KR20180004028A - Simultaneous analytical kit for diagnosis of disease using enzyme activity detection and immunological detection - Google Patents

Abstract

The present invention relates to a simultaneous analytical kit for diagnosis of diseases using enzyme activity detection and immunological detection, and specifically, to an analytical kit for identification of risk factors causing hemolytic anemia due to glucose-6-phsphate dehydrogenase (G6PD) activity deficiency and for checking whether a person is infected with malaria at the same time, wherein the simultaneous analytical kit of the present invention makes data through visual observation or image quantitative analysis for results of screening the G6PD activity deficiency to determine the activity level of G6PD, and simultaneously can also analyze malaria infection without additional sample preparation.

Description

Technical Field [0001] The present invention relates to a kit for simultaneous analysis of diseases using enzymatic activity measurement and immunological measurement,

The present invention relates to a kit for simultaneous diagnosis of disease diagnosis using enzyme activity measurement and immunological measurement, and a simultaneous assay method for disease diagnosis using enzyme activity measurement and immunological measurement, and more particularly, To a simultaneous assay kit and a simultaneous assay method capable of quickly and accurately screening and visualizing a specific enzyme activity in blood and diagnosing malaria infection at the same time, using the blood sample of a person having the enzyme.

Glucose-6-phsphate dehydrogenase (G6PD) is an important enzyme that converts carbohydrates into energy and protects red blood cells from side effects that may occur during infection or drug treatment.

G6PD deficiency is the most common enzyme deficiency disease and is inherited as X-chromosomal recessive disorder, and it is known that there is a difference in severity according to race and sex. In normal condition, only 3% of the enzyme is used. Therefore, most patients do not have symptoms. However, when oxidative stress is caused by infection, medicine, etc., NADPH production disorder accumulates intracellular oxidants and causes hemolysis.

G6PD acts on the hexose monophosphate (HMP) pathway as glucose-6-phosphate is converted to 6-phosphate gluconate, and coenzyme NADP is reduced to NADPH. When G6PD is deficient, NADPH can not be produced. Glutathione Glutathione (GSH) metabolism can not start.

GSH in red blood cells can only be converted to GSH reduced by NADPH to neutralize the oxidizing agent produced in red blood cells by infection or certain drugs. In the red blood cells lacking G6PD, oxidants such as over ^- oxygen (O ^2- ) and hydrogen peroxide are accumulated, and the hemoglobin is oxidized to become methemoglobin by the oxidizing agent, and as it progresses to sulfhemoglobin, (Heinz) body ', which are attached around the red blood cell membrane, causing hemolysis.

Unlike other cells in the body, red blood cells are the metabolic pathway that produces NADPH, and the pathway using G6PD is unique. It is relatively vulnerable to oxidative stress as compared with other cells. When exposed to oxidative stress, red blood cell hemolytic anemia appear.

Because the enzyme activity is transmitted through the X-chromosome, the activity is reduced to less than 5% in men and in homozygous females, and various levels of enzyme activity are reported in women with heterozygous females. G6PD deficiency is the most common cause of hemolysis and jaundice in neonates in erythrocyte enzyme deficiency. When a newborn with G6PD deficiency develops acidosis due to infection, hypoxia, dehydration, or exposure to drugs, hemolysis occurs and transfusion is required do. The major factors that cause severe G6PD deficiency symptoms include overexposure to chemicals such as anti-malarials, aspirin, phenacetin, sulfa, naphthalene, and bacterial or viral infections.

Malaria is recognized as the most important disease among the six tropical diseases selected by the World Health Organization (WHO). Approximately 40% (2.4 billion) of the world 's population live in the malaria epidemic, with about 3 to 500 million patients each year, of which more than 1 million die.

Malaria is an acute febrile infection caused by the parasitic infestation of genus Plasmodium parasites in red blood cells and hepatocytes. The female mosquitoes, which are infected with malaria parasites, are injected with sporozoite by injecting sporozoite into the body. Since it is a blood-related disease, it can be infected even when the blood of an infected person is transfused.

Malaria disease in humans has been additionally confirmed to be infectious from monkey fever, in addition to the four species of tropical fever, triad fever, quadruple fever, and ovoid fever. Malaria is transmitted through incubation period after infection, fever and fever persists for several days, and after repeated chills, fever, fever, and fever, periodic recurrence occurs. In the case of tropical fever malaria, the mortality rate is 10% or more if not treated and the mortality rate is 0.4% ~ 4.0% when treated.

