KR102341169B1 - Reagent kit for detecting glycated albumin ratio and detection method of glycated albumin ratio using thereof - Google Patents

Abstract

The present invention relates to a reagent kit for measuring the glycated albumin ratio capable of diagnosing the presence or absence of diabetes and a method for measuring the glycated albumin ratio using the same, and more particularly, to gold nanoparticles to which an albumin antibody is bound, and a boronic acid derivative to which a dye is bound. It relates to a reagent kit for measuring the glycosylated albumin ratio comprising a reagent kit and a method for measuring the glycosylated albumin ratio using the same.
The reagent kit for measuring the glycosylated albumin ratio of the present invention includes (a) albumin detection particles to which an albumin antibody specifically binding to albumin is bound; and (b) a reagent kit for measuring a glycosylated albumin ratio comprising a boronic acid derivative to which a dye specifically detecting glycosylated albumin is bound.
The reagent kit for measuring the glycosylated albumin ratio according to the present invention includes "albumin detection particles to which an albumin antibody specifically binding to total albumin is bound" and "boronic acid derivative to which a dye specifically binding to glycosylated albumin is bound" Therefore, the concentration of each sample can be measured individually without interfering with the inherent absorption wavelength of each dye by measuring the sample with a signal of a different dye, so that the ratio of glycated albumin in the blood can be easily calculated.

Description

A reagent kit for measuring the glycated albumin ratio and a method for measuring the glycated albumin ratio using the same {Reagent kit for detecting glycated albumin ratio and detection method of glycated albumin ratio using thereof}

The present invention relates to a reagent kit for measuring the glycated albumin ratio capable of diagnosing diabetes mellitus and a method for measuring the glycated albumin ratio using the same, and more particularly, to gold nanoparticles to which an albumin antibody is bound and a boronic acid derivative to which a dye is bound. It relates to a reagent kit for measuring the glycosylated albumin ratio comprising: and a method for measuring the glycosylated albumin ratio using the same.

Diabetes mellitus is a metabolic disease that occurs due to an abnormality in insulin, which plays a role in regulating blood sugar. Type 1 diabetes, which occurs when cells that produce insulin are destroyed due to an abnormality in the immune system, and insulin secretion is insufficient or not used effectively. It is classified according to the cause, such as type 2 diabetes.

Diabetes mellitus is characterized by high blood sugar in which the concentration of glucose in the blood rises. Failure to control blood sugar can lead to complications such as diabetic retinopathy, kidney disease, and foot lesions, so the importance of blood sugar management in diabetic patients is increasing. .

Although glucose is used as a conventional marker for diabetes measurement, there is a problem in that an error according to measurement time and fluctuation according to a patient's condition may appear directly because the blood glucose value before and after a meal fluctuates greatly. In addition, glucose oxidase used for glucose measurement is vulnerable to environmental influences such as pH and other interfering substances contained in the blood, and may affect the activity of the enzyme by generating hydrogen peroxide.

Therefore, recently, glycated hemoglobin (HbA1c) has been used as a biomarker for measuring blood glucose that can be measured more accurately than glucose and is more stable. Since glycated hemoglobin is stable until red blood cells are destroyed, it is used as an index indicating the average blood sugar level for 2-3 months, and is used to investigate the progress of diagnosis and treatment of diabetes. However, it is known that the glycosylated hemoglobin measurement method is not suitable for patients with some diseases, such as end-stage chronic renal failure, in which blood sugar cannot be maintained steadily, or for patients with abnormal red blood cells.

Albumin is a protein present not only in blood but also in major organs and body fluids. Depending on the concentration of glucose in the blood, glycated albumin is sometimes formed. Albumin has a glucose binding rate 10 times higher than that of hemoglobin, so glycated albumin responds more sensitively to changes in blood sugar than glycated hemoglobin. The average blood glucose level can be monitored. Therefore, it is useful as an important blood glucose control indicator in patients with end-stage chronic renal failure, iron deficiency anemia, and diabetic patients with mutant hemoglobin.

