KR20180040853A - Method for lyophilization liver enzyme and lipid - Google Patents

Abstract

The present invention relates to a freeze-drying method for concentrated liver enzyme test reagent and lipid test reagent. According to the present invention, the freeze-drying method (1) for concentrated liver enzyme test reagent and lipid test reagent comprises: (S100) a test reagent liquid preparation step for including a forming agent in concentrated liver enzyme test reagent and lipid test reagent to prepare a test reagent liquid (L); (S200) an injection step for injecting a fixed amount of the test reagent liquid (L) prepared in the test reagent liquid preparation step (S100) into a cuvette (C); (S300) a test reagent liquid freeze-drying step for freezing the cuvette (C) into which the test reagent liquid (L) has been injected in the injection step (S200) at -50°C under vacuum and heating the same under specific conditions; and (S400) a hermetical packaging step for hermetically packaging the cuvette (C) having finished the test reagent liquid freeze-drying step (S300). According to the present invention, the freeze-drying method facilitates preservation and storage of test reagents that are used together with a sample collected from a subject in order to measure concentrations of liver function enzymes and lipids in a body, and subsequently, improves accuracy of the test, and thus has the advantage of improving the technique for measuring concentrations of liver function enzymes and lipids in a body.

Description

[0001] The present invention relates to a method for lyophilizing liver enzymes and lipids,

The present invention relates to a method for lyophilization of a concentrated hepatic enzyme assay reagent and a lipid assay reagent, and more particularly, to a method for lyophilization of serum and plasma collected from an examinee in order to measure the concentration of hepatic functional enzyme and lipid content And a method for lyophilizing a liver reagent and a lipid test reagent under specific conditions to improve the ease and accuracy of the storage and testing of the test reagent.

Examination of biochemical items using blood is an important index to diagnose basic human diseases.

In general, biochemical tests in hospitals are centered on large biochemical automated testing equipment in clinical laboratories.

In the case of biochemical test reagents, test reagents in the form of dry powder have been frequently used because of preservation / storage of test reagents. However, recently, due to the development of technology, Is a liquid reagent that can be applied to automated testing equipment.

Unlike conventional large-scale biochemical testing equipment, these liquid type biochemical cartridges are aimed at obtaining fast and accurate test results by using a small amount of sample (serum and plasma of an examinee) as a small automated testing equipment.

However, biochemical cartridges that are ready-to-use as a liquid form and a sample taken from an inspector are not only difficult to handle in storage and transportation, but also have a thorough It requires hygienic management.

As a prior art related to a concentrated hepatic enzyme assay reagent and a method for freeze-drying a lipid assay reagent, as shown in Fig. 6 (a), the "liver function test method" of Korean Patent Registration No. 10-0444564 Hereinafter referred to as " Prior Art 1 ") comprises the steps of treating a blood sample passing through the liver of a patient and a blood sample collected every predetermined time after introducing an effective amount of a bile acid derivative of the coloring or fluorescence into the vein of the patient, The blood sample is treated so as to obtain plasma containing the bile acid derivative, the color or fluorescence of the bile acid derivative is measured in the plasma, the measurement value obtained in the step of measuring the color or fluorescence of the bile acid derivative in the plasma is adjusted to the plasma reduction curve The plasma reduction curve obtained from a subject with a known liver function is compared with the plasma reduction curve obtained above to determine the patient ' s liver function And evaluating the color or fluorescence of the bile acid derivative in each sample by the steps of:

As another prior art, as shown in Fig. 6 (b), a method of rapidly analyzing and examining cholesterol and triglycerides with a small amount of sample of Korean Patent Laid-Open Publication No. 10-2015-0094710 (hereinafter referred to as " Prior Art 2 ') is a reagent, assay, kit, device and system for rapidly measuring cholesterol and cholesterol fractions from a blood sample. Total cholesterol, low density lipoprotein cholesterol and high density lipoprotein cholesterol are cholesterol variants can be measured by a single analytical test using kinetic measurements under conditions that convert sub-species into detectable results at a definable (rate) rate, After initiation, measurements were made at different time points to detect lipolytic enzymes, cholesterol esterase, cholesterol oxidase and peroxidase Can be used in combination to produce a colored product in an amount proportional to the amount of converted cholesterol and a method for calculating the level of ultra-dense lipid protein cholesterol, including triglyceride measurement, So that the cholesterol values can be determined more precisely and accurately at a lower cost than can be obtained by performing a number of different analytical tests with various reaction mixtures and the ratio of cholesterol sub-fraction to total cholesterol can be determined .

However, in the prior arts 1 to 2, the sample collected from the tester is mixed with the test reagent of the test item to derive the test result, but the state of the test reagent mixed with the sample is different from that of the present invention There is a problem that the storage of the test reagent and the ease of the test process are deteriorated as described above.

Korean Patent Registration No. 10-0444564 (Aug. 2004) Korean Patent Publication No. 10-2015-0094710 (2015.08.19)

SUMMARY OF THE INVENTION Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art,

It is an object of the present invention to provide a freeze-drying method for a specific test reagent which has excellent linearity, reproducibility and accuracy in the measurement of liver enzymes and lipid concentration.

It is another object of the present invention to provide a method for preparing a test reagent liquid containing a specific molding agent in a concentrated liver function assay reagent and a lipid test reagent for lyophilization, .

Further, it is an object of the present invention to provide an inspection reagent excellent in easiness of preservation and storage.

In order to accomplish the above object, the present invention has been made to solve the above problems,

A method for lyophilization of a concentrated hepatic enzyme assay reagent and a lipid assay reagent,

Enriched hepatic enzymatic assay Reagent and lipid assay Reagent containing a formative agent in a reagent to produce an assay reagent liquid Liquid preparation step;

An injection step of injecting a predetermined amount of the test reagent liquid prepared in the test reagent liquid preparation step into the cuvette;

Freezing the cuvette in which the test reagent liquid is injected at a temperature of minus 50 < 0 > C under vacuum, and heating the cuvette under specific conditions;

And a vacuum packing step of vacuum-packing the cuvette having completed the liquid freeze-drying step of the test reagent, wherein the test reagent mixed with the serum and the plasma obtained from the examinee in the measurement of the concentration of liver enzymes and lipids in the body is lyophilized State.

The test reagent liquid freeze-drying step consists of a vacuum of 0 to 20 [millitorr] pressure. The test reagent liquid is frozen at a temperature of 50 [° C] and slowly heated to a temperature of 30 [ So that the test reagent is freeze-dried.

It should be understood, however, that the terminology used herein is for the purpose of describing the claimed invention in its broadest possible form and should not be construed in a limiting sense. It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the embodiments described in the present specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention, and not all of the technical ideas of the present invention are described. Therefore, various equivalents It should be understood that water and variations may be present.

As described above, according to the present invention,

Concentrated Liver Functional Enzyme Assay Used to Measure Concentration of Liver Functional Enzymes and Lipids Content The lyophilization of reagents and lipid test reagents ensures that linearity, reproducibility and accuracy of the test are improved.

In addition, solid preparation of the concentrated test reagent is accomplished by preparing a test reagent liquid containing a specific molding agent for lyophilization of a concentrated hepatic enzyme assay reagent and lipid assay reagent.

Further, since the safety of the test reagent is secured by the molding agent and the vacuum packaging, the activity is maintained.

