile 9 9 Lys-lys 10 10 Ser-cys 11 11 Ser-lys 12 14 Leu-ala 13 18 Ser-arg 14 13 Met-aps 15 16 Thr-gln 16 12 Gln-his 17 15 Thr-glu 18 17 Met-ala 19 19 Glu-his 20 20 Cys-val 21 22 Gly-glu 22 25 Glu-his 23 21 Arg-Arg 24 28 Ser-Val 25 24 Val-His 26 23 Pro-Ala 27 26 Arg-Asn 28 27 Val-Ala 29 29

Through the comparison of Table 3, it can be seen that the experiment using the blood of the chicken showed almost the same results as in PBS (see FIG. 5). In the experiment it was confirmed that the same results as in the PBS buffer in the actual blood.

1 is a basic conceptual view of the present invention. Even if a specific antibody is included or not, it is possible to detect a specific substance by making a database of such binding characteristics using both characteristics that most proteins bind with respect to various substances and that the binding is different from substance to substance. Shows the method.

2 shows a method for analyzing a substance by reacting a sample to be detected with a probe composed of various kinds of amino acid dimers or derivatives thereof.

3 and 4 show the results of binding experiments consisting of 29 amino acid dimers to a short peptide, which is a type of protein (antigen) of AI virus, and a probe consisting of 30 amino acid dimers to protein (antigen) of SARS virus. The patterning results of the binding experiments are shown.

5 shows a comparison of patterns when using PBS and when using chicken blood.

Claims (10)

Patterning and database binding affinity data with a plurality of probes composed of amino acid dimers or derivatives thereof and specific proteins of known viruses, Measuring a binding affinity by contacting a plurality of probes composed of the amino acid dimer or a derivative thereof with a sample to be detected; Comparing the measured binding affinity with the database; Confirming the presence or absence of a specific protein by the comparison, Method for analyzing a specific protein comprising a. The method of claim 1, wherein the amino acid dimer or derivative thereof is a compound of Formula 1 [Formula 1]

Wherein X and X 'are each independently hydrogen; Biotin; Streptavidin; Avidin; Or a functional group comprising a biotin, streptavidin or avidin and a linker which binds the biotin, streptavidin or avidin to the main structure of the compound of Formula 1; n is 0 or 1; R and R 'are each independently selected from the groups listed below, and in the case of R or R' is -CH ₂ -CH ₂ -CH ₂ -and -CH ₂ -SS-CH _2-, the carbon to which the substituent is attached Of hydrogen is eliminated:

. The method according to claim 2, wherein the amino acid dimer or derivative thereof Phe-Tyr, Tyr-Trp, Phe-Phe, Phe-Trp, Phe-Leu, Phe-Arg, Gln-Trp, Trp-Phe, Phe-Ala, Trp -Gln, Ala-Ile, Phe-Met, Cys-Phe, Val-Met, Tyr-Gln, Leu-Val, Arg-Phe, Ile-Met, Val-Ala, Lys-Phe, Ser-Cys, Ser-Asp To analyze a particular protein selected from the group consisting of Asp-Ser, Ser-Glu, Glu-Ser, Asp-Cys, Asp-Glu, Cys-Glu, Cys-Asp, Glu-Asp, and derivatives thereof. Way. The method according to claim 2 or 3, wherein the linker is polyethylene glycol (PEG), DNA, C1-C20 alkylene, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), carbo Nyldiimidazole (CDI), Sulfosuccinimidyl (Sulfo-NHS), Isocyanate derivatives, Acyl azide derivatives, N-hydroxysuccinimides (NHS), Sulfonyl chloride derivatives, Aldehyde derivatives, and epoxy derivatives Method for analyzing a specific protein is selected from the group consisting of. The method of claim 4, wherein the linker is sulfosuccinimidyl. The method of claim 2, wherein the contacting of the plurality of probes consisting of the amino acid dimer or derivative thereof with the sample to be detected is Immobilizing a plurality of probes composed of the amino acid dimer or derivative thereof or the sample to be detected on a surface of a substrate; And Contacting the plurality of probes or the sample to be detected consisting of the amino acid dimer or derivative thereof with the fixed probe or sample, Method for analyzing a specific protein comprising a. The method of claim 6, further comprising, after contacting the plurality of probes consisting of the amino acid dimer or derivative thereof with the sample to be detected, contacting the secondary capture material conjugated with the labeling material. How to analyze specific proteins. The method of claim 1, wherein measuring the binding affinity is using an HRP-TMB assay. The method of claim 1, wherein the virus is avian influenza virus or SARS virus. The protein of claim 1, wherein the known viral protein is selected from the group consisting of H5N1 hemagglutinin N-terminus, H5N1 hemagglutinin intermediate domain, SARS envelope peptide C-terminus, SARS substrate peptide C-terminus, and combinations thereof Method of analyzing a particular protein.

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