Korean Patent Application Laid-Open No. 2004-0018893 discloses a method for measuring the concentration of glycated hemoglobin using an immunochromatographic method. Hemoglobin antibody, hemoglobin containing glycated hemoglobin, and glycated hemoglobin antibody are bonded to a nitrocellulose membrane in a sandwich method. For total hemoglobin, the red color of hemoglobin is absorbed through absorption spectroscopy. After quantifying each, the method of measuring the ratio is adopted. The price of the glycosylated hemoglobin antibody is significantly higher than that of the hemoglobin antibody, which increases the unit price of the kit. In addition, the method of measuring the absorbance of hemoglobin and the fluorescence of the dye attached to the glycated hemoglobin, respectively, and measuring the ratio, has disadvantages in that it is necessary to measure each in different methods and use a relatively expensive fluorescence analyzer.

Korea Patent Registration No. 2018-0032513 discloses a method for measuring the ratio of glycated albumin in albumin in blood or plasma by measuring the reflectivity of "silica nanoparticles encapsulating dye-boronic acid". Using a dye that selectively binds to glycosylated albumin may be helpful in measuring the ratio, but the error caused by measuring the signal of total albumin and the signal of glycosylated albumin reacted in other cartridges and calculating the ratio may be large. have.

Ikeda et al. disclosed an enzyme-boronic acid immunoassay (ELIBA) using an albumin antibody and boronic acid conjugated with a peroxidase enzyme (Ikeda et al, Clin Chem. 44(2):256-263, 1998). . Although these methods showed high selectivity and accuracy, their development as a low-cost rapid kit is limited due to the fact that it takes a lot of time (30 to 90 minutes) and requires an instrument capable of measuring fluorescence. Therefore, there is an urgent need to develop a method for measuring glycated albumin more simply and accurately.

It is an object of the present invention to provide a reagent kit for measuring the glycosylated albumin ratio, which can measure the glycosylated albumin ratio simply and accurately, and a method for measuring the glycosylated albumin ratio using the same.

In order to achieve the above object, the present invention provides an albumin detection particle bound to (a) an albumin antibody specifically binding to albumin; and (b) a reagent kit for measuring a glycosylated albumin ratio comprising a boronic acid derivative to which a dye specifically detecting glycated albumin is provided.

In the present invention, the albumin detection particle to which the albumin antibody is bound and the boronic acid derivative to which the dye for detecting glycated albumin is bound has an absorption wavelength that does not interfere with each other.

In the present invention, the albumin detection particles are gold nanoparticles, polystyrene particles, quantum dot particles, upconversion nanoparticles, and silica particles having a dye fixed therein having an absorption wavelength of 450 to 650 nm that can be optically measured. characterized in that it is selected.

In the present invention, the albumin detection particle is characterized in that it has a diameter of 5 ~ 500nm.

In the present invention, the boronic acid derivative to which the dye is bound is characterized in that it is a yellow dye having an absorption wavelength of 400 to 430 nm or a blue dye having an absorption wavelength of 600 to 660 nm.

The present invention also includes (a) albumin detection particles to which an albumin antibody specifically binding to albumin is bound; and (b) a method for measuring the glycosylated albumin ratio using a reagent kit for measuring the glycosylated albumin ratio comprising a boronic acid derivative to which a dye specifically detecting glycosylated albumin is provided.

The present invention also comprises the steps of (a) adding serum or plasma solution to albumin detection particles to which an albumin antibody specifically binding to albumin is bound; (b) administering the reactant to the albumin antibody-immobilized membrane of the cartridge; (c) administering the dye-bound boronic acid derivative to the membrane and then washing the membrane with a washing solution; and (d) measuring the optical reflectivity of the membrane with an optical instrument to calculate the ratio of total albumin and glycated albumin.

In the present invention, the optical device measures the optical reflectance by simultaneously irradiating the wavelength of the albumin detection particle to which the albumin antibody specifically binding to total albumin is bound and the wavelength of the boronic acid derivative to which the dye is bound with a light source. do it with

The reagent kit for measuring the glycosylated albumin ratio according to the present invention includes "albumin detection particles to which an albumin antibody specifically binding to total albumin is bound" and "boronic acid derivative to which a dye specifically binding to glycosylated albumin is bound" Therefore, the concentration of each sample can be measured individually without interfering with the inherent absorption wavelength of each dye by measuring the sample with a signal of a different dye, so that the ratio of glycated albumin in the blood can be easily calculated.