In addition, it is a very effective invention that enables the preservation / storage of a vacuum-packed cuvette by storing a lyophilized test reagent and facilitating hygienic inspection by preventing freezing and drying from microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram of a method for freeze-drying a concentrated hepatic enzyme assay reagent and a lipid assay reagent of the present invention.
FIG. 2 is a table showing specific conditions of the liquid reagent freeze-drying step of the reagent in the freeze-drying method of the concentrated hepatic enzyme assay reagent and the lipid assay reagent of the present invention.
FIG. 3 shows an embodiment of a cuvette used in the method of the present invention for lyophilization of a reagent for assaying liver function enzymes and a reagent for lipid analysis.
FIG. 4 is a table showing the items and components of the hepatic functional enzyme assay reagent of the concentrated hepatic enzyme assay reagent and lipid assay reagent of the present invention.
FIG. 5 is a table showing the items and components of a lipid assay reagent of the concentrated liver function enzyme assay reagent and lipid assay reagent of the present invention.
FIG. 6 shows a representative diagram of the prior art for the method of freeze-drying a concentrated hepatic enzyme assay reagent and a lipid assay reagent of the present invention.

Hereinafter, the function, structure and action of the concentrated hepatic enzyme assay reagent and lipid-assay reagent of the present invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram of a method for freeze-drying a concentrated hepatic enzyme assay reagent and a lipid assay reagent of the present invention.

As shown in FIG. 1, the present invention relates to a method for freeze-drying (1) a concentrated hepatic enzyme assay reagent and a lipid assay reagent,

A test reagent liquid preparation step (S100) for preparing a test reagent liquid (L) containing a molding agent in a concentrated hepatic enzymatic test reagent and a lipid test reagent;

An injecting step (S200) of injecting a predetermined amount of the test reagent liquid (L) prepared in the test reagent liquid preparation step (S100) into the cuvette (C);

(S300) of freezing the cuvette (C) injected with the test reagent liquid (L) in a vacuum state at a temperature of minus 50 ° C and heating the cuvette (C) in a specific condition in the injecting step (S200);

And a vacuum packing step (S400) of vacuum packing the cuvette (C) in which the liquid reagent freeze-drying step (S300) is completed. And the test reagent mixed with the test reagent is in a lyophilized state.

The test reagent liquid (L) in the test reagent liquid preparation step (S100) is prepared by adding a molding agent to each of the corresponding test reagents. The molding agent included in the test reagent liquid is a polyethylene glycol (PEG), a dextran, , Bovine serum albumin (BSA), so as to maintain the activity of the enzyme and substrate of the test reagent liquid (L) and liver enzyme test reagent and lipid test reagent.

Meanwhile, AST, ALT and GGT are the test items constituting the assay reagent for hepatic functional enzyme of the present invention. FIG. 4 is a table showing the amounts of raw materials of the hepatic functional assay reagent for measuring the hepatic functional enzyme content. In other words,

AST catalyzes the transamination of the amino acids of el-aspartic acid and alpha-ketoglutarate to produce glutamate and oxaloacetate, which are abundant in the myocardium, liver, brain and other tissues, and are thus used in the diagnosis and treatment of liver and heart disease.

The principle of measurement is that when the serum and plasma are reacted with the concentrated substrate enzyme solution (R1, R2) injected into the lyophilized cuvette (C), the catalytic reaction of AST generates glutamic acid and oxaloacetate, It becomes maleate by the lyase dehydrogenase and at the same time NADH is oxidized to NAD.

Therefore, the activity of AST in serum and plasma is determined by measuring the decrease in absorbance at a measurement wavelength of 340 [nm] by oxidation of NADH.

The concentrated test reagent of the present invention for testing AST is divided into an enzyme reagent (R1) and a substrate reagent (R2)

The enzymatic reagent R1 contained 0.25 mg of Tris (TRISMA BASE) buffer, 4.6 mg of L-Aspartic acid, 0.14 ku of Malate dehydrogenase, 0.37 ku of LDH (lactate dehydrogenase) ],

The substrate reagent R2 is composed of 0.25 mg of Tris (TRISMA BASE) buffer, 0.006 mg of NADH (β-Nicotinamide adenine dinucleotide), and 0.62 mg of alpha-ketoglutarate.