Methods for diagnosing malaria include peripheral blood donation (blood test), total enzyme chain reaction, and fluorescent staining. These diagnostic methods are mainly diagnostic methods that can be done in the laboratory. Due to the nature of the disease, the malaria antigen test technique is used to make a quick and simple diagnosis at the site. This method is a method of detecting and diagnosing specific antigens produced by malaria parasites.

However, when primaquine, pamaquine, chloroquine, etc. are used as a malaria treatment for G6PD genetic mutation, the accumulation of oxidizing substances in erythrocytes accelerates the side effects caused by erythrocyte disruption It is often the case that it is induced and, in severe cases, it dies. Therefore, at the same time as the diagnosis of malaria, an easy way to obtain information on the types of therapies available is needed.

Korean Patent No. 10-0559370 (Mar. 3, 2006) discloses a malaria diagnostic kit comprising a monoclonal antibody specific for malaria lactate dehydrogenase and aldolase.

Accordingly, the present inventors developed an assay kit capable of quickly and accurately screening and visualizing infection or enzyme activity. As a result, a simultaneous assay kit was prepared for comparing the color of the test pad and the control pad to identify the enzyme activity with a clear color change, and simultaneously diagnosing the infection. The glucose-6-phosphate dehydrogenase glucose-6-phsphate dehydrogenase (G6PD) activity deficiency, and can be used as an assay kit for diagnosing malaria.

It is an object of the present invention to provide a kit for simultaneous diagnosis of disease diagnosis using enzyme activity measurement and immunological measurement.

It is another object of the present invention to provide a simultaneous analysis method for disease diagnosis using enzyme activity measurement and immunological measurement.

The present invention relates to a kit for simultaneous diagnosis of disease diagnosis using enzymatic activity measurement and immunological measurement, and a simultaneous assay method for disease diagnosis using enzyme activity measurement and immunological measurement, wherein the specific enzyme activity in blood is screened and read visually And analysis of malaria diagnosis markers can be carried out at the same time.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention relates to a kit for simultaneous analysis of disease diagnosis using enzyme activity measurement and immunological measurement comprising a dye compound and at least one detection line to which an antibody is immobilized.

The basic principle of the invention is that the conversion of the formazan dye sample using the activity of glucose-6-phsphate dehydrogenase (G6PD) and the detection of the pLDH of malaria detected by immunochromatography antibody-binding reactions of disease markers such as plasmodium lactate dehydrogenase, Histidine Rich Protein II (HRPII), and Aldolase are performed simultaneously using an optimized single sample mixture. The enzymatic reaction result can be determined from a pad in which a capillary phenomenon is induced, which is selected from the group consisting of cellulose, glass fiber and polyester.

In the case of a diagnostic device for use in conjunction with a disease test, the monoclonal antibody to the diagnostic device can be used for G6PD screening in a white cellulose or nitrocellulose membrane, which is a stationary phase. The result of the enzyme reaction is observed by observing the color of the whole test area and detecting the presence of antibody-antigen-antibody conjugate in the region where antigen-antibody conjugate is immobilized, The presence or absence of a test line can be determined to determine whether or not the disease is present.

The kit may further include a sample pad for injecting a sample on the upstream side of the test pad.

The dye compound may be contained in a sample pad or a dye pad. For example, the dye compound may be pretreated with a sample pad, or may be pretreated with a separate pad, lyophilized, and then positioned as a dye pad between the sample pad and the test pad But is not limited thereto.

The sample pad may be selected from the group consisting of glass fiber, cellulose and polyester, and may be, for example, glass fiber, but is not limited thereto.

The dye pad may be selected from the group consisting of cellulose, glass fiber and polyester, and may be, for example, cellulose, but is not limited thereto.

In an embodiment of the present invention, when the dye compound is pretreated on the sample pad without the dye pad, the sample pad may further include a buffer, a surfactant, or a mixture thereof as a substance to be pretreated.

The buffer was washed with 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffered saline (PBS) buffer And / or a Tris buffer.

The surfactant may utilize Triton X-100 and / or Tween 20, but is not limited thereto.

As used herein, the term "detection line" means a line prepared for an analyte or a labeling substance to arrive at an analysis kit in an assay kit using immunochromatography. For example, the presence or concentration of the analyte can be confirmed by fixing the antibody specific to the antigen to be analyzed on the test pad to prepare a detection line, but the present invention is not limited thereto.