In addition, since the reagent kit for measuring the glycosylated albumin ratio according to the present invention contains a dye that can label and distinguish total albumin and glycosylated albumin, it is simply administered with an optical analyzer through administration of a washing solution to the measurement cartridge without a separate separation process It has the advantage of being able to measure glycated albumin.

1 is an explanatory diagram illustrating a method for preparing an albumin detection particle/antibody conjugate to which an albumin antibody specifically binding to total albumin according to the present invention is bound.
2 is a flowchart illustrating a method for measuring glycated albumin and total albumin using the reagent kit and optical instrument for measuring the glycated albumin ratio of the present invention.
3 is a graph showing the K/S value of glycated albumin measured according to the present invention.
(A: K/S substitution value of glycated albumin % reflectance value measured by irradiation with blue light source, B: K/S substitution value of total albumin % reflectance value measured by irradiation with red light source, C: ratio of glycated albumin in total albumin K/S ratio (A/B))

In the present invention, "albumin detection particle to which an antibody that specifically binds to total albumin" and "a boronic acid derivative to which a dye that specifically binds to glycated albumin is bound" is a sandwich language comprising a membrane to which an albumin antibody is immobilized. When applied to the assay, albumin and glycated albumin can be measured individually without interfering with their intrinsic absorption wavelength by measuring albumin detection particles and the signal of the dye bound to the glycated albumin, respectively, so the ratio of glycated albumin in the blood can be measured. We wanted to confirm that it can be easily calculated.

In the present invention, a cartridge comprising (a) albumin detection particles to which an albumin antibody is bound, (b) a boronic acid derivative bound to a dye, and (c) a membrane and an absorption pad to which the albumin antibody is immobilized is prepared, respectively, and then the albumin antibody The albumin detection particles bound to were reacted with the plasma sample, the reaction solution was absorbed into the membrane of the cartridge, and the dye-bound boronic acid derivative was reacted.Then, the cartridge was put into an optical device, and the optical reflectance of all albumin was measured. By measuring the optical reflectivity of glycated albumin, it was confirmed that the ratio of glycated albumin could be measured.

Accordingly, the present invention in one aspect (a) an albumin detection particle to which an albumin antibody specifically binding to albumin is bound; And (b) relates to a reagent kit for measuring the glycosylated albumin ratio comprising a boronic acid derivative to which a dye specifically detecting glycosylated albumin is bound.

1 is an explanatory view showing a method for producing an albumin detection particle to which an albumin antibody specifically binding to total albumin according to the present invention is bound.

In the present invention, the albumin detection particles have different absorption wavelengths depending on the size and type of particles, but gold nanoparticles, polystyrene particles, upconversion nanoparticles and silica particles having an absorption wavelength in the range of 450 to 650 nm, and silica particles having a dye therein. It can be used, and it is preferable to use a diameter of 5 to 500 nm. The dyes fixed to the inside of the silica particles are Corona green, Hoechst 33258, Newport green PDX, Oil red O, Pseudo-purpurine (Pseudo- purpurine), calcein, etc. are preferable.

In the present invention, the albumin antibody is an antibody capable of binding to general albumin and total albumin, and as a blocker for binding to gold nanoparticles, casein, bovine serum albumin, ethanolamine (Ethanolamine) may be exemplified, but is not limited thereto.

For reference, the blocker is a material that binds the antibody to the gold nanoparticles and then fills the area to which the antibody is not attached on the surface of the gold nanoparticles, and serves to minimize non-specific reactions.

In addition, the buffer used to prepare the albumin detection particle to which the total albumin-binding antibody is bound is Hepes buffer (HEPES), carbonate buffer (Carbonate buffer), phosphate buffered saline (Phosphate buffered saline), Tris buffer (Tris) buffer), glycine buffer, etc. may be exemplified, but the present invention is not limited thereto.

In the present invention, the dye-bonded boronic acid derivative may be a yellow dye having an absorption wavelength of 400 to 430 nm or a blue dye having an absorption wavelength of 600 to 660 nm. As the yellow dye, tartrazine, cilenterazine, Sunset Yellow FCF, and the blue dye are xylene cyanol, toluidine blue, and the like. Examples may be provided, but the present invention is not limited thereto.

