KR100722844B1 - Methods and kits for in situ measurement of enzyme activity and amount using single measurement system - Google Patents

Abstract

The present invention provides phosphorus for measuring the activity and amount of enzyme in a sample in a single measurement system. A method and kit for measuring in situ , the method of the present invention comprising the steps of: (a) contacting a sample with a capturing agent that is capable of binding to the enzyme of interest and immobilized on a solid phase matrix; (b) measuring the activity of the enzyme of the assay bound to the capture agent; (c) contacting a detection antibody specific for the enzyme of interest to which the capture agent is bound; And (d) detecting the antigen-antibody complex formed in step (c). According to the present invention, in measuring the activity and amount of the enzyme, there is no need for a separate measurement system. In addition, according to the present invention, the sample measuring the activity and the amount is the same, and the activity and the amount of the enzyme is measured in the same system, it is measured in such a way that there is almost no gap in time, precisely the activity and amount of the enzyme Can be obtained at the same time.

Enzyme, active, amount, in situ, simultaneous, measurement

Description

Methods and Kits for In Situ Measurement of Enzyme Activity and Amount Using Single Measurement System}

1A is a concentration standard curve of glutathione peroxidase 1 (GPx1) measured using the kit of the present invention.

Figure 1b shows the results of measuring the activity of glutathione peroxidase 1 (GPx1) in erythrocytes according to the present invention. Hemolysates of various dilutions were used as samples.

Figure 1c shows the results of measuring the amount of GPx1 in red blood cells in accordance with the present invention. Hemolysates of various dilutions were used as samples.

Figure 2a is a concentration standard curve of thioredoxin 1 (Trx 1) measured using the kit of the present invention.

Figure 2b shows the results of measuring the activity of thioredoxin 1 (Trx 1) in the sample according to the present invention.

Figure 2c shows the results of measuring the amount of Trx1 in the sample according to the present invention.

The present invention provides phosphorus for measuring the activity and amount of enzyme in a sample in a single measurement system. A method and kit for in situ measurement are provided.

Determination of the activity and amount of enzymes is an essential experimental procedure in the biotechnology field of enzymes. Many studies have already been made on how to measure the activity and amount of enzymes.

For example, as a method of measuring the amount of enzymes, a biuret reaction method (Itzhaki, RF et al., Anal. Biochem . 9: 401-410 (1964)), a lowry reaction method (Lowry, OH et al., J) ... Biol Chem 193: 265-275 (1951)), UV absorbance measuring method (Warburg, O. et al, Biochem Z. 310:.. 384-421 (1941)) and a die-bonding method (Bradford, MM et al, Anal Biochem 72:... there is a well-known, such as 248-254 (1976)).

Methods for measuring the activity of enzymes are roughly divided into stopped methods and continuous methods, and continuous methods include uncoupling methods and coupling methods. Methods for measuring enzyme activity are described in Robert K. Scopes, Protein Purification , 2nd ed. Springer-Verlag New York Inc. (1988); Crabtree, B. et al., In Techniques in the life sciences , Vol. B211, pp. 1-37, Elsevier / North-Holland, Amsterdam. (1079); Scopes, RK Anal. Biochem. 49: 73-87 (1972); And Garcia-Carmona, F. et al., Anal. Biochem . 113: 286-291 (1981).

However, according to the enzyme analysis method developed so far, the activity and amount of the enzyme are measured in different assay systems. According to this conventional technique, since the activity measurement and the measurement of the amount of the enzyme are carried out independently, not only the identity of the sample to be measured is difficult to be ensured, but also a large amount of the sample is required for such a measurement. In addition, according to the prior art, the measurement procedure is cumbersome and time consuming due to the two operations. In addition, it was not clear how to distinguish the amount of protein with and without enzyme activity.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors developed a research efforts result, in-situ (in situ) measurement method capable of measuring the activity and amount of enzyme in a single measurement system (single measurement system) in order to address the old need in the art are, for the present invention It was completed.

It is therefore an object of this invention to provide a a-situ (in situ) measurement method for measuring the activity and amount of enzyme sample in a single measurement system.

