CN114480565A - Stable high-sensitivity glycated albumin determination kit with strong anti-interference capability - Google Patents
Stable high-sensitivity glycated albumin determination kit with strong anti-interference capability Download PDFInfo
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Abstract
The invention relates to the technical field of medical detection, in particular to a stable and high-sensitivity glycated albumin determination kit with strong anti-interference capability, which comprises a first reagent and a second reagent, wherein the ratio of the first reagent to the second reagent is 4: 1; the first reagent comprises 20-200 mmol/L buffer solution, 1-5 g/L proteinase K, 0.1-1 g/L4-aminoantipyrine, 0.05-0.1 g/L nitroxide free radical oxide, 5-50 KU/L catalase, 0.5-5 g/L nonionic surfactant and 0.1-1 g/L preservative; the second reagent comprises 20-200 mmol/L buffer solution, 1-10 g/L ethylene glycol bisaminoethyl ether tetraacetic acid, 0.5-5 g/L nonionic surfactant, 10-100 g/L glycerol, 10-100 g/L polyalcohol, 0.5-5 g/L sodium aniline sulfonate, 0.1-0.5 g/L hydroxyl-2, 4, 6-triiodobenzoic acid, 5-20 ku/L fructosamine oxidase, 10-30 ku/L peroxidase, 20-50 mu mol/L potassium ferrocyanide and 0.1-1 g/L preservative; the glycated albumin can be stably measured using the first reagent and the second reagent.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a high-sensitivity glycated albumin determination kit which is stable and has strong anti-interference capability.
Background
Glycated serum albumin (GA) is a product obtained after serum albumin is glycated by glucose, namely, serum protein and glucose are subjected to non-enzymatic glycosylation reaction, wherein 90% of glycated serum protein is formed by combining with lysine in a serum protein chain, and more than 90% of glycated albumin is obtained by saccharification, and the half-life of glycated albumin is short, so that the measured concentration of glycated albumin can reflect the blood sugar level of a human body in the past 2-3 weeks and is not influenced by temporary blood sugar fluctuation, and therefore, GA has important significance in the diagnosis and treatment of diabetes; the conventional method for monitoring the glycated albumin mainly comprises an affinity chromatography method, an NBT method, a protease method and the like, wherein the protease method has the advantages of good specificity, no pollution to a biochemical analyzer and the like, and is the mainstream of the development of the current domestic automatic detection kit, but the kit using the method generally has the problem of poor stability and needs to be improved.
Disclosure of Invention
The invention aims to provide a high-sensitivity glycated albumin assay kit which is stable and has strong anti-interference capability, and can stably assay glycated albumin.
In order to achieve the purpose, the invention provides a stable and high-sensitivity glycated albumin assay kit with strong anti-interference capability, which comprises a first reagent and a second reagent, wherein the ratio of the first reagent to the second reagent is 4: 1;
the reagent I comprises: 20-200 mmol/L buffer solution, 1-5 g/L proteinase K, 0.1-1 g/L4-aminoantipyrine, 0.05-0.1 g/L nitroxide free radical oxide, 5-50 KU/L catalase, 0.5-5 g/L nonionic surfactant and 0.1-1 g/L preservative;
the second reagent comprises: 20-200 mmol/L buffer solution, 1-10 g/L ethylene glycol bis-aminoethyl ether tetraacetic acid, 0.5-5 g/L nonionic surfactant, 10-100 g/L glycerol, 10-100 g/L polyalcohol, 0.5-5 g/L sodium anilinesulfonate, 0.1-0.5 g/L hydroxyl-2, 4, 6-triiodobenzoic acid, 5-20 ku/L fructosamine oxidase, 10-30 ku/L peroxidase, 20-50 mu mol/L potassium ferrocyanide and 0.1-1 g/L preservative.
The preparation method of the reagent I comprises the following steps: mixing the buffer solution, the nonionic surfactant, the preservative, the 4-aminoantipyrine and the nitroxide free radical oxide into purified water to obtain a first mixed solution;
adding the proteinase K and the catalase into the first mixed solution, stirring until the proteinase K and the catalase are completely dissolved, adding purified water, uniformly mixing, standing, and filtering insoluble substances to obtain the reagent I.