ALT catalyzes the transamination of L-alanine and alpha-ketoglutarate to produce glutamic acid and pyruvic acid, which are abundant in the liver, kidney and other tissues, and are thus used in the diagnosis and treatment of liver disease.

The principle of measurement is that glutamic acid and pyruvic acid are produced by the catalytic reaction of ALT when the serum and plasma are reacted with the concentrated substrate enzyme solution (R1, R2) injected into the lyophilized cuvette (C), and pyruvic acid is converted to lactic dehydrogenase And the NADH is oxidized to NAD.

Therefore, the activity of ALT in serum and plasma is determined by measuring the decrease in absorbance at a measurement wavelength of 340 [nm] by oxidation of NADH.

The concentrated test reagent of the present invention for testing ALT is divided into an enzyme reagent (R1) and a substrate reagent (R2)

The enzyme reagent R1 is concentrated to 0.25 mg of Tris (hydroxymethyl) amino methane, 4.76 mg of L-Alanine and 0.4342 ku of Latate dehydrogenase,

The substrate reagent R2 is composed of 0.24 mg of Tris (hydroxymethyl) amino methane, 0.0661 mg of NADH and 0.6136 mg of α-ketoglutaric acid disodium salt.

Γ-GTP of GGT is an enzyme that degrades gamma-glutamyl peptides into other peptides or amino acids that are abundant in the kidney, pancreas, liver or other tissues due to the activity of gamma-glutamylase. Significant clinical biochemical parameters are considered to be important parameters in observing alcoholic liver disease and its treatment trends.

The principle of measurement is that the serum and plasma react with the concentrated buffer solution (R1) injected into the lyophilized cuvette (C) and the substrate reagent (R2) to form an L-gamma -glutamyl-3-carboxy-4-nitroaniline substrate glutamylglycylglycine and 5-amino-2-benzoic acid are liberated as a result of the action of? -GTP, and the ratio of 5-amino-2-benzoic acid is 405 [nm ] To measure the activity of [gamma] -GTP in serum and plasma.

The concentrated test reagent of the present invention for inspecting GGT is divided into a buffer solution (R1) and a substrate reagent (R2)

The buffer (R1) is concentrated to 1.11 [mg] in glycylglycine buffer (pH 8.14)

The substrate reagent (R2) is constituted by concentrating L-gamma -glutamyl-3-carboxy-4-nitroaniline to 0.64 [mg].

Further, the test items constituting the lipid test reagent are TCHO, TG, HDL-C and LDL-C, which will be described in more detail with reference to the table of the raw material amount of the lipid test reagent for measuring the lipid content in the body shown in FIG. ,

The measurement principle of TCHO is that when serum and plasma are added to concentrated enzyme reagent (R1) and substrate reagent (R2) injected into a lyophilized cuvette (C), cholesterol ester in serum and plasma is hydrolyzed to become cholesterol Cholesterol esterase (CHE) produces fatty acids. Cholesterol is already present in serum and plasma and is oxidized by CO (carbon monoxide) to produce hydrogen peroxide.

Hydrogen peroxide is quantitatively oxidized and condensed by 4 - amino - antipyrine, TOOS and POD enzymes, and the amount of total cholesterol in serum and plasma is determined by absorbance measurement at a measuring wavelength of 546 [nm].

The concentrated test reagent of the present invention for testing TCHO is divided into an enzyme reagent (R1) and a substrate reagent (R2)

The enzyme reagent R1 was concentrated to HEPES 1.192 [mg], POD 0.1 [ku], CHE 0.09 [ku] and ASOD 0.182 [ku]

The substrate reagent R2 is composed of HEPES 1.192 [mg], COD 0.24 [ku], and TOOS 0.064 [mg].

TG is a lipid of serum composed of triglyceride (triglyceride), cholesterol, phospholipid, free fatty acid and trace amount of fat-soluble substance such as fat soluble vitamin and carotene.