For the boronic acid derivative to impart selectivity to glycated albumin, it is recommended to use 4-carboxylicphenyl boronic acid (CPBA) and 3-aminophenyl boronic acid (APBA). Preferably, 4-carboxylicphenyl boronic acid (CPBA) uses CPBA because it has relatively high thermal stability compared to 3-aminophenyl boronic acid (APBA). It is preferable to do

In the present invention, the cartridge for measuring the glycated albumin ratio includes an absorbent pad that absorbs a reaction solution (sample) and a membrane to which the albumin antibody is fixed, and includes a housing surrounding them.

The absorbent pad can use polypropylene, sponge, cotton, cellulose fiber, etc., and the membrane is made of glass fiber, cellulose fiber, nitrocellulose ( Nitrocell μlose), polyester sulfone, nylon membrane, etc. commonly used can be used without limitation.

The cartridge according to the present invention can measure the glycosylated albumin ratio based on the immunological method for measuring total albumin and the boronic acid affinity method for measuring glycated albumin, and both the immunolateral flow measurement method and the immune vertical measurement method are applicable.

According to another aspect, the present invention provides an albumin detection particle to which (a) an albumin antibody specifically binding to albumin is bound; And (b) relates to a method for measuring the glycosylated albumin ratio using a reagent kit for measuring the glycosylated albumin ratio comprising a boronic acid derivative to which a dye specifically detecting the glycosylated albumin is bound.

In another aspect, the present invention comprises the steps of: (a) adding a serum or plasma solution to albumin detection particles to which an albumin antibody specifically binding to albumin is bound; (b) administering the reactant to the albumin antibody-immobilized membrane of the cartridge; (c) administering the dye-bound boronic acid derivative to the membrane and then washing the membrane with a washing solution; and (d) measuring the optical reflectivity of the membrane with an optical instrument to calculate the ratio of total albumin and glycated albumin.

2 is a flowchart illustrating a method for measuring glycated albumin and total albumin using the reagent kit and optical instrument for measuring the glycated albumin ratio of the present invention.

As shown in FIG. 2 , a sample (serum or plasma solution) is reacted with “albumin detection particles to which an albumin antibody that specifically binds to total albumin is bound”, and then the reactant is applied to the membrane on which the albumin antibody of the cartridge is immobilized. After administration, the dye-bound boronic acid derivative is administered to the membrane, the unbound dye is washed, and the optical reflectance of the membrane can be measured with an optical instrument.

In this case, the optical device simultaneously irradiates the wavelength of the albumin detection particle to which the albumin antibody specifically binding to total albumin is bound and the wavelength of the boronic acid derivative to which the dye specifically detects glycated albumin is irradiated with a light source to obtain optical reflectivity can be measured.

The optical device can be used without any particular limitation as long as it can measure optical reflectivity using optical properties, and the wavelength of the albumin detection particle to which the albumin antibody specifically binding to total albumin is bound and the "dye-bound boronic acid" The wavelength of the derivative is simultaneously irradiated with a light source (e.g., blue (620nm) and red (425nm) can

The ratio of glycated albumin can be calculated by the following formula by calculating the relative amount of glycated albumin according to the total amount of albumin.

* glycosylated albumin ratio (%) = glycosylated albumin/total albumin

In general, when the ratio of the glycated albumin is 16% or more, diabetes can be diagnosed.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of reagent kit and cartridge for measuring the glycosylated albumin ratio

1-1-1: Preparation of gold nanoparticles bound to albumin antibody

10 ml of gold nanoparticles (colloidal gold) and 200 μl of an albumin antibody solution (Abcam, ab10241) were mixed with a magnetic stirrer for 1 hour. After mixing, 200 μl of 0.01M ethanolamine was additionally added, and then mixed and reacted with a magnetic stirrer for 1 hour. After the reaction, it was washed three times with carbonate buffer under the conditions of 13,000 × g, 30 minutes using a centrifuge, and then stored refrigerated at 4°C. As a result of measuring the absorption wavelength of the prepared gold nanoparticles, it was found that it was 525 nm.

1-1-2: Preparation of albumin antibody-conjugated polystyrene particles

0.5 ml of dispersed polystyrene particles were placed in a 2 ml microcentrifuge tube, 0.1M borate buffer was filled in the tube, and then mixed well. After centrifuging the solution until a pellet was formed, the supernatant was removed with a micropipette, and 0.1M borate buffer was added to the solution from which the supernatant was removed, mixed well and centrifuged. After the final centrifugation, the supernatant was removed and the pellet was released after the process of adding 1 ml of 0.1M borate buffer.