The kit may include at least two detection lines, and each detection line may be one in which different antibodies are immobilized.

The test pad is a pad to which the analysis result is to be confirmed. A reaction product in which the sample reacts with the dye compound is developed and can be confirmed through an inspection area that can be confirmed from the appearance after assembling the plastic housing. For example, The analysis result may be confirmed by a change in color identified in the detection line, or the analysis result may be confirmed as the antibody labeled on the detection line appears. However, the present invention is not limited thereto.

The test pad may be a nitrocellulose membrane, but is not limited thereto.

The kit may further include a control pad on the downstream side of the test pad for determining whether the color of the test pad changes.

The check pad is a check object to check the analysis result, and it can be confirmed whether the check is valid or not. The verification pad can be confirmed through a check area which can be confirmed from the appearance after assembling the plastic housing. For example, The degree of change can be confirmed by contrasting with the color of the control pad, but is not limited thereto.

The control pad may be selected from the group consisting of cellulose, glass fiber and polyester, and may be, for example, cellulose, but is not limited thereto.

The kit may additionally include an absorbent pad on the downstream side of the control pad.

The absorbent pad may be, but is not limited to, cellulose.

The analyte to be analyzed using the kit refers to a biological material, biological sample or specific component in the sample having a specific activity that is the object of analysis to identify one of the results such as presence, absence, or concentration.

The analyte may be at least one selected from the group consisting of an antigen, an enzyme, a protein, a carbohydrate, a lipid and a nucleic acid, and may be, for example, an enzyme, but is not limited thereto.

These enzymes include glucose-6-phsphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine-beta-hydroxylase, glutathione synthesis (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyltransferase (gamma-glutamyltransferase), as well as enzymes such as glutathione synthetase, aspartate aminotransferase -glutamyl transferase (GGT), and may be, for example, G6PD, but is not limited thereto.

The analyte may be a toxic chemical, an environmental hormone or a neurotransmitter.

As used herein, the term "toxic chemical" means a harmful chemical having acute toxicity, fouling toxicity, mutagenicity and carcinogenicity, and includes cadmium, cadmium compounds, chlorine, hydrogen chloride, fluorine, silicon fluoride, lead, Nitrogen oxides, and nitrogen oxides.

The term " environmental hormone " in this specification means an industrial substance corresponding to an exogenous endocrine disrupting chemical substance and may be one or more selected from the group consisting of insecticides, pesticides, organic heavy metals, dioxins and synthetic estrogens used as pharmaceuticals have.

As used herein, the term "neurotransmitter" refers to a substance that is secreted in a neuron, which acts to increase or decrease the potential of adjacent neurons.

The analyte may be, but is not limited to, one which is present in the body fluid of the specimen or is separated from the body fluids of the specimen.

The specimen may be an animal, such as a mammal, rodent, reptile, or bird, and may be, for example, a mammal, but is not limited thereto.

As used herein, the term "dye compound" refers to a substance that exhibits color so that the analysis result can be visually confirmed according to the specific activity with the analyte to be reacted.

The dye compound may be selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride INT 2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3-bis- (2-methoxy- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carbanilide (XTT) Nitro blue tetrazolium (NBT), 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS), tetrazolium, tetrazolium chloride ( tetrazolium chloride) and 2,3,5-triphenyl-2H-tetra Tetrazolium chloride (TTC), and may be, for example, 2,6-dichlorophenolindophenol, but it is not limited thereto. no.

According to one embodiment of the present invention, when the blood lacking G6PD is used as a sample, DCPIP is not reduced when it reacts with DCPIP, which is a dye compound, so that the blue color is maintained. As a result, hemoglobin and blue dye are mixed Cyan or brown.

The kit may further comprise nicotinamide adenine dinucleotide phosphate (NADP), an NADP derivative, or an electron transfer agent that reduces NADP to NADPH.

The NADP derivative may be selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate, β-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, nicotinamide 1, N6- ethenoadenine dinucleotide and nicotinamide 1, and N6-ethenoadenine dinucleotide phosphate. However, the present invention is not limited thereto.

As used herein, the term "electron transfer agent" refers to a substance that contributes to the NADP + -reduced response to NADPH.

The electron transfer agent may be 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase, Meldola Blue, and may be, for example, PMS, but is not limited thereto.

