KR100673835B1 - Method for assaying protein using surface plasmon resonance - Google Patents

Abstract

A method for assaying protein is provided to be able to analyze extremely small amount of protein by amplifying a surface plasmon resonance(SPR) signal using a non-labeled antibody through a sandwich technique, thereby being variously applied to a disease detecting sensor, proteomics study and the like. The method comprises the steps of: (a) forming a self-assembled mono-molecule layer having carboxyl group and hydroxyl group on the surface of a metal thin film on a SPR chip, where the metal is selected from the group consisting of Au, Ag, Cu and Al; (b) biotining the self-assembled mono-molecule layer of the SPR chip and then combining it with avidin or streptavidin; (c) fixing the avidin or streptavidin of the SPR chip on a target protein using a specific and biotined primary antibody; (d) reacting the fixed antibody of the SPR chip with a biological sample such as tissue, cell, whole blood, serum, plasma, saliva, sputum, and urine including the target protein; (e) reacting the SPR chip with a specific and non-labeled secondary antibody; and (f) measuring the refractive index change of the SPR chip on the material fixed on the metal thin film surface to calculate the amount of the target protein.

Description

Method for assaying protein using surface Plasmon Resonance

1 is a conceptual diagram of a sensor chip manufacturing process for amplifying an SPR signal using a secondary antibody, (a) forming a self assembled monolayer on a gold thin film, and (b) a carboxyl group of a SAM to increase orientation Biotinylation, (c) immobilizing streptavidin in biotin to increase the immobilization site of the protein, (d) immobilizing a detection antibody capable of binding to the antigenic protein as a sample to streptavidin, (e) flows the biotinylated antigen sample, and (f) flows the unlabeled antibody again to amplify the SPR signal and enhance sensitivity.

Figure 2 is a graph showing the SPR value that is changed in real time by the immune response of the antigen-antibody according to the PSA-ACT concentration, (a) flowing the PSA-ACT antigen complex to the biotinylated PSA-ACT monoclonal antibody complex, (b) stopping the infusion of the PSA-ACT antigen complex, (c) flowing the PSA-ACT polyclonal antibody complex back into the PSA-ACT antigen complex, and (d) stopping the infusion of the PSA-ACT polyclonal antibody complex Represents a step.

Figure 3 is a graph showing the difference between the linear range and the primary and secondary response of the immunological assay.

The present invention relates to a method for quantifying proteins by amplifying Surface Plasmon Resonance (SPR) signals through sandwich assays using primary and non-labeled secondary antibodies specific for the target protein.

Methods for quantitative analysis of specific proteins include Enzyme Immunoassay (EI), Enzyme-Linked Immunosorbent Assay (ELISA), and Radioimmunoassay (RIA). Among them, ELISA is a method of binding an enzyme to an antibody to confirm an antigen-antibody reaction. The method is simple, inexpensive, and can be analyzed in large quantities, making it one of the most widely used antigen-antibody assays. . The ELISA method is widely used because it is a very sensitive reaction such as radioimmunoassay (RIA) and does not use radioactivity as in RIA. However, this method requires a large amount of sample to analyze, it takes a long time and has a multi-step process. RIA has the highest sensitivity, but safety is a problem in that it uses radioactive materials.

Isotope measurement in protein quantification is a safety problem, enzyme reaction measurement is difficult to analyze the samples present in various concentrations because of the narrow analysis range, fluorescence method is to bind the expensive fluorescent material to the detection protein again There is a problem that should be used. In order to solve these problems, a label-free detection method using SPR optically is preferred.

SPR sensor technology uses a phenomenon that causes a signal change when a biological material such as a protein is bonded to the sensor surface, and is used in biosensors or biochips. The reason that SPR is suitable for use as a biosensor is because the SPR phenomenon is very sensitive to the physical and chemical changes occurring near the surface of the metal film (nearly 500 nm or less) as a change in resonance angle or wavelength. This is because the processing can be applied to various fields. In addition, SPR can measure the binding affinity in real time with the optical principle alone without any damage or modification to the sample, and without any markers using radioactive materials or fluorescent materials, compared to other methods. . With these advantages, SPR has been evaluated as a useful technique in the field of biosensor for biomolecule measurement and used for protein analysis. However, biosensors using SPR have a low sensitivity for analyzing low concentration biomaterials.