After 400 μl of 1 mg/ml HSA antibody was added to the above solution, it was reacted with a mixer at room temperature for 17 hours. After the reaction, the supernatant was removed by centrifugation, and 1 ml of 0.1M borate buffer containing BSA at a concentration of 10 mg/ml was added to re-disperse the pellet. The BSA-added solution was reacted with a mixer at room temperature for 30 minutes. After centrifugation for 5-6 minutes, the supernatant was removed. 10 mg/ml BSA, 0.1% NaN ₃ , 1 ml of 0.1M PBS containing 5% glycerol was added to release the pellet. As a result of measuring the absorption wavelength of the prepared polystyrene particles, it was found that 475 nm.

1-1-3: Preparation of quantum dot particles bound to albumin antibody

0.985 g of cadmium perchlorate hexahydrate (Cd(ClO ₄ ) ₂ .6H ₂ O) and 125 ml of distilled water were dissolved in a three-neck flask, and then 5.7 mM thioglycolic acid (HSCH ₂ COOH, TGA) was added and stirred. The pH was then titrated with 1M NaOH until the solution became clear. Next, the solution was refluxed at 100° C. or less to replace TGA with CdTe nanocrystals, and then, bubbles were removed with nitrogen gas for 20 minutes.

_{A tellurium precursor (H 2} Te) was prepared by adding 15-20 ml of 0.5M sulfuric acid to 0.2 g of Al ₂ Te ₃ lump, and then bubbles were removed with nitrogen for 20 minutes, and the product was stored at 4°C.

To prepare silica quantum dot particles (SiQDs), 1.5 ml of 48 mM (3-mercapto propyl) trimethoxysilane (MPS) was added to 10 ml CdTe quantum dot solution, and after stirring the mixture for 2 hours, the solution was diluted with 2 L of distilled water. Dialysis was carried out for 1 hour.

Then, 2 ml of sodium silicate was added to the mixture to start the silica polymerization reaction, and the surface silica shell was grown for 12 hours. After the reaction was completed by adding 2 ml of ethanol, centrifugation (15,110 × g, 4 2 min) to remove the supernatant. The quantum dot particle pellet was dispersed by adding 1 ml of distilled water to ultrasonic treatment for 30 minutes to complete the preparation.

100 μl of the quantum dot particle solution and 100 μl of the antibody solution with a concentration of 400 μg/ml were mixed at 37° C. for 30 minutes to attach the antibody to the quantum dot particles, and centrifuged (15110 × g, 4° C., 10 minutes) to centrifuge the unreacted antibody. was removed and the pellet of the quantum dot particle-antibody complex was dispersed in 200 μl PBS and stored at 4° C. to complete the preparation. As a result of measuring the absorption wavelength of the prepared quantum dot particles, it was found that it was 590 nm.

1-1-4: Preparation of silica particles with dye immobilized inside albumin antibody-bound

A stirred reaction product of xylene cyanol and silica precursor (Dye-Pre_Si) in a 1L round bottom flask containing 150 ml of cyclohexane, 33.5 ml of Triton X-100 and 35.3 ml of n-hexanol Solution <Korea Patent No. 10-2055341 Example 1-1-1> 9.25 ml and TEOS (tetraethyl orthosilicate) 2.7 ml were added, and the mixture was uniformly mixed for 1 hour using a stirrer.

After that, 1.5 ml of 15-30% aqueous ammonia (NH ₄ OH) was added, and after reacting at room temperature for 20 to 30 hours, 300 ml of ethanol was added to terminate the reaction, and using a centrifuge at 3900 rpm for 15 minutes. After washing with ethanol 4 times and DI washing 3 times, it was placed in an oven at 60° C. and dried to prepare "silica nanoparticles with dye immobilized therein".

100 μl of a solution of polyethyleneimine and polyvinyl alcohol in a 1:100 ratio was mixed with 5 ml of “silica nanoparticles with dye fixed therein” dispersed in ethanol and stirred for 17 hours After preparing the surface-modified silica nanoparticles, the antibody is immobilized in the same manner as the antibody immobilization method of the upconverted nanoparticles in 1-1-3 above. As a result of measuring the absorption wavelength of the prepared silica particles having the dye fixed therein, it was found that it was 630 nm.