The term "dye reducing agent" in this specification means a substance that reduces the dye compound.

The dye reducing agent may be ascorbic acid, but is not limited thereto.

According to one embodiment of the present invention, the reactant reacted with the dye compound DCPIP to analyze the sample is developed on the test pad and the control pad, and DCPIP contained in the reactant by the pretreated ascorbic acid in the control pad is blue The color of the test pad is compared with the color of the control pad to confirm the result of the analysis.

The antibody specifically binds to at least one member selected from the group consisting of plasmodium lactate dehydrogenase (pLDH), histidine rich protein II (HRPII), and aldolase And may be, for example, specifically binding to pLDH, but is not limited thereto.

The antibody may be labeled with at least one labeling substance selected from the group consisting of radioactive isotopes, dyes, magnetic beads, gold nanoparticles, celerium, cellulose beads, silica beads, silver beads and dendrimers, , Fluorescent dyes or gold nanoparticles, but are not limited thereto.

The kit may be used to quantitatively analyze an analyte contained in a sample using a reader after performing the analysis. Specifically, the marker material displayed on the detection line is photographed with a camera provided in the reader, and then the intensity of the color of the recognized region can be measured and utilized for analysis. For example, fluorescent materials or gold nanoparticles can be used as a labeling substance to quantitatively analyze with a reader, but the present invention is not limited thereto.

The kit may further include a check line on the test pad to determine the validity of the test.

Since the control line appears when the analysis is performed irrespective of the presence or concentration of the analyte, if an indication appears on the control line during the analysis, the result of the analysis through the detection line can be considered as valid.

According to one embodiment of the present invention, the reactant reacted with the dye compound DCPIP to analyze the sample is developed on the test pad and the control pad, and DCPIP contained in the reactant by the pretreated ascorbic acid in the control pad is blue The color of the test pads is compared with the color of the control pad to determine the presence or absence of the analyte that specifically reacts with the dye compound. Can be confirmed.

In addition, according to one embodiment of the present invention, a reaction product reacted with an antibody labeled with a fluorescent dye to analyze a sample is developed on a test pad, and an antibody and a reactant immobilized at a detection line position of the test pad are immobilized on an antibody- Antigen-antibody binding. In this case, analysis results of the presence and concentration of the analyte specifically binding to the antibody through the detection line can be confirmed.

Another aspect of the present invention is a simultaneous assay method for disease diagnosis using enzyme activity measurement and immunological measurement comprising the steps of:

A sample injection step of reacting a sample with an analytical reagent containing a dye compound and an antibody;

Measuring an activity of the reagent for analysis and a color change appearing in a reaction product of the sample; And

A step of diagnosing a disease in which the reagent for analysis and the reactant of the sample are tested for binding of the antibody.

The analyte may be at least one selected from the group consisting of an antigen, an enzyme, a protein, a carbohydrate, a lipid and a nucleic acid, and may be, for example, an enzyme, but is not limited thereto.

The enzyme may be selected from the group consisting of glucose-6-phsphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine-beta-hydroxylase, glutathione (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyltransferase (ALT) as well as enzymes such as glutathione synthetase, aspartate aminotransferase gamma-glutamyl transferase (GGT), and may be, for example, G6PD, but are not limited thereto.

The dye compound may be selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride INT 2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3-bis- (2-methoxy- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carbanilide (XTT) Nitro blue tetrazolium (NBT), 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS), tetrazolium, tetrazolium chloride ( tetrazolium chloride) and 2,3,5-triphenyl-2H-tetra Tetrazolium chloride (TTC), and may be, for example, 2,6-dichlorophenolindophenol, but it is not limited thereto. no.

The analytical reagent may further include nicotinamide adenine dinucleotide phosphate (NADP), an NADP derivative, or an electron transfer agent that reduces NADP to NADPH.

The NADP derivative may be selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate phosphate, beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, nicotinamide 1, N6- ethenoadenine dinucleotide and nicotinamide 1, and N6-ethenoadenine dinucleotide phosphate (nicotinamide 1, N6-ethenoadenine dinucleotide phosphate).

The electron transfer agent may be 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase, Meldola Blue, and may be, for example, PMS, but is not limited thereto.

The analytical reagent may be a mixture or a compound comprising at least one selected from the group consisting of 2,6-dichlorophenolindophenol, NADP, and G6PD. The sample may be absorbed into the solid support or the solid support, lyophilized, and then dispensed in a predetermined amount.