The measurement of triglyceride in serum is an important test for the diagnosis of arteriosclerosis, coronary artery disease and diabetes. The principle of measurement is that triglyceride in serum is decomposed into glycerol and fatty acid by the action of lipoprotein lipase (LPL). Glycerolkinase GK) and ATP act on α-Glycerol-3-phosphate and ADP.

At this time, α-Glycerol-3-phosphate is α-Glycerol-3-phosphate- oxidase (GPO) of H ₂ O ₂ generating a H ₂ O ₂ by the action and the generation of the 4-Aminoantipyrine presence of Peroxidase (POD) (4-AAP) and ADPS are measured by colorimetry at a measurement wavelength of 546 [nm].

The concentrated test reagent of the present invention for testing TG is divided into an enzyme reagent (R1) and an enzyme reagent (R2)

The enzyme reagent R1 contained PIPES 1.512 [mg], glycerol kinase (GK) 0.3 [ku], ATP 0.109 [mg], GPO 0.8 [ku], ascorbic acid oxidase 0.101 [ku], 4-aminoantipyrine 0.0106 [mg ],

The enzyme reagent R2 is composed of PIPES 1.512 [mg], peroxidase (POD) 0.272 [ku], and lipoprotein lipase (LPL) 80 [ku].

It has been reported that serum total cholesterol levels have been associated with coronary heart disease for a long time. Recently, it has been reported that the HDL-C level and the incidence of coronary heart disease are closely related, so that high density lipoprotein cholesterol ) Is a risk factor for the development of coronary heart disease and is an important test for diagnosing this disease.

Although selective chemical precipitation has been widely used to quantify HDL-C, this method is not suitable for large-scale analytical laboratories due to the separate precipitation procedure.

Therefore, the present invention is freeze-dried test reagent for quantitative test, and does not require a precipitation operation using a specific surfactant, and can be applied to various automatic analyzers.

Cholesterol, LDL, cholesterol and cholesterol are produced by CHE, COD and POD enzymes under specific conditions. Cholestenone and H ₂ O ₂ are produced by the enzymes of Chylomicron, VLDL and LDL in the first step. H ₂ O ₂ is decomposed into water and oxygen by catalase.

The remaining HDL-Cholesterol in step 2 forms a pigment by CHE, COD and POD enzymatic reaction in the presence of a specific surfactant. The change of absorbance of this dye is measured and the concentration of HDL-Cholesterol is measured at a measurement wavelength of 546 [nm] .

The concentrated test reagent of the present invention for testing HDL-C is divided into an enzyme reagent (R1) and an enzyme reagent (R2)

The enzyme reagent R1 is concentrated to HEPES 1.192 [mg], POD 0.1 [ku], ASOD 0.182 [ku], COD 0.08 [ku] and DSBmT 0.064 [mg]

The enzyme reagent R2 is composed of HEPES 1.192 [mg], CHE 0.27 [ku], and 4-aminoantipyrine 0.0342 [mg].

In the case of LDL-C, blood total cholesterol is closely related to cardiovascular disease, and it is an important test to assess the blood level of LDL-C and the risk of developing coronary vascular disease.

Recently, it has been tested by beta-quantification method which is a combination of ultracentrifugation and chemical precipitation. However, this method is not suitable for general examination because it has many manual and high machine dependence.

Therefore, the Friedewald method has been used as the most general LDL calculation method. However, since this calculation method calculated from total cholesterol depends on the precision and accuracy of TG and HDL-C, it is impossible to measure pure LDL concentration and TG is 250 [mg / And it has limitations in that it can be used only in the fasting state.

Therefore, in the present invention, the LDL-C value is calculated from the equation according to the analysis of TG and TCHO by the measurement principle.

In this case,

LDL-C = TOTAL CHOLESTEROL - HDL CHOLESTEROL - TG / 5, and the unit is [mg / dL].