1-2: Preparation of dye-conjugated boronic acid derivatives

According to the method described in Example 1 of Korean Patent Registration No. 2029798, silica nanoparticles encapsulating a yellow dye-boronic acid derivative was prepared.

1-2-1: Synthesis of yellow dye encapsulated silica nanoparticles (YD@SNP)

Add 135.0 ml of cyclohexane, 31.8 ml of Triton X-100, 32.4 ml of n-hexanol, 6.12 ml of 0.1M tartrazine, and 2.7 ml of TEOS (tetraethyl orthosilicate) into a 1L round bottom flask and stirrer was uniformly mixed for 1 hour. After adding 1.08 ml of 25-30% aqueous ammonia (NH ₄ OH) and reacting at room temperature for 24 hours, 200 ml of ethanol was added to terminate the reaction. Using a centrifuge, washing with ethanol 4 times at 3800 rpm for 15 minutes and After DI washing 3 times, it was placed in an oven at 60° C. and dried.

1-2-2: Amination of yellow dye encapsulated silica nanoparticles (YD@SNP)

For cross-linking between YD@SNP and the carboxyl group of CPBA, the hydroxyl group (-OH) on the surface of YD@SNP was substituted with a primary amine group. That is, 100 mg of YD@SNP was put into 100 ml of ethanol, dispersed for 30 minutes using an ultrasonic disperser, and then 1 ml of APTES (3-Aminopropyltriethoxysilane) was added to a stirrer and reacted at room temperature for 2 hours. After the reaction, the reaction mixture was prepared in a centrifuge 4 times of ethanol wash and three times DI was washed and then placed in a 60 ℃ oven dried aminated YD @ SNP (YD @ SNP- NH 2) under the condition of 3800rpm, 15 minutes using a .

1-2-3: Conjugation of Aminated Yellow Dye Encapsulated Silica Nanoparticles (YD@SNP-NH _{2 ) and CPBA}

Carbodiimide cross-coupling method using 1-ethyl-3 [3-dimethyl aminopropyl] carbodiimide hydrochloride (EDC), which is a cross-linking agent that connects a carboxyl group and a primary amine group, in order to impart binding ability with glycated albumin As a glycated albumin binding material, 4-carboxylicphenyl-boronic acid (CPBA) was immobilized on the surface of _{silica nanoparticles (YD@SNP-NH 2 ) encapsulating an amination yellow dye.}

That is, in an environment where light is blocked, 3.48 mM CPBA is dissolved in 0.1 M MES (2-(Morpholino) ethanes μlfonic acid) buffer (pH 6.0) to activate the carboxyl functional group of CPBA, and the final concentration of EDC is 1 mM. After adding and reacting with stirring for 30 minutes, YD@SNP-NH ₂ was added and reacted at room temperature on a stirrer for 10 to 20 hours.

After completion of the reaction, using a centrifuge, ethanol washing 4 times and DI washing 3 times at 3800 rpm for 15 minutes, and then lyophilized at room temperature or freeze-dried to encapsulate yellow dye-boronic acid (YD@SNP-CPBA) ) was prepared.

As a result of analysis using a scanning electron microscope and dynamic light scattering method, it was confirmed that nanoparticles having a uniform shape and a size of about 35 to 45 nm were synthesized.

1-3: Cartridge manufacturing through the combination of the membrane and the housing to which the antibody is not immobilized

After bonding the membrane (nitrocellulose) and the absorbent pad (cellulose fiber), the housing (upper and lower case) was combined to manufacture a cartridge.

1-4: Manufacture of a cartridge for measuring the glycosylated albumin ratio

15 μl of albumin antibody solution (abcam, ab83465) was loaded onto the membrane of the cartridge prepared in steps 1-3 with a pipette and dried in a drying oven at 37° C. for 10 hours to prepare a reagent kit for measuring the glycosylated albumin ratio.

Example 2: Measurement of glycosylated albumin using reagent kit for measurement of glycosylated albumin ratio

2-1: Reaction of total albumin with specific reagents

200 μl of the "albumin antibody-bound gold nanoparticles" prepared in Example 1 was placed in a transparent tube, and the ratio (%) of glycated albumin was measured with an Olympus AU400 analyzer using a standard reagent (Asahi Kasei GA-L). After adding 5 μl of the plasma sample, it was mixed by shaking 10 times. 25 μl of the reaction solution was placed on the membrane on which the albumin antibody of the cartridge was immobilized and absorbed for 15 seconds.