The antibody specifically binds to at least one member selected from the group consisting of plasmodium lactate dehydrogenase (pLDH), histidine rich protein II (HRPII), and aldolase And may be, for example, specifically binding to pLDH, but is not limited thereto.

The antibody may be labeled with at least one labeling substance selected from the group consisting of radioactive isotopes, dyes, magnetic beads, gold nanoparticles, celerium, cellulose beads, silica beads, silver beads and dendrimers, , Fluorescent dyes or gold nanoparticles, but are not limited thereto.

The kit may further include a check line on the test pad to determine the validity of the test.

Since the control line appears when the analysis is performed irrespective of the presence or concentration of the analyte, if an indication appears on the control line during the analysis, the result of the analysis through the detection line can be considered as valid.

Since the simultaneous assay method for disease diagnosis using the enzymatic activity measurement and immunological measurement of the present invention relates to a kit for simultaneous analysis of disease diagnosis using enzyme activity measurement and immunological measurement as another aspect of the present invention, And this is omitted in order to avoid the excessive complexity described herein.

The kit for simultaneous assay of disease diagnosis using the enzyme activity measurement and immunological measurement according to the present invention can accurately screen specific enzymatic activity in the blood and visually read it so that it can effectively cause hemolytic anemia due to G6PD activity deficiency It can be used for identification of risk factors, and analysis of malaria diagnosis markers can be performed at the same time. Therefore, screening tests for disease selection and drug selection can be performed in parallel.

1A is a schematic diagram showing the structure of a kit for simultaneous diagnosis of disease diagnosis using enzyme activity measurement and immunological measurement.
FIG. 1B is a photograph of a kit for simultaneous diagnosis of disease using enzymatic activity measurement and immunological measurement according to an embodiment of the present invention.
FIG. 2 is a photograph showing that the degree of change in hue of the test pad is increased according to the enzyme activity.
FIG. 3 is a photograph showing the result of the test of the sample showing the G6PD activity deficiency according to the level.
FIG. 4 is a graph showing a standard curve based on quantitative analysis results of samples showing G6PD activity deficiency by concentration. FIG.
FIG. 5 is a graph showing a standard curve based on the results of quantitative analysis of infection-mimicking samples by concentration of malic acid lactate dehydrogenase.
FIG. 6 is a photograph showing the time taken to perform simultaneous analysis of disease diagnosis using enzyme activity measurement and immunological measurement using the kit of the present invention. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Preparation of reaction solution

Sample pad Pretreatment solution (1 x PBS 32 mL, 461 uL of D-glucose 6 phosphate disodium salt hydrate, 250 mg / mL G6P), 50 mg / mL NADP (b-Nicotinamide adenine dinucleotide phosphate hydrate, Sigma, N5755) 192 uL, 480 uL of 100 mg / mL maleimide (sigma, 129585), 1-methoxy-5-methylphenazium methyl sulfate (hereinafter referred to as PMS, sigma M8640) Dichlorophenolindophenol (DCPIP), which changes from blue to colorless as NADP + is reduced to NADPH, and 4- (2-hydroxyethyl) -1-piperidine for stabilization 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (hereinafter referred to as HEPES) buffer and a surfactant were prepared.

Example 2. Preparation of assay kit

As shown in FIG. 1A, a sample pad, a dye pad, a conjugation pad, an NC membrane, a test pad, and an absorption pad are arranged on the support plate from upstream to downstream As shown in Fig. A control pad can additionally be attached between the test pad and the absorbent pad to further clarify the enzyme activity measurement results.

The sample pad is made of glass fiber material and corresponds to 1.92 mg per sheet. 1 ml of PBS, 461 μL of 250 mg / mL G6P (D-glucose 6 phosphate disodium salt hydrate, Sigma G7250), 192 μL of 50 mg / mL NADP (b-Nicotinamide adenine dinucleotide phosphate hydrate, Sigma, N5755) 480 μL of maleimide (Sigma, 129585) and 96 μL of 20 mg / mL 1-methoxy-5-methylphenazinium methyl sulfate (PMS, Sigma M8640) Mixed and infiltrated, and lyophilized.

The dye pad infiltrated DCPIP (2,6-dichlorophenolindophenol) into the cellulosic (Ahlstrom, ap 31) pad at 10 ug / ml and was freeze-dried because DCPIP was sensitive to high temperature.