The preparation method of the reagent II comprises the following steps: mixing the buffer solution, the ethylene glycol bis (aminoethyl ether) tetraacetic acid, the non-ionic surfactant, the glycerol, the polyol, the sodium salt of the aniline sulfonic acid, the hydroxy-2, 4, 6-triiodobenzoic acid, the potassium ferrocyanide and the preservative into purified water to obtain a second mixed solution;
and adding the fructosamine oxidase and the peroxidase into the second mixed solution, stirring until the fructosamine oxidase and the peroxidase are completely dissolved, adding purified water, uniformly mixing, standing, and filtering insoluble substances after standing to obtain the reagent II.
Wherein the buffer solution in the reagent I is an alkyl sulfonic acid buffer solution in Good's biological buffer solution with the pH value of 7.5-8.5.
Wherein the buffer solution in the second reagent is Good's biological buffer solution with the pH value of 7.5-8.5.
Wherein the nonionic surfactants in the first reagent and the second reagent are both one or two of polyoxyethylene lauryl ether and alkylphenol polyoxyethylene ether.
Wherein the preservative in the first reagent and the second reagent is one or two of methyl chloro isothiazolinone and methyl isothiazolinone.
The sodium salt of aniline sulfonic acid in the reagent II is one of sodium salt of N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-N-bis (4-sulfobutyl) -3-methylaniline and sodium salt of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline.
Wherein, the polyalcohol in the reagent II is one of hexahydric alcohol and dihydric alcohol.
Wherein the nitroxide oxide in the first reagent is one or the combination of two of nitroxide piperidinol and 3-oxo-2-phenyl-4, 4, 5, 5-tetramethyl imidazoline-1-oxygen.
The invention discloses a high-sensitivity glycated albumin assay kit which is stable and has strong anti-interference capability, and the principle is as follows: proteinase K decomposes glycated albumin in a human serum (or plasma) sample to produce glycated amino acids; then in the presence of a protease inhibitor, the glycated amino acid is converted into a glucuronone, an amino acid and hydrogen peroxide by the action of a fructosamine amino acid oxidase; under the action of peroxidase, carrying out Trinder reaction on hydrogen peroxide, 4-aminoantipyrine and 3-hydroxy-2, 4, 6-triiodobenzoic acid to generate red quinonimine, and measuring the absorbance change of the red quinonimine to obtain the concentration of the glycated albumin in the sample; the method is characterized in that the rapid enzymolysis of the glycated albumin is realized by adopting serine protease proteinase-proteinase K with high specificity and good stability under a low metal ion interference system through the assistance of a specific nonionic surfactant, and a reducing endogenous substance which seriously interferes with the trinder reaction in a sample is masked by using a specific nitroxide free radical oxide; the influence of oxide on 4-aminoantipyrine in the reagent is eliminated through a certain amount of catalase, so that the long-time stability of a reagent test blank is realized; under the condition that ethylene glycol bisaminoethylether tetraacetic acid inhibits the activity of proteinase K, glycated albumin is accurately determined by the high specificity effect of relatively cheap and efficient lysine type fructosamine amino acid oxidase on glycated amino acid, and a Trinder reaction color development system with high sensitivity and good stability is established by means of the combination of sodium anilinesulfonate and hydroxy-2, 4, 6-triiodobenzoic acid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a method of preparing the reagent of the present invention.
FIG. 2 is a flow chart of a method for preparing reagent two according to the present invention.
FIG. 3 is an anti-interference experimental table of the stable, strong anti-interference and high-sensitivity glycated albumin assay kit of the present invention.
FIG. 4 is a table showing the correlation between the clinical results of example 1 and the same type of test agent.
FIG. 5 is a table showing the correlation between the clinical results of example 2 and the same type of test agent.
FIG. 6 is a table showing the correlation between the clinical results of example 3 and the same type of test agent.