Against this background, the inventors bound the self-assembled monolayer of SPR via avidin or streptavidin to capture the target protein with the primary antibody and detected the surface plasma resonance (SPR) signal when detected using the unlabeled secondary antibody. As the refractive index change is increased, the SPR signal can be amplified, and thus, the present invention was completed by confirming that the protein existing at low concentration can be measured with high sensitivity.

One object of the present invention is to capture a target protein with a primary antibody bound to the self-assembled monolayer of SPR via avidin or streptavidin and to detect the captured protein using an unlabeled secondary antibody. A method of quantifying protein by amplifying a signal.

In one embodiment, the present invention provides a method for forming a self-assembled monolayer on a surface of a metal thin film of a surface plasmon resonance (SPR) chip; (2) biotinylating the self-assembled monolayer of the SPR chip and binding it to avidin or streptavidin; (3) immobilizing avidin or streptavidin of the SPR chip with the primary antibody specific for the target protein and biotinylated; (4) reacting the immobilized antibody of the SPR chip with a biological sample comprising the target protein; (5) reacting the SPR chip with a secondary antibody specific and unlabeled for the target protein; And (6) calculating a target protein amount by measuring a change in refractive index of a material immobilized on the surface of the metal thin film of the SPR chip, and a method for quantifying the protein by amplifying the SPR signal by a sandwich analysis method. .

SPR is a method of sensitively measuring the change in refractive index of a metal thin film. When a biochemical such as a protein is bound to a metal surface, SPR can detect a changed amount and quantify a protein. As the metal exhibiting such a phenomenon, metals having an easy dielectric emission and negative dielectric constant are mainly used, such as gold, silver, copper, aluminum, and the like. Among them, silver showing the sharpest SPR resonance peak and gold showing excellent surface stability are commonly used. The resonance angle at which surface plasmon resonance occurs, that is, the angle at which the reflected light is minimized, results in an effective refractive index that changes as the mass of the metal thin film surface layer dielectric increases or the structure changes, resulting in a change in the resonance angle. Therefore, using the SPR principle, which can measure the change of these materials in an optical way, biochemical reactions, such as selective bonding or separation between various biochemicals, can be transformed into resonance angles through appropriate chemical modification of the metal thin film surface layer. Sensitive SPR sensors can be used as highly sensitive biochemical sensors. SPR has the advantage of directly measuring the binding of a biological sample without a separate signal amplification, but when measuring a very small amount of biological sample requires a separate signal amplification technology.

The present invention utilizes a sandwich assay using two antibodies to amplify the SPR signal.

In the present invention, the term "primary antibody" is an antibody immobilized in a fixed orientation through a self-assembled monolayer and avidin or streptavidin on the surface of the metal thin film of the SPR chip, capture the target protein (capture) do.

SAM's reactor on the SPR surface will have non-specific and random binding with reactors present at various sites of the primary antibody. We controlled the orientation of the antibody by binding the primary antibody with the reactor of SAM via avidin-biotin or streptavidin-biotin. Biotin can specifically bind to the reactor of the antibody, eg, the primary amino group of the antibody, and avidin or streptavidin can have a constant orientation since it has four binding sites for biotin.

In the present invention, the term "secondary antibody" is an antibody that serves to detect a target protein trapped in the primary antibody, the refractive index changes when using the secondary antibody than when not using the secondary antibody Will increase. Therefore, when a target protein is detected using a secondary antibody, the SPR signal can be amplified and a small amount of protein can be detected with high sensitivity.