The conjugation pad was prepared from cellulose material. 0.25 mg of anti-malaria pLDH antibody (Anti-Malaria pLDH antibody, Median diagnostic, cat. # 12001) as a monoclonal antibody specific to plasmid lactate dehydrogenase (pLDH), the first antibody used for malaria detection, ) Was reacted with Alexa Fluro 647 (Thermo Fisher Scientific) fluorescent dye selected for 1 hour. The polymer with fluorescent dye labeling was immobilized on the conjugate pad, followed by lyophilization.

The test pad was prepared as a nitrocellulose membrane (NC membrane).

The anti-malaria pLDH antibody was dispensed onto the nitrocellulose membrane in the form of a thin line to display the detection line on the test pad. After dispensing, the dispensed protein was immobilized in a dehumidifying device maintained at a temperature of 25 and a humidity of 35 to 50% for 2 hours. Subsequently, a second antibody (goat anti mouse IgG, Arista, Cat # ABGAM-0500) was dispensed and dried under the same conditions as those for the detection line method in order to display a control line on the test pad.

The absorbent pad was prepared and attached to a cellulose pad.

The control pad was prepared by infiltrating AP31 pad with 1 M ascorbic acid (Sigma, 255564), followed by hot air drying at 35 ° C for 2 hours, and cutting to a length of 10 mm.

As shown in FIG. 1B, the assay kit of the present invention was completed by inserting and supporting the support plate into a plastic housing.

Example 3. Visual analysis of risk factors for hemolytic anemia

Blood samples were prepared from 100% to 10% of enzyme activity.

Specifically, 150 uL of 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (hereinafter referred to as HEPES) buffer was prepared, and 5 ul Or 5 μl of G6PD-deficient blood were mixed to prepare a normal sample and a G6PD deficient sample. Then, a kit for assaying enzyme activity was used in which a control pad was placed on the downstream side of the test pad without including the conjugation pad, the detection line and the control line .

As can be seen from FIG. 2, the enzyme activity assay kit of the present invention can be analyzed in comparison with the color of the control pad because the degree of change of the color of the test pad increases according to the activity of the enzyme.

In the sample (normal blood) with an enzyme activity of 100%, both the test region (test pad) and the control region (control pad) were red with the same blood. The normal blood contains G6PD, so pink is produced by the reduction of the DCPIP dye, resulting in a red color mixed with hemoglobin.

However, when a sample with an enzyme activity of 10% (blood of a person at risk of hemolytic anemia due to G6PD deficiency) is reacted, the DCPIP dye is not reduced to maintain its blue color, and hemoglobin and blue dye are mixed to form a turquoise . As a result, it was confirmed that the test region appeared green and the control region was red, so that it could be distinguished from normal blood clearly.

Figure 3 shows the difference in color change due to differences in the degree of G6PD deficiency. Depending on the degree of deficiency, it can be seen that not only green but also brown can appear.

Example 4. Quantitative analysis of risk factors for hemolytic anemia

150 uL of 10 mM HEPES buffer was prepared, and G6PD was added at 10.1, 8.33, 6.55, 4.78. 3, 2.45, 1.9. 1.35, and 0.8 U / gHb, respectively, and they were added to the assay kit of the present invention. The results obtained from the assay kit were measured using a Hubi-Quan Pro (Humasis) reader capable of quantifying the image through image analysis, and the results are shown in FIG. 4 and Table 1.

The HubiKan Pro Reader is a reader that captures images with a camera and then performs quantitative measurements on a specific range of colors. Therefore, the intensity of the color appearing in the pads recognized by the reader in the analysis kit was measured and utilized for analysis.

U / g Hb Test first Test second Test third Average Standard Deviation Coefficient of variation (%) 10.10 3982.34 3741.16 3834.67 3852.72 99.29 3 8.33 3483.64 3312.86 3365.98 3387.49 71.36 2 6.55 2894.69 3008.79 3048.08 2983.85 65.06 2 4.78 2406.38 2856.18 3021.32 2761.29 259.86 9 3.00 1505.56 1808.09 1264.51 1526.05 222.39 15 2.45 1581.90 1140.82 1001.25 1241.32 247.47 20 1.90 762.60 637.83 926.67 775.70 118.28 15 1.35 476.86 561.23 447.21 495.10 48.30 10 0.80 33.02 138.40 17.31 62.91 53.76 85

Image analysis results were converted to G6PD enzyme activity values and shown in FIG. 4 as a two-step primary standard curve across enzyme depletion to normal range.