FIG. 7 is a table showing the accuracy of the stable and highly interference-resistant glycated albumin assay kit of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, it is to be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships illustrated in the drawings, and are used merely for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, are not to be construed as limiting the present invention. Further, in the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Referring to fig. 1 to 7, the present invention provides a stable and highly interference-resistant assay kit for glycated albumin with high sensitivity, comprising: the reagent kit comprises a first reagent and a second reagent, wherein the ratio of the first reagent to the second reagent is 4: 1;
the reagent I comprises: 20-200 mmol/L buffer solution, 1-5 g/L proteinase K, 0.1-1 g/L4-aminoantipyrine, 0.05-0.1 g/L nitroxide free radical oxide, 5-50 KU/L catalase, 0.5-5 g/L nonionic surfactant and 0.1-1 g/L preservative;
the second reagent comprises: 20-200 mmol/L buffer solution, 1-10 g/L ethylene glycol bis-aminoethyl ether tetraacetic acid, 0.5-5 g/L nonionic surfactant, 10-100 g/L glycerol, 10-100 g/L polyalcohol, 0.5-5 g/L sodium anilinesulfonate, 0.1-0.5 g/L hydroxyl-2, 4, 6-triiodobenzoic acid, 5-20 ku/L fructosamine oxidase, 10-30 ku/L peroxidase, 20-50 mu mol/L potassium ferrocyanide and 0.1-1 g/L preservative.
Further, the buffer solution in the reagent I is an alkyl sulfonic acid buffer solution in Good's biological buffer solution with the pH value of 7.5-8.5.
Further, the buffer solution in the second reagent is Good's biological buffer solution with the pH value of 7.5-8.5.
Further, the nonionic surfactants in the first reagent and the second reagent are both one or two of polyoxyethylene lauryl ether and alkylphenol polyoxyethylene ether.
Further, the preservative in the first reagent and the second reagent is one or two of methyl chloro isothiazolinone and methyl isothiazolinone.
Further, the sodium salt of aniline sulfonic acid in the second reagent is one of sodium salt of N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-bis (4-sulfobutyl) -3-methylaniline and sodium salt of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline.
Further, the polyol in the second reagent is one of hexahydric alcohol and dihydric alcohol.
Further, the nitroxide oxide in the first reagent is one or a combination of two of nitroxide piperidinol and 3-oxo-2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxygen.
In the present embodiment, the principle is: proteinase K decomposes glycated albumin in a human serum (or plasma) sample to produce glycated amino acids; then in the presence of a protease inhibitor, the glycated amino acid is converted into a glucuronone, an amino acid and hydrogen peroxide by the action of a fructosamine amino acid oxidase; under the action of peroxidase, carrying out Trinder reaction on hydrogen peroxide, 4-aminoantipyrine and 3-hydroxy-2, 4, 6-triiodobenzoic acid to generate red quinonimine, and measuring the absorbance change of the red quinonimine to obtain the concentration of the glycated albumin in the sample; the method is characterized in that rapid enzymolysis of the glycated albumin is realized by adopting serine protease proteinase-proteinase K with high specificity and good stability under a low metal ion interference system through the assistance of a specific nonionic surfactant, and a reducing endogenous substance which seriously interferes with a trinder reaction in a sample is masked by using a specific nitroxide radical oxide; the influence of oxide on 4-aminoantipyrine in the reagent is eliminated through a certain amount of catalase, so that the long-time stability of a reagent test blank is realized; under the condition that ethylene glycol bisaminoethylether tetraacetic acid inhibits the activity of proteinase K, the glycated albumin is accurately measured by the high specificity effect of the relatively cheap and efficient lysine type fructosamine amino acid oxidase on the glycated amino acid, and a Trinder reaction color development system with high sensitivity and good stability is established by means of the combination of sodium anilinesulfonate and hydroxy-2, 4, 6-triiodobenzoic acid; in a stability load experiment that the reagent I and the reagent II are placed at 37 ℃ for 7 days and a storage stability experiment that the reagent I and the reagent II are stored at 4-8 ℃ for 12 months, the appearance, the sensitivity, the accuracy and the linear range of the reagent I and the reagent II have no obvious change, and completely meet the requirements of clinical detection. The invention can adopt a manual testing method and can also detect samples through analysis instruments such as a full-automatic biochemical analyzer, and the like, and specifically adopts the following calculation method:
further, the preparation method of the reagent I comprises the following steps:
s101, mixing the buffer solution, the nonionic surfactant, the preservative, the 4-aminoantipyrine and the nitroxide free radical oxide into purified water to obtain a first mixed solution;
adding purified water accounting for about 80% of the total amount required by the preparation of the first reagent into a mixing container, accurately weighing and sequentially stirring to dissolve the buffer solution, the nonionic surfactant, the preservative, the 4-aminoantipyrine and the nitroxide free radical oxide in full batch into the mixing container, and continuously stirring for 5 minutes after complete dissolution; whether the pH value is 8.0 +/-0.05 or not is tested, and otherwise, the pH value is adjusted to be within the range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution, so that a first mixed solution is prepared.