Another object of the present invention is to provide an in situ measurement kit for measuring the activity and amount of enzyme in a sample in a single measurement system.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides an in situ measurement method for measuring the activity and amount of an enzyme in a sample in a single measurement system comprising the following steps: (a) Binding capacity to the enzyme of interest Contacting the sample with a capturing agent which is present and immobilized on a solid matrix; (b) measuring the activity of the enzyme of the assay bound to the capture agent; (c) contacting a detection antibody specific for the enzyme of interest to which the capture agent is bound; And (d) detecting the antigen-antibody complex formed in step (c).

The present invention relates to in-situ (in situ) measurement method capable of measuring the activity and amount of enzyme in a single measurement system (single measurement system).

The term “ in situ ” as used herein referring to a measurement method means measuring both the activity and the amount of enzyme while using the same enzyme immobilized on the capture agent in a single measurement system. That is, according to the present invention, the enzyme of the analysis target may be in situ measurement method because it maintains the same phase during the measurement process. In addition, the method of the present invention can also be expressed as a method (simultaneous quantitating method) for simultaneously measuring the activity and the amount of the enzyme.

The term “single measurement system” means, for example, when using a matrix (eg, a microplate) in which a capture agent is bound on a surface, as the reaction system, the substrate is used as it is when measuring the activity and amount of the enzyme.

Samples to be applied to the present invention are not particularly limited and include viruses, bacteria, cells or tissue-derived extracts, lysates or purified products, blood, plasma, serum, lymph, bone marrow fluid, saliva, ocular fluid, semen, Brain extracts, spinal fluid, joint fluid, thymic fluid, ascites or amniotic fluid, but are not limited thereto. Preferably, the sample applied to the present invention is a virus, bacteria, cell or tissue-derived extract, lysate or purified product, blood, plasma, serum or lymph, most preferably a virus, bacteria, cell or Tissue-derived extract or lysate (eg erythrocyte lysate).

The two most important elements in the measurement system of the present invention are the capturing agent and the detection antibody.

As used herein, the term “capture agent” refers to a substance that captures and immobilizes an enzyme to be analyzed. The trapping agent may be any substance as long as it has a binding affinity for the enzyme to be analyzed, a substance having specificity or no specificity to the enzyme to be analyzed, and preferably a substance having both affinity and specificity. to be. Non-limiting examples of capture agents include antibodies, receptors, streptavidin (or avidin), aptamers, lectins, DNA, RNA, ligands, coenzymes, inorganic ions, cofactors ), Sugars, lipids or substrates. Preferably, the capture agents are antibodies, streptavidin (or avidin) and aptamers, more preferably antibodies, even more preferably antibodies having both binding capacity and specificity for the enzyme, most preferably the enzyme It is a monoclonal antibody having both binding capacity and specificity for.

In the present invention, the capture agent is immobilized on the surface of the solid matrix. Available as matrices to which the capture agent is immobilized can be any conventionally used in the art, including but not limited to hydrocarbon polymers such as polystyrene and polypropylene, glass, metals and gels. The matrix of the solid phase is provided in the form of dipsticks, microtiter plates, particles (eg beads), affinity columns and immunoblot membranes (eg polyvinylidene fluoride membranes), protein chips or test tubes, cuvettes. (See US Pat. Nos. 5,143,825, 5,374,530, 4,908,305 and 5,498,551). Most preferably, the solid matrix is a microtiter plate.

In the present invention, the analyte is an enzyme. The enzyme is not particularly limited and includes, but is not limited to, redox enzymes, transferases, hydrolases, lyases, isomerases, and ligases.

According to the present invention, a sample requiring measurement of the activity and amount of an enzyme to be analyzed is contacted with a capture agent to bind the enzyme to the capture agent. Samples may be purified and purified, but in the present invention, even if a biological sample (eg, cell-derived extract or lysate) is directly reacted with the capture agent without purification, the activity and amount of the enzyme can be accurately measured.

When the enzyme of interest is bound to the capture agent, the activity of the enzyme is shown in step (b). Determination of the activity of the enzyme bound to the capture agent can be carried out in a protocol suitable for the type of enzyme according to various enzyme activity measurement methods known in the art (see Robert K. Scopes, Protein Purification , 2nd ed. Springer-). Verlag New York Inc. (1988). Basically, enzyme activity is measured by measuring the amount of product produced by treating a substrate with an enzyme bound to the capture agent. Enzyme activity measurement methods include largely stopped methods and continuous methods, and continuous methods include uncoupling methods and coupling methods.

Determination of enzyme activity includes, for example, (i) a direct or indirect assay using various indicators, and (ii) a coupling assay using indicator enzymes and indicators.