Example 5. Concurrent Diagnosis of Malaria and Risk Factors for Hemolytic Anemia

The results obtained with the assay kit of the present invention were measured using a Hubi-Quan Pro fluorescence reader (Humasis) capable of quantifying through image analysis. The reader can utilize the kit for quantitative confirmation by converting the fluorescence value obtained from the analysis kit into an intensity value by converting it into a quantitative value.

Analysis of malleia lactate dehydrogenase added to the mixed reaction mixture at concentrations of 50, 200, and 500 ng / mL using the imitation blood sample of Malia infection confirmed that a standard curve according to G6PD analysis can be obtained As shown in Fig.

When the clinical samples containing the malaria parasites were measured, a test was performed to determine whether the concentrations ascertained by the microscopic observation were the same. A small amount of 100, 400 and 3200 parasites / uL concentrations of cultured malarial protozoa were spiked into the blood, repeated five times, and analyzed with a Huviwan pro fluorescent reader.

Expected values (parasites / uL) Test first Test second Test third Exam Four Exam 5th Average 100 99.16 95.15 104.85 97.75 101.41 99.66 400 424.53 417.59 414.67 416.48 406.07 415.87 3200 3183.83 3311.34 3269.14 3291.26 3214.68 3254.05

Example 6. Diagnosis of Malaria and Confirmation Time of Concurrent Test for Hemolytic Anemia Risk Factor

The time taken for analysis using the kit for simultaneous assay of disease diagnosis using the enzyme activity measurement and immunological measurement of the present invention was measured.

Unlike the case where the fluorescence reader is used, the assay kit of the present invention can visually confirm G6PD deficiency and malaria infection using gold nanoparticles instead of using a fluorescent substance for immunological reaction of an antigen antibody, The color intensity of the nanoparticles can be quantitatively analyzed using a gold reader (HUBI, Humasis).

Specifically, as shown in FIG. 6, a kit (PF / PV) containing an antibody specifically binding to the four left lactate dehydrogenase enzymes was incubated with the normal blood (N), G6PD deficient blood (D), malaria lactate dehydrogenase (N + PV) containing 500 ng / mL, G6PD deficient blood (D + PV) containing 500 ng / mL malaria lactate dehydrogenase and the right four kits were treated with lactate dehydrogenase (PF) containing no antibody to the same sample as the left.

As can be seen from FIG. 6, since the analysis using the kit for simultaneous analysis of disease diagnosis using the enzyme activity measurement and immunological measurement of the present invention can be performed at the elapse of 5 minutes after the sample is injected, It can be analyzed more quickly than the fact that it can be read out after 10 minutes or more after the injection.

The assay kit of the present invention shows that the activity of G6PD and the activity of malaria in the presence of malaria lactate dehydrogenase can be confirmed by detecting the red blood cells in the normal blood, the G6PD deficient blood in the blue color, The kit can be analyzed simultaneously for diagnosis.

Claims (19)