The studies for amplifying the SPR signal before the present invention are as follows. Korean Patent Publication No. 2005-00581854 discloses an SPR signal by capturing a protein to be analyzed as a primary antibody, binding it to a secondary antibody, binding an enzyme such as HRP, and adding a substance that reacts with the enzyme to form a precipitate on the chip surface. Amplification method is described. WO 01/09388 describes a method for capturing analytes, such as proteins, with primary antibodies and amplifying SPR signals using colloidal metal nanoparticles conjugated with secondary antibodies. The present invention directly measures the change in SPR refractive index due to the binding of the target protein with its specific secondary antibody binding, but the prior patents differ from the present invention in that they form a precipitate or amplify the SPR signal using metal nanoparticles. There is. In addition, the prior art has a disadvantage that the procedure is complicated and time-consuming compared to the present invention, in particular, the method of amplifying the SPR signal using the metal nanoparticles, when binding the secondary antibody to the metal nanoparticles The binding strength of the secondary antibody that binds to the target protein can be weakened, the binding specificity of the metal nanoparticles and non-specific adsorption occurs with the protein other than the target protein has a problem of low reliability of the result.

However, the present invention can easily quantify even a small amount of protein by overcoming the problems of the prior patent. The inventors examined the extent to which the SPR signal was amplified using the PSA-ACT antigen. As a result of analyzing the PSA-ACT antigen by each concentration using the SPR biosensor, the SPR signal value increased as the concentration of PSA-ACT increased only when the antigen was injected (a-c section in FIG. 2). Injecting 1 ng / ml of antigen concentration into a flow cell yields an RU value of 8.5, 14 RU at 10 ng / ml, 28 RU at 100 ng / ml, 64 RU at 500 ng / ml, and 112 RU at 1000 ng / ml Had In the SPR system, when the RU value is more than three times that of the blank sample (the sample without the antigen added), the detection limit value can be defined. Therefore, 21 RU is three times the RU value of 7 when only the blank sample is flowed. The results provide only results that are within the range of the above protein concentration. Therefore, it is difficult to analyze the PSA-ACT protein having a concentration of 100 ng / ml or less based on the immune response of the antigen-antibody alone.

However, when PSA polyclonal antibody was injected after formation of the PSA-ACT antigen and PSA-ACT monoclonal antibody complex, 78 RU at 1 ng / ml PSA-ACT antigen concentration and 87 RU at 100 ng / ml concentration, 100 ng 128 RU at a concentration of / ml, 345 RU at a concentration of 500 ng / ml, and 615 RU at a concentration of 1000 ng / ml (CD section in FIG. 2). When not reacted with the secondary PSA-ACT antibody, the detection limit was up to 100 ng / ml, but when reacted with the secondary PSA-ACT antibody, it was detectable up to 1 ng / ml of PSA-ACT. Thus, the protein of the present invention can be easily quantitated even in trace amounts at a level of 1 ng / ml.

In addition, the self-assembled monolayer of the SPR chip of the present invention is characterized by having a carboxyl group (-COOH) (Receptor) and a hydroxyl group (-OH) (Spacer). When SAM is reformed into the reactor, nonspecific adsorption of SAM surface and proteins in the sample is minimized when biological sample is injected into the SPR chip. Thus, under conditions where nonspecific adsorption between SAM and protein is minimized, only the target protein can be specifically bound by the primary antibody and amplified by the secondary antibody to accurately quantify the target protein.

Looking at each step of quantifying the protein by amplifying the SPR signal using the two antibodies of the present invention in detail.

First, using a primary antibody immobilized on the surface of the metal thin film of the SPR chip to capture the target protein to be analyzed, corresponding to the first to third steps.

The first step is to form a self-assembled monolayer on the surface of the metal thin film of the surface plasmon resonance (SPR) chip.

As used herein, the term "self assembled monolayer (SAM)" refers to a regularly ordered organic molecular film produced on the surface of the SPR chip, preferably a carboxyl group (-COOH) and a hydroxyl group ( -OH) In a specific embodiment, an EG6 oligomer whose end group is substituted with a carboxyl group (-COOH) HS (CH2) 11 (OH2CH2) 6OCH2COOH and an EG3 oligomer whose end group is substituted with a hydroxyl group (-OH) HS (CH 2) 11 (OH 2 CH 2) 3 OH is mixed in an ethanol solution in an appropriate ratio, preferably 1:20, and more preferably in a ratio of 1 to 10 to form a self-assembled thin layer having a carboxyl group and a hydroxyl group on the SPR sensor chip. To manufacture.