In the method of (i), the indicator compound is reacted directly or indirectly with the enzyme reaction product. For example, a method of detecting fatty acids using Rhodamine B fluorescent staining agent in lipase assay (see US Patent No. 5,914,245), protons, electrons, ion transfers and pH changes generated during enzymatic reactions can be used as staining agents. Sein, Oregon Green, Rhodol, Thymol Blue, Methyl Orange, Bromocresol Blue), and the like.

The activity of an enzyme that does not produce a product of color development or fluorescence can be carried out by coupling an indicator with an additional enzyme that produces an observable product. In addition, coupling assay methods can be used to amplify the activity of the analyte. For example, even in the case of an enzyme that produces a chromogenic product such as NADH or NADPH, a coupling enzyme is used to generate a more chromogenic product (e.g., formazan) and detect the same. Enzyme activity is measured.

According to a specific embodiment of the present invention, the activity of glutathione peroxidase is measured in a coupling manner. Indirectly by reducing the oxidized glutathione (GSSG) produced by the catalysis of glutathione peroxidase with a coupling enzyme glutathione reductase and measuring the absorbance of NADP ⁺ formed in this process at 340 nm using a spectrophotometer. The activity of glutathione peroxidase of the analyte is measured.

The activity of the enzyme of interest bound to the capture agent is measured and then the amount of enzyme is measured in that system, ie in situ . Substances used to measure the activity of the enzyme are removed from the measurement system (preferably by washing) and then contacted with a detection antibody specific for the enzyme of interest bound to the capture agent. , Antigen-antibody complexes are formed. The amount of antigen-antibody complex formed is then measured.

The detection antibody used in the present invention is an antibody showing binding specificity for the enzyme to be analyzed, and both polyclonal antibodies and monoclonal antibodies can be used, preferably monoclonal antibodies. When an antibody is used as the capture agent, the detection antibody should be an antibody that acts on an epitope different from the antibody as the capture agent.

Enzyme-specific antibodies can be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816, 56) or phage antibody library method (Clackson et al, Nature , 352: 624-628 (1991); and Marks et al, J. Mol . Biol . , 222: 58, 1-597 (1991)). Can be. General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting an enzyme antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

The detection or measurement of antigen-antibody complex formation can be carried out by directly detecting the antigen-antibody formed, but preferably by using a label that generates a detectable signal. According to a preferred embodiment of the present invention, the detection antibody is bound to a label that generates a detectable signal or to an affinity substance. The label may include enzymes (eg alkaline phosphatase, β-galactosidase, horseradish peroxidase, β-glucosidase and cytochrome P ₄₅₀ ), radioactive substances (eg, C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET). In order to facilitate detection, affinity substances that can be bound to the detection antibody include biotin. Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

When a radioisotope is used as a label binding to a detection antibody, the antigen-antibody complex formed in the final process of the present invention can be detected by measuring radioactivity.

When an enzyme catalyzing a color reaction is used as a label binding to a detection antibody, the substrate used for the enzymatic reaction can be added to the measurement system of the present invention to measure the reaction product to detect the antigen-antibody complex. For example, when alkaline phosphatase is used as a label, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS-B1) as substrates chromogenic reaction substrates such as -phosphate) and enhanced chemifluorescence (ECF) can be used, and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) when horseradish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine) Substrates such as ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and o -phenylenediamine (OPD) can be used.

When an affinity substance is bound to a detection antibody, the antigen-antibody complex is detected using a substance bound to a label (eg, a color reaction-catalyst) that generates a detectable signal to a partner that binds the affinity substance. can do. For example, in the case of using a biotin-coupled detection antibody, a streptavidin-coupled color reaction-catalyst (eg, horseradish peroxidase) is treated to the antigen-antibody complex, and then the color reaction substrate is reacted. By detecting the resulting signal, the antigen-antibody complex is detected.

According to a preferred embodiment of the present invention, the method of the present invention is used for the analysis of enzymes catalyzing the redox reaction. Specific examples of the redox enzymes of the present subject matter include glutathione peroxidase and thioredoxin.

According to a preferred embodiment of the present invention, the method of the present invention is carried out to determine the activity and amount of glutathione peroxidase, more preferably glutathione peroxidase 1 (GPx1), most preferably human glutathione peroxidase 1.