2-2: Reaction of glycosylated albumin with specific reagents

25 μl of the "dye-coupled boronic acid derivative" prepared in Example 1 was added to the membrane on which the albumin antibody of the cartridge was immobilized, and then absorbed for 15 seconds. After absorption, 25 μl of the washing solution was administered to the cartridge and washed for 15 seconds.

2-3: Total albumin and glycosylated albumin measurement

The cartridge was placed in an optical instrument (Epithod ^® 616 analyzer, Dixgen), and the optical reflectance of total albumin in red and the optical reflectivity of glycated albumin in blue were measured.

2-4: Measurement of glycated albumin using K/S value

The ratio (%) of glycated albumin was determined by comparing the optical reflectance of glycated albumin and total albumin measured in 2-3. The % reflectance (%R) measured from each wavelength was used by converting the K/S value, which is a quantitative indicator of how much colored material is present on the surface, and converting the % reflectivity into K/S value. The formula to do is as follows:

K/S=

(K = absorption coefficient, S = scattering coefficient)

As described in Figure 3, the % reflectance value obtained by irradiating a blue light source (620 nm) representing the amount of glycated albumin and the % reflectance value obtained from a red light source (425 nm) representing the amount of total albumin are respectively K/S values. After substitution with , the amount of glycated albumin could be measured by calculating their ratio. 3 (c) is a value obtained by substituting K/S for the reflectance of glycated albumin measured with a blue light source by a value obtained by substituting a K/S value for the reflectivity of all albumin measured with a red light source. It is a graph showing the ratio of (K/S ratio value). From this, it was found that as the ratio of glycated albumin contained in the blood increased, the amount of glycated albumin (K/S ratio value) combined with the dye-bound boronic acid derivative was relatively increased.

In addition, "albumin antibody-bound polystyrene particles", "albumin antibody-bound quantum dots", and "albumin antibody-bound silica particles with a dye immobilized inside" may be used instead of "albumin antibody-bound gold nanoparticles" It was found that the same results were obtained in both cases.

Therefore, it was confirmed that the method for measuring glycated albumin according to the present invention can be widely used for diagnosing diabetes.

Above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it will be clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. . Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (8)

(a) albumin detection particles to which an albumin antibody specifically binding to albumin is bound; and
(b) a reagent kit for measuring the glycosylated albumin ratio comprising a boronic acid derivative to which a dye specifically detecting glycosylated albumin,
The albumin detection particle is selected from the group consisting of quantum dot particles having an absorption wavelength of 450 to 650 nm that can be optically measured, upconversion nanoparticles and silica particles having a dye fixed therein,
The dye bound to the boronic acid derivative is a reagent kit for measuring the glycosylated albumin ratio, characterized in that it is a yellow dye having an absorption wavelength of 400 to 430 nm or a blue dye having an absorption wavelength of 600 to 660 nm.
The reagent kit for measuring the glycosylated albumin ratio according to claim 1, wherein the albumin detection particle to which the albumin antibody is bound and the boronic acid derivative to which a dye for detecting glycated albumin is bound have absorption wavelengths that do not interfere with each other.
delete The reagent kit for measuring the glycosylated albumin ratio according to claim 1, wherein the albumin detection particles have a diameter of 5 to 500 nm.
delete delete (a) adding serum or plasma solution to albumin detection particles to which an albumin antibody specifically binding to albumin is bound;
(b) administering the reactant to the albumin antibody-immobilized membrane of the cartridge;
(c) administering the dye-bound boronic acid derivative to the membrane and then washing the membrane with a washing solution; and
(d) measuring the optical reflectivity of the membrane with an optical instrument to calculate the ratio of total albumin and glycosylated albumin.
The method of claim 7, wherein the optical device measures the optical reflectance by simultaneously irradiating the wavelength of the albumin detection particle to which the albumin antibody specifically binding to total albumin is bound and the wavelength of the boronic acid derivative to which the dye is bound with a light source. A method for measuring the glycated albumin ratio, characterized in that

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