The second step is to biotinylate the SAM of the SPR chip and bind it with avidin or streptavidin. More specifically, the carboxyl groups of the self-assembled monolayer are biotin hydrazide and EDC (1-ethyl-3- (3-dimethylaminopropyl)- carbodiimide hydrochloride) can be used for biotinylation. EDC interacts with the carboxyl group to make the carboxyl group reactive with the amino group of hydrazide. Thereafter, avidin or streptavidin solution is injected into the flow cell of the SPR system and fixed to the SAM.

The third step is the immobilization of avidin or streptavidin of the SPR chip with the primary antibody specific for the target protein and biotinylated.

To biotinylate only a specific region of the primary antibody, the lysine residues or N-terminal primary amine groups (-NH) of the protein are biotinylated. Biotinylation of proteins can be achieved using NHS-ester of biotin, and sulfo-NHS-biotin was used in specific embodiments of the present invention. Biotinylated primary antibodies are injected into the flow cell of the SPR system to immobilize with avidin or streptavidin.

Next, the captured target protein is amplified using a secondary antibody to quantify the protein, which corresponds to the fourth to sixth steps.

Step 4 is to react the immobilized antibody of the SPR chip with a biological sample containing the target protein.

In the present invention, the term "biological sample" refers to a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, which the target protein to be analyzed contains. The process of separating proteins from biological samples is carried out using known processes.

The fifth step is to react the SPR chip with a secondary antibody that is specific and unlabeled for the target protein.

The sixth step is to calculate the amount of the target protein by measuring the change in the refractive index of the material immobilized on the surface of the metal thin film of the SPR chip.

The concentration of a target protein in a biological sample of unknown concentration can be determined by measuring the RU value of a standard sample which knows the concentration of the protein. The concentration-specific RU values obtained by regression analysis of the RU values of the standard sample are converted into a function of linear relevance. This can be obtained through a general program, and the slope and intercept of the equation can be estimated by the method of least square over n data pairs. The concentration of the analyte can be calculated by substituting the RU value of an unknown (unknown concentration) sample into a fitted regression equation obtained in this way.

Without effective amplification of the SPR signal, detection and quantification of proteins cannot be made significant. For example, the prostate diagnostic marker PSA protein is diagnosed as prostate cancer when it is normal and above when it has a value of 4 ng / ml or less. Although it cannot be analyzed, amplification of the SPR signal by the method of the present invention enables protein analysis and further diagnoses cancer.

Therefore, the method of the present invention can effectively amplify the SPR signal, it can be applied to a variety of disease diagnostic sensors, proteomics research, etc. that must analyze a trace amount of protein.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

< Example  1> Primary Response Measurement of Protein PSA-ACT Complex

<1-1> Formation of Self-assembled Monolayer Using Ethylene Glycol on Gold Thin Film Surface

EG ₆ oligomer HS (CH ₂ ) ₁₁ (OH ₂ CH ₂ ) ₆ OCH ₂ COOH (Cosbiotech, Korea) and terminal group whose terminal group is substituted with carboxyl group (-COOH) for antibody immobilization of gold thin film chip SPR by mixing EG ₃ oligomer HS (CH ₂ ) ₁₁ (OH ₂ CH ₂ ) ₃ OH (Cosbiotech, Korea) substituted with a hydroxyl group (-OH) in a 0.5 mM pure ethanol solution at a ratio of 1: 9. A self-assembled thin film was formed on the sensor chip. And the residue that was not immobilized on the surface of the thin film was washed sequentially with ultra pure distilled water (18.2 mΩ / cm) and pure ethanol.