Oxidative stress is associated with the pathogenesis of aging and various diseases. Oxidative stress can be characterized by an imbalance between the production and destruction of free radicals such as superoxide anions, hydrogen peroxide, lipid peroxides and peroxynitrite. Inactivation of reactive oxygen species by enzymes is mainly accomplished by glutathione peroxidase, superoxide dismutase and catalase (1). The extent of damage by reactive oxygen species is generally associated with an increase or decrease in one or more free radical scavenging enzymes, one of which is glutathione peroxidase (GPx). In mammalian cells, glutathione peroxidase builds a major antioxidant defense system (2, 3). In various diseases, various amounts of GPx are observed in tissues. In mice, the lack of glutathione peroxidase 1 (GPx1) results in abnormal vascular and cardiac function and structure (4). GPx1 activity has been identified in liver, lung, platelets and erythrocytes (5, 6).

According to one specific embodiment of the present invention for measuring the activity and amount of glutathione peroxidase (preferably GPx1), the present invention is carried out as follows.

First, a sample (preferably erythrocyte debris) is contacted with a glutathione peroxidase-specific antibody (trapping agent) coated on the surface of the microtiter plate. The activity of glutathione peroxidase bound to the capture agent is then measured.

The activity of glutathione peroxidase is preferably carried out according to the coupling assay method. By reducing the oxidized glutathione (GSSG) produced by the catalysis of glutathione peroxidase with the coupling enzyme glutathione reductase and measuring the absorbance of NADP ⁺ formed in this process at 340 nm using a spectrophotometer, Indirectly, the activity of glutathione peroxidase of the analyte is measured. The rate of absorbance reduction at 340 nm directly indicates the activity of glutathione peroxidase in the sample. The amount of glutathione peroxidase is then measured by washing the microtiter plate, treating the detection antibody and then detecting the antigen-antibody complexes formed.

According to a preferred embodiment of the present invention, the method of the present invention is carried out to determine the activity and amount of thioredoxin, more preferably thioredoxin 1 (Trx 1), most preferably human thioredoxin 1 .

According to one specific embodiment of the present invention for measuring the activity and amount of thioredoxin, the present invention is carried out as follows.

First, the sample is contacted with a thioredoxin-specific antibody (trapping agent) coated on the surface of the microtiter plate. Then, the activity of thioredoxin bound to the capture agent is measured.

The activity of thioredoxin is preferably performed according to an uncoupling assay method. For example, in the presence of a reducing agent (reduced type dithiothreitol (DTT)), a sample (a thioredoxin activity assay subject) and a protein which can be reduced by thioredoxin are reacted to form a reduced protein. Subsequently, the activity of thioredoxin is analyzed by measuring the absorbance of the reduced protein at 650 nm. The microtiter plate is then washed, treated with a detection antibody and the amount of thioredoxin in the sample is measured by detecting the antigen-antibody complexes formed.

According to another aspect of the invention, the invention provides an in situ measurement kit for measuring in a same measurement system the activity and amount of an enzyme in a sample comprising: (i) a solid substrate, ( Ii) a capture agent immobilized on the substrate and having a binding capacity to the enzyme of interest, (iii) a detection antibody that specifically binds to the substrate of the enzyme of interest, and (iii) the enzyme of interest.

The kit of the present invention is intended to implement the method of the present invention, the content in common between the two is omitted in order to avoid excessive complexity of the present specification.

When the kit of the present invention is prepared for carrying out the activity of the enzyme in a coupling assay mode, the kit of the present invention may further include a coupling enzyme and a substrate for the coupling reaction. When a color reaction reaction-catalyst enzyme is bound to the detection antibody, the kit of the present invention may further include a substrate of the enzyme. When avidin is bound to the detection antibody, the kit of the present invention may further include streptavidin (or avidin) to which a color reaction-catalyst enzyme is bound. Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

According to the prior art, a separate measurement system was used to measure the activity and the amount of the enzyme. However, according to the present invention, the activity and amount of enzyme can be measured in situ (or simultaneously) in a single measurement system. That is, according to the present invention, in measuring the activity and amount of the enzyme, there is no need for a separate measurement system. In addition, if the activity and amount of the enzyme is measured by a separate system as in the prior art, since the activity and amount of the enzyme are measured in a spatially and temporally spaced manner, it is strictly a measurement value for the same sample and enzyme. Can not. However, according to the present invention, the sample measuring the activity and the amount is the same, and the activity and the amount of the enzyme is measured in the same system, and also measured in such a manner that there is almost no interval in time, precisely the activity and amount of the enzyme Can be obtained at the same time. In addition, according to the method of the present invention, the convenience and accuracy of the measurement of the enzyme can be achieved with only a small amount of the sample.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example  One