A dye compound, and at least one detection line to which the antibody is immobilized; and an immunological measurement. The kit for simultaneous diagnosis of diseases according to claim 1, wherein the dye compound is contained in a sample pad or a dye pad, using enzyme activity measurement and immunological measurement. The kit for simultaneous diagnosis of diseases according to claim 1, wherein the detection line is included in an examination pad. The kit for simultaneous diagnosis of diseases according to claim 1, wherein the kit comprises at least two detection lines, and each detection line is immobilized with a different antibody. The method of claim 1, wherein the enzyme is glucose-6-phsphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine-beta-hydroxyase dopamine beta hydroxylase, glutathione synthetase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma Wherein the enzyme is at least one selected from the group consisting of gamma-glutamyl transferase (GGT), and an immunological assay. The method according to claim 1, wherein the dye compound is selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride INT 2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3-bis- (2- Methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5 (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium- -carbanilide (XTT), Nitro blue tetrazolium (NBT), 3- (4,5-dimethylthiazol-2-yl) -5- (3- carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS), tetrazolium ), Tetrazolium chloride and 2,3,5- Wherein the at least one enzyme is at least one selected from the group consisting of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) Kits. The kit according to claim 1, wherein the kit further comprises an activity of measuring nicotinamide adenine dinucleotide phosphate (NADP), an NADP derivative or an NADPH-releasing electron transfer agent, A kit for simultaneous analysis of disease diagnosis using The method of claim 7, wherein the NADP derivative is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate beta-nicotinamide adenine dinucleotide phosphate (beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide wherein the enzyme activity is at least one selected from the group consisting of nicotinamide 1 and nicotinamide 1 and N6-ethenoadenine dinucleotide phosphate (Nicotinamide 1, N6-ethenoadenine dinucleotide phosphate). To Simultaneous analysis kit for diagnosis of disease. 8. The method of claim 7, wherein the electron transfer agent is selected from the group consisting of 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase Diaphorase) and Meldola Blue. The kit for simultaneous analysis of diseases using enzyme activity measurement and immunological measurement. The antibody of claim 1, wherein the antibody is selected from the group consisting of Plasmodium lactate dehydrogenase (pLDH), Histidine Rich Protein II (HRPII), and Aldolase A kit for simultaneous analysis of disease diagnosis using enzyme activity measurement and immunological measurement. The antibody of claim 10, wherein the antibody is labeled with at least one labeling substance selected from the group consisting of radioactive isotopes, dyes, magnetic beads, gold nanoparticles, cerenium, cellulose beads, silica beads, silver beads and dendrimers Kit for simultaneous analysis of disease diagnosis using enzyme activity measurement and immunological measurement. Simultaneous assay of disease diagnosis using enzymatic activity measurement and immunological measurement including the following steps:
A sample injection step of reacting a sample with an analytical reagent containing a dye compound and an antibody;
Measuring an activity of the reagent for analysis and a color change appearing in a reaction product of the sample; And
A step of diagnosing a disease in which the reagent for analysis and the reaction product of the sample are bound to the antibody. 13. The method of claim 12, wherein the enzyme is selected from the group consisting of glucose-6-phsphate dehydrogenase (G6PD), lactate dehydrogenase, glucose oxidase, dopamine beta hydroxylase, glutathione synthetase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma Wherein the enzyme activity is at least one selected from the group consisting of gamma-glutamyl transferase (GGT), and simultaneous assay of disease diagnosis using immunological measurement. The method according to claim 12, wherein the dye compound is selected from the group consisting of 2,6-dichlorophenolindophenol (DCPIP), 2- (4-iodophenyl) -3- (4-nitrophenyl) 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride INT 2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,3-bis- (2- Methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5 (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium- -carbanilide (XTT), Nitro blue tetrazolium (NBT), 3- (4,5-dimethylthiazol-2-yl) -5- (3- carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS), tetrazolium ), Tetrazolium chloride and 2,3,5- Wherein the at least one enzyme is at least one selected from the group consisting of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) . 13. The method of claim 12, wherein the analyzing reagent further comprises an nicotinamide adenine dinucleotide phosphate (NADP), an NADP derivative, or an electron transfer agent that reduces NADP to NADPH. Simultaneous Analysis of Disease Diagnosis Using. 16. The method of claim 15, wherein the NADP derivative is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate beta-nicotinamide adenine dinucleotide phosphate (beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide wherein the enzyme activity is at least one selected from the group consisting of nicotinamide 1 and nicotinamide 1 and N6-ethenoadenine dinucleotide phosphate (Nicotinamide 1, N6-ethenoadenine dinucleotide phosphate). To Yonghan Diseases simultaneous analysis. 16. The method of claim 15, wherein the electron transfer agent is selected from the group consisting of 1-methoxy-5-methylphenazium methyl sulfate (PMS), phenzaine methosulfate, diaphorase Diaphorase, and Meldola Blue. The method of simultaneous assay for disease diagnosis using enzyme activity measurement and immunological measurement. 13. The method according to claim 12, wherein the antibody comprises at least one selected from the group consisting of Plasmodium lactate dehydrogenase (pLDH), Histidine Rich Protein II (HRPII), and Aldolase Wherein the enzyme activity is measured by an enzyme activity assay and immunological assay. The antibody of claim 12, wherein the antibody is one labeled with a labeling substance selected from the group consisting of radioisotopes, dyes, magnetic beads, gold nanoparticles, cerenium, cellulose beads, silica beads, silver beads and dendrimers Phosphorus, enzyme activity and immunological measurement.

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