<1-2> Biotinylation of Carboxyl Group and PSA-ACT Antibody

Biotin was synthesized at the EG end carboxyl group to have even orientation of Streptavidin (Sigma Aldrich, USA). Biotinylation of self-assembled thin films with terminal groups on the solid surface is carried out in a similar manner as in solution (O'Shannessey, DJ, Quarles, RH, J. Immunol. Methods 99: 153- 161. (1987)). First, the prepared surface was washed with 0.1 M MES (2-2-morpholinoethane sulfonic acid) (Sigma Aldrich, USA) buffer (pH = 4.7-5.5). And 65 μl of 100 mg / ml EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) (Uppsala, Sweden) and biotin hydrazide (Rockford, IL, USA) to activate the carboxyl group and conjugate the biotin (50 mM) was soaked for 12 hours in a solution. At this time the temperature was room temperature and slightly shaken. Then, the reacted chip surface was dried with high purity nitrogen gas.

Also, using the EZ-link sulfo-NHS-LC-Biotinylation Kit (Rockford, Il, USA)) to biotinylate monoclonal PSA-ACT antibodies (Emeryville, CA, USA) to be immobilized to streptavidin antibodies, The protocol was performed sequentially.

<1-3> Immobilization of Streptavidin and PSA-ACT Monoclonal Antibodies Using SPR Sensor

Chips performed up to this procedure were mounted in an SPR biosensor system (BIACORE 2000). The flow rate of the solution in this procedure was carried out at 5 μl / ml, all experiments were carried out at a temperature of 25 ℃. Streptavidin solution (dissolved in HBS buffer at 20 μg / ml, pH = 7.4) was injected into flow cell 2 (Fc2) of the SPR system for 7 minutes for streptavidin immobilization. In addition, the injection of streptavidin was performed once more to maximize process efficiency. Fc1 was used as a reference cell to see a more accurate response signal. The SPR signal was obtained by subtracting Fc1 from Fc2. After immobilization of streptavidin, unreacted surfaces were flowed for 7 minutes using 1M ethanolamine-HCl (Uppsala, Sweden) to reduce nonspecific adsorption.

Next, biotinylated PSA-ACT monoclonal antibody (20 mg / ml in HBS buffer) was flowed on the surface of the SPR sensor chip for about 7 minutes. Afterwards, to wash the biotinylated PSA-ACT monoclonal antibody that is not immobilized to streptavidin, a mixed solution of 50 mM NaOH solution and 1 M NaCl solution was flowed for 2 minutes. At the end of the experimental procedure, an SPR sensor chip capable of detecting PSA, an indicator of prostate cancer, was completed.

<1-4> Measurement of PSA-ACT Antigen

In this procedure, the flow rate of all solutions was maintained at 5 μl / min. PSA-ACT antigen (Emeryville, CA, USA) was added to HBS at pH 7.4 (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) (Uppsala, Sweden) buffer 1, 10, 100, 500, Diluted at a concentration of 1000 ng / ml, and injected into Fc2 for 2 minutes, respectively, and immobilized using the antigen-antibody reaction. In this experiment, injection of 0 ng / ml PSA-ACT analyte meant that only HBS buffer was flushed to capture the blank. In order to reuse the chip after measuring the immune response, the mixed solution of 50 mM NaOH and 1 M NaCl was flowed into the sensor chip for 2 minutes, and the chip was used again.

As a result of analyzing the PSA-ACT antigen by each concentration using the SPR biosensor, as shown in the ac section of Figure 2, it can be seen that the SPR signal value increases as the PSA-ACT concentration increases when only the antigen is injected. there was. When 1 ng / ml of PSA-ACT antigen was flowed into the flow cell, the RU value was 8.5, 14 RU at 10 ng / ml concentration, 28 RU at 100 ng / ml, 64 RU at 500 ng / ml, and 1000 ng / In ml, a value of 112 RU was obtained. However, according to BIACORE, the SPR system defined that the RU value should be more than three times that of the blank sample (the sample without the antigen added). As a result of the experiment, when the blank sample is only flowed, the value of RU is 7, so that the value of 21 RU should be more than tripled. Therefore, it was confirmed that the detection limit when only the antigen was flowed was 100 ng / ml.