Experimental Materials and Methods

Experimental material

UV-spectrophotometer (Molecular device) and deionized water system (Mirae ST) were used. β-NADPH (Sigma N7505); Glutathione peroxidase (Lab Frontier, Cat. No. LF-P0071); Reduced glutathione (Sigma G4251); t -butylated hydroperoxide ( t- BHP) (Sigma B2633); Na ₂ EDTA (USB) was used. 0.1 M Tris buffer / 1 mM EDTA (pH 7.0) as the diluent solvent and 1 mM t- BHP were used as the initiator.

Preparation of red blood cells

Blood was collected using EDTA as an anticoagulant, the blood was centrifuged at 3,000 rpm for 10 minutes at 4 ° C. to spin-down red blood cells, and then buffy coat (red blood cell-derived material between the precipitated red blood cells and plasma) was removed. . The erythrocyte precipitate was washed with 10 volumes of chilled brine at 4 ° C., then centrifuged at 4 ° C. for 10 minutes at 3,000 rpm and the brine at the top was removed. This process was repeated once. Erythrocytes were disrupted by adding 10 volumes of chilled deionized water to the obtained red blood cells. If the analysis was performed on the same day, the lysates were stored on ice, otherwise frozen and stored at -70 ° C. The concentration of lysate in erythrocytes was measured to determine the appropriate concentration fold.

LF- GPx1  activity ELISA Of kit  making

Based on a sandwich ELISA (enzyme-linked immunosorbent assay), the LF-GPx1 activity ELISA kit of the present invention was prepared to measure the activity and amount of glutathione peroxidase 1 (Gpx1) in situ . In a well of a 96-well plate, a monoclonal antibody against human GPx1 (Lepfrontier, Cat. No. LF-MA0090) was coated with a capture antibody. The kit includes a detection antibody, Labfrontier, Cat. No. Biotin-conjugated human GPx1-specific monoclonal antibodies of LF-MA0091 were included, horseradish peroxidase (HRP) -conjugated Immunopure Avidin (Pierce) and TMB (3,3,5,5-tetramethylbenzidine; Bio-Fx, TMBC- 1000-01). In addition, the kit of the present invention, NADPH (Sigma, N7505), GSH (Sigma, G4251), GSSG reductase (LabFrontier, LF-P00071, Yeast form) and T-butylated hydroperoxide for measuring the activity of Gpx1 Also included was an LF-GPx1 activity ELISA kit.

LF- GPx1  Erythrocytes using activity ELISA GPx1  Activity analysis

Measurement of glutathione peroxidase-like activity in this experiment was carried out by measuring the ability to catalyze the reaction between hydroperoxide and glutathione in aqueous buffer at physiological pH. The oxidation of GSH to GSSG was indirectly assessed by measuring spectrophotometer oxidation of NADPH in the presence of glutathione reductase. The reaction proceeded at room temperature in a Molecular device, and the absorbance was measured at 340 nm.

The reaction was carried out using the LF-GPx1 activity ELISA kit containing 100 mM Tris buffer (pH 7.0) described above. To the kit of the present invention, 5.5 μl of red cell lysate obtained in the above process was first added, followed by reaction at 25 ° C. by adding 0.2 mM NADPH, 1 mM GSH, 0.2 unit GSSG reductase, and 1 mM hydroperoxide. The decrease in absorbance at 340 nm was recorded over time. The reaction rate was evaluated at a time point where the decrease in absorbance was constant for at least 20 minutes. One unit of GPx1 is the amount of enzyme capable of oxidizing 1 nmole NADPH to NADP ⁺ at 25 ° C.

LF- GPx1  Erythrocytes using activity ELISA GPx1  dose

After completing the above activity measurements, the plates were washed with PBST to remove all substrates and GSSG reductase. Subsequently, 100 μl of the detection antibody biotin-conjugated human GPx1-specific monoclonal antibody (2 μg / ml) was treated and reacted at 37 ° C. for 60 minutes. The plates were then washed with PBST, treated with 100 μl of HRP-conjugated Immunopure Avidin (0.5 μg / ml), followed by 100 μl of TMB, and the reaction proceeded at 25 ° C. for 3 minutes to allow signals to come out. . 100 μl of 1 NH ₂ SO ₄ was treated to terminate the reaction, and the absorbance at 450 nm was measured. In order to calculate the concentration of GPx1 from the measured absorbance, a GPx1 concentration standard curve (0-100 ng / ml) was obtained (FIG. 1A).