As described above, the present invention provides a method of measuring the concentration of liver enzymes and lipids in blood serum and plasma obtained from an examinee, wherein the test reagent liquid (L) comprising liver enzyme assay reagent and lipid assay reagent is lyophilized AST, ALT and GGT, which are the test items of hepatic enzymes, are applied to the measurement wavelengths of 340 nm, 340 nm, and 405 nm, respectively, as described above by KINETIC TEST. TG, TG, HDL-C, and LDL-C were measured at endpoints of 546 [nm] at a wavelength of 546 [nm] The resultant value is calculated.

AST, ALT, and GGT were detected with a spectrophotometer at a measurement wavelength of 340 nm and a measurement wavelength of 405 nm, respectively, and the lipid test items TCHO, TG, and HDL- C and LDL-C are detected using a measurement wavelength of 546 [nm].

The test reagent liquid freeze-drying step (S300) is performed by freezing the test reagent liquid (L) at a temperature of 50 [占 폚] under a vacuum of 0 to 20 [millitorr] Keep the temperature [hr] and freeze-dry the test reagent.

That is, the present invention lyes the test reagent liquid (L) by a specific lyophilization method comprising temperature and time as shown in FIG.

Thus, liver enzyme assay reagent and lipid assay reagent composed of specific enzyme reagent and substrate reagent are molded with specific molding agent and lyophilized to maximize the easiness of preservation / storage and accuracy of test result.

In the present invention, as a concentrated test reagent, the test can be performed with an ultra-minute amount of serum and plasma from 0.5 to 5.0 [ul] from an examiner, and the reagent for assaying liver function enzymes and the reagent for lipid reaction, (C) is injected with 50 [mu] l and lyophilized.

Therefore, when measuring the hepatic functional enzyme and lipid content, 50 [ul] of purified water is respectively injected into the lyophilized cuvette (C) before the serum collected from the examinee and the plasma are mixed, whereby the lyophilized test reagent liquid ).

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined in the appended claims.

Therefore, it is to be understood that the present invention may be embodied in many other specific forms without departing from the spirit or essential characteristics thereof, and the embodiments are to be considered in all respects as illustrative and not restrictive.

The present invention is a method for lyophilizing a concentrated hepatic enzyme assay reagent and a lipid assay reagent and is applied to an assay for detecting the concentration of liver enzymes and lipids in the body. Therefore, various inspection bodies such as hospitals and public health centers, It can be applied to contribute to the promotion of various industries.

1: Freeze-dried method of concentrated hepatic enzyme assay reagent and lipid test reagent
S100: Test reagent liquid preparation step S200: Injection step
S300: Liquid freeze-drying step S400: Vacuum packaging step
C: Cuvette L: Test reagent liquid

Claims (3)

In the lyophilization method (1) of a concentrated hepatic enzyme assay reagent and a lipid assay reagent,
A test reagent liquid preparation step (S100) for preparing a test reagent liquid (L) containing a molding agent in a concentrated hepatic enzymatic test reagent and a lipid test reagent;
An injecting step (S200) of injecting a predetermined amount of the test reagent liquid (L) prepared in the test reagent liquid preparation step (S100) into the cuvette (C);
(S300) of freezing the cuvette (C) injected with the test reagent liquid (L) in a vacuum state at a temperature of minus 50 ° C and heating the cuvette (C) in a specific condition in the injecting step (S200);
And a vacuum packing step (S400) of vacuum packing the cuvette (C) in which the liquid reagent freeze-drying step (S300) is completed. And the freeze-dried test reagent mixed with the test substance is in a lyophilized state. The method according to claim 1,
Wherein the test reagent liquid freeze-drying step (S300) comprises a vacuum of 0 to 20 millitorr pressure. The method according to claim 1,
In the liquid freeze-drying step (S300) of the test reagent, the test reagent liquid (L) is frozen at a temperature of 50 [° C] and slowly heated to maintain the temperature at 30 [° C] And a method for lyophilization of reagents and lipid test reagents.

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