< Example  2> Secondary Reaction Measurement of Protein PSA-ACT Complex Using Unlabeled Secondary Sandwich Technique

The immune response of the antigen-antibody alone could not analyze PSA-ACT proteins with concentrations below 100 ng / ml. These detection limits reveal serious limitations in disease diagnosis using SPR sensors. For example, a PSA protein that is elevated when prostate cancer develops has a normal range of 4 ng / ml or less and can be diagnosed as prostate cancer when it is over, with a detection limit of 100 ng / ml or less. This reveals a serious problem that cannot be detected even if the cancer has actually advanced. Another cancer marker, AFP (alpha-fetoprotein, liver cancer), has a normal range of 15 ng / ml or less, and other cancer markers have a normal range of 100 ng / ml or less. To make a diagnosis, the detection limit must be lowered.

Therefore, in order to solve the above problems, a method for amplifying the SPR signal by reacting the secondary antibody, which is unlabeled in the SPR sensor system, with the antigen again.

PSA polyclonal antibody (20 μg / ml in HBS buffer) (Emeryville, CA, USA) to reduce the detection limit by amplifying SPR signal values after the immune reaction between the PSA-ACT antigen and the PSA-ACT monoclonal antibody. Was injected into Fc2 at a flow rate of 10 μl / min for 2 minutes.

As a result, as shown in the c-d section of Figure 2, it can be seen that the value of the SPR signal is much amplified when the secondary antibody is reacted again than when only the antigen flowed. The unlabeled secondary PSA polyclonal antibody was given 78 RU for the chip immobilized with 1 ng / ml of PSA-ACT antigen concentration, and 87 RU for 100 ng / ml for 10 ng / ml. 128 RU, 500 ng / ml was 345 RU, and finally 1000 ng / ml was 615 RU. This was much higher than the RU value (7 RU) seen when a blank sample was inserted.

3 also shows the linear range of the immunological assay, which is based on the SPR signal value using the sandwich technique of immobilizing the secondary antibody back to the reacted antigen-antibody rather than the slope of the SPR signal value that reacted only with the antigen and antibody (0.1013). The slope (0.5377) was confirmed to be about 5 times higher.

< Example  3> SPR  Nonspecific binding assay on chip

It was tested how much non-specific adsorption of proteins other than the PSA antigen occurs in the chip prepared by the method of Example 1. Fibrinogen (Sigma-Aldrich, USA), IgG (Sigma-Aldrich, USA) and BSA (Sigma-Aldrich, USA) were each 20 μg / ml on the surface of the biotinylated PSA antibody immobilized on streptavidin. The amount was poured and measured with an SPR sensor. As a result, as shown in FIG. 4, the RU value of fibrinogen was measured as 20.5, the RU value of IgG was 13.7, and the RU value of BSA was 12.9. These values are almost negligible, much smaller than when the target protein, PSA antigen, was given away.

Therefore, when the surface of the SPR chip was modified using EG-COOH (Receptor) and EG-OH (Spacer), nonspecific adsorption with the protein could be minimized.

As described above, the measurement method of the present invention can amplify the SPR signal by using a sandwich technique using an unlabeled antibody, and can be applied to various diseases such as disease diagnosis sensors and proteomics studies that require analysis of trace proteins. Can be used in a way.

Claims (5)

(1) forming a self-assembled monolayer on the surface of the metal thin film of the surface plasmon resonance (SPR) chip; (2) biotinylating the self-assembled monolayer of the SPR chip and binding it to avidin or streptavidin; (3) immobilizing avidin or streptavidin of the SPR chip with the primary antibody specific for the target protein and biotinylated; (4) reacting the immobilized antibody of the SPR chip with a biological sample comprising the target protein; (5) reacting the SPR chip with a secondary antibody specific and unlabeled for the target protein; And (6) calculating the amount of the target protein by measuring the change in refractive index for the material immobilized on the metal thin film surface of the SPR chip; How to quantify protein by amplifying SPR signal by sandwich assay. The method of claim 1 wherein the metal is selected from the group consisting of gold, silver, copper and aluminum. The method of claim 1 wherein the self-assembled monolayer has a carboxyl group (-COOH) and a hydroxyl group (-OH). The method of claim 1 wherein the concentration of the target protein is between 1 ng / ml and 1000 ng / ml. The method of claim 1, wherein the biological sample is tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

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