Experiment result

1b and 1c show the results of measuring the activity and amount of GPx1 in erythrocytes using the LF-GPx1 activity ELISA kit of the present invention. Experiments were performed on hemolysates of various dilutions. Activity is expressed in mU / mg hemolyte. As can be seen in Figure 1b, the kit of the present invention shows a hemolyte concentration-dependently increased value in the hemolyte dilution range (36-fold dilution difference range) of 1: 4 to 1: 144, accurate analysis results You can see that it shows. Experimental results showed that GPx1 activity at dilution 1: 4, 1: 8, 1:16, 1:35, 1:71 and 1: 144 was 2.94 mU / mg, 2.49 mU / 0.5 mg, 1.79 mU / 0.25 mg, respectively. , 1.15 mU / 0.125 mg, 0.81 mU / 0.0625 mg and 0.65 mU / 0.03125 mg. In addition, the concentrations of GPx1 in the hemolytes of dilution 1: 4, 1: 8, 1:16, 1:35, 1:71 and 1: 144 were analyzed as 7.13, 6.97, 5.33, 3.25, 0.92 and 0.295 ng, respectively. It became. The hemolyte dilution, 1: 4, 1: 8, 1:16, 1:35, 1:71, and 1: 144 in FIG. 1C are 1 mg / 0.1 mL, 0.5 mg / 0.1 mL, 0.25 mg / 0.1, respectively. ML, 0.125 mg / 0.1 mL, 0.062 mg / 0.1 mL and 0.031 mg / 0.1 mL RBC hemolyte.

Example  2

Experimental Materials and Methods

Experimental material

UV-spectrophotometer (Molecular device) and deionized water system (Mirae ST) were used. Insulin (Sigma I5500), human thioredoxin (Rapfrontier, Cat.No. LF-P0001), and DTT (USB) were used. 50 mM Tris buffer / 1 mM EDTA (pH 7.5) as the diluting solvent and 1.6 mM dithiothreitol (DTT) as the initiator were used.

LF- Trx  1 activity ELISA

Based on the sandwich ELISA, the LF-Trx 1 activity ELISA kit of the present invention was prepared to measure the activity and amount of thioredoxin 1 (Trx 1) in situ . In a well of a 96-well plate, a monoclonal antibody against human Trx 1 (Lapfrontier, Cat. No. LF-MA0055) was coated with a capture antibody. The kit includes a detection antibody, Labfrontier, Cat. No. Biotin-conjugated human Trx 1-specific monoclonal antibodies of LF-MA0077 were included and HRP-conjugated Immunopure Avidin (Pierce) and TMB were included. In addition, the kit of the present invention includes TT-Trx1 activity by including DTT, insulin (Sigma, I5500) and recombinant human-thioredoxin (Lapfrontier, Cat.No. LF-P0001) for measuring the activity of Trx 1. ELISA kits were prepared.

LF- Trx  Recombinant human-using 1 activity ELISA Trx  1 activity analysis

Determination of thioredoxin-like activity in this experiment was carried out using a spectrophotometer on reduced insulin (Insulin- (SH) ₂ ) produced by catalyzing thioredoxin in the presence of reduced DTT at 650 nm. By measuring OD, the activity of thioredoxin is analyzed. The rate of OD increase at 650 nm directly indicates the activity of thioredoxin in the sample. Reaction progress was made at room temperature in a Molecular device, and OD was measured at 650 nm.

The reaction was carried out using the LF-Trx 1 activity ELISA kit with 50 mM Tris buffer (1 mM EDTA, pH 7.5). To the kit of the present invention, 100 μl of recombinant thioredoxin protein (from 5 ng serial dilution), 1.6 μm DTT and 200 μg of insulin were added and reacted at 25 ° C. The OD increase at 650 nm was recorded over time. Reaction rate evaluation was performed at a time point where the OD increase was constant for at least 30 minutes. The definition of 1 unit of Trx 1 is the amount that changes A ₆₅₀ by 1 per minute at 25 ° C. in the insulin reduction assay described above.

LF- Trx  Recombinant human-using 1 activity ELISA Trx  1 dose

After completing the activity measurements described above, the plates were washed with PBST to remove all substrates and DTT. Subsequently, 100 μl of the detection antibody biotin-conjugated human Trx 1-specific monoclonal antibody (0.5 μg / ml) was treated and reacted at 37 ° C. for 60 minutes. The plates were then washed with PBST, treated with 100 μl of HRP-conjugated Immunopure Avidin (0.5 μg / ml), followed by 100 μl of TMB, and the reaction proceeded at 25 ° C. for 3 minutes to allow signals to come out. . 100 μl of 1 NH ₂ SO ₄ was treated to terminate the reaction, and the absorbance at 450 nm was measured. In order to calculate the concentration of Trx 1 from the measured absorbance, a Trx 1 concentration standard curve (0-50 ng / ml) was obtained (FIG. 2A).

Experiment result

2b and 2c show the results of measuring the activity and amount of recombinant Trx 1 using the LF-Trx 1 activity ELISA kit of the present invention. Experiments were performed on recombinant Trx 1 at various concentrations. Activity is expressed in U / mg. As can be seen in Figure 2b, the kit of the present invention shows a concentration-dependently increased value in the range of recombinant Trx 1 dilution of 5 ng to 0.078 ng, showing that the accurate analysis results. As a result, the activity of Trx 1 at concentrations of 5, 2.5, 1.25, 0.625 and 0.3125 ng was analyzed to be 38, 56, 80, 112 and 128 U / mg, respectively. In addition, as can be seen in Figure 2c, as the concentration of recombinant Trx 1 increases O.D. The value also shows a pattern that increases proportionally.

Thus, it can be seen that the kit of the present invention can accurately measure activity and amount simultaneously in the same system.

As noted above, the present invention provides methods and kits for in situ determination of activity and amount of enzyme in a single measurement system. According to the present invention, in measuring the activity and amount of the enzyme, there is no need for a separate measurement system. In addition, according to the present invention, the sample measuring the activity and the amount is the same, and the activity and the amount of the enzyme is measured in the same system, it is measured in such a way that there is almost no gap in time, precisely the activity and amount of the enzyme Can be obtained at the same time. In addition, according to the present invention, it is possible to achieve the convenience and accuracy of the enzyme measurement with only a small amount of the sample.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Reference

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4.Forgione MA, Cap A, Liao R, Moldovan NI, Eberhardt RT, Lim CC, Jones J, Goldschmidt-Clermont PJ, Loscalzo J. Heterozygous cellularglutathione peroxidase deficiency in the mouse: abnormalities in vascular and cardiac function and structure. Circulation. 2002 Aug 27; 106 (9): 1154-8.

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Claims (6)

In-situ (in situ) measurement method for measuring the activity and amount of enzyme sample, comprising the steps of a single measurement system: (a) contacting the sample with a capturing agent capable of binding to the enzyme of interest and immobilized on a solid matrix; (b) measuring the activity of the enzyme of the assay bound to the capture agent; (c) contacting a detection antibody specific for the enzyme of interest to which the capture agent is bound; And (d) detecting the antigen-antibody complex formed in step (c). The method of claim 1, wherein the sample is a virus, bacteria, cell or tissue-derived extract, lysate or purified product, blood, plasma, serum, lymph, bone marrow fluid, saliva, ocular fluid, semen, brain extract, spinal fluid , Joint fluid, thymic fluid, ascites and amniotic fluid is selected from the group consisting of in situ measurement method. The method of claim 1, wherein the capture agent, antibody, receptor, streptavidin, avidin, aptamer, lectin, DNA, RNA, ligand, coenzyme, inorganic ion, cofactor, sugar in situ measurement method in that the, or lipid matrix according to claim. According to claim 1, wherein situ measurement method in which the capture agent is characterized in that the antibody. According to claim 1, wherein situ measurement method for the detection antibody, it is combined a label that generates a detectable signal, or wherein the affinity substance is bonded. The activity of the sample in the enzyme comprising the following and in situ assay kit the amount to measure in a single measurement system: (ⅰ) matrix, (ⅱ) the solid phase is immobilized on said matrix, and having a binding affinity for the enzyme of the analyte A detection antibody that specifically binds a capture agent, (iii) a substrate of the enzyme of interest, and (iii) an enzyme of interest.

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