S102, adding the proteinase K and the catalase into the first mixed solution, stirring until the proteinase K and the catalase are completely dissolved, adding purified water, uniformly mixing, standing, and filtering insoluble substances to obtain a first reagent;
accurately weighing the protease K and the catalase in full batch, adding the protease K and the catalase into the first mixed solution, and slowly stirring for 10 minutes until the protease K and the catalase are completely dissolved, wherein violent stirring and a magnetic stirrer are avoided; and (3) quantifying with purified water to a preparation amount, uniformly stirring, standing and balancing the solution for 15 minutes, and filtering insoluble substances with a 0.8-micron-aperture nylon filter membrane to obtain the reagent I.
Further, the preparation method of the reagent II comprises the following steps:
s201, mixing the buffer solution, the ethylene glycol bis-aminoethyl ether tetraacetic acid, the non-ionic surfactant, the glycerol, the polyol, the sodium salt of aniline sulfonic acid, the hydroxy-2, 4, 6-triiodobenzoic acid, the potassium ferrocyanide and the preservative into purified water to obtain a second mixed solution;
adding purified water accounting for about 80% of the total amount required by the preparation of the reagent I into a mixing container, accurately weighing and sequentially stirring to dissolve the buffer solution, the ethylene glycol bis-aminoethyl ether tetraacetic acid, the nonionic surfactant, the glycerol, the polyol, the sodium salt of aniline sulfonic acid, the hydroxy-2, 4, 6-triiodobenzoic acid, the potassium ferrocyanide and the preservative in a full batch into the mixing container, and continuously stirring for 5 minutes after complete dissolution; the pH was tested to determine whether it was 8.0. + -. 0.05 or not, otherwise adjusted to within this range with 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution to obtain a second mixed solution.
S202, adding the fructosamine amino acid oxidase and the peroxidase into the second mixed solution, stirring until the fructosamine amino acid oxidase and the peroxidase are completely dissolved, adding purified water, uniformly mixing, standing, and filtering insoluble substances after standing to obtain a reagent II;
accurately weighing the fructosamine oxidase and the peroxidase in full batch, adding into the second mixed solution, and slowly stirring for 10 minutes until completely dissolving, wherein violent stirring and a magnetic stirrer are avoided; and (3) quantifying with purified water to a preparation amount, uniformly stirring, standing and balancing the solution for 15 minutes, and filtering insoluble substances with a 0.8-micron-aperture nylon filter membrane to obtain the reagent II.
For a better understanding of the present invention, several specific manufacturing processes are described below.
Example 1: the reagent I adopts MES buffer solution with 50mol/LPH value of 8.0, 2g/L proteinase K, 0.3g/L, 4-aminoantipyrine, 0.2g/L nitroxide radical piperidinol, 20KU/L catalase, 1g/LTriton X-100 and 0.5g/LPROClin300 as components; the preparation method comprises the following steps: adding purified water accounting for about 80% of the total amount required by the preparation of the reagent I into a mixing container, respectively and accurately weighing and sequentially stirring and dissolving a whole batch of MES, Triton X-100, 4-aminoantipyrine and nitroxide radical piperidinol into the mixing container, continuously stirring for 5 minutes after complete dissolution, and adjusting the pH value to be within the range of 8.0 +/-0.05 by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution; accurately weighing and adding a full batch of proteinase K and catalase, and slowly stirring for 10 minutes until the proteinase K and the catalase are completely dissolved, so that violent stirring and a magnetic stirrer are avoided; accurately weighing and adding ProClin300 in full batch, stirring for dissolving, quantifying with purified water to a preparation amount, stirring uniformly, standing and balancing the solution for 15 minutes, and filtering insoluble substances with a 0.8 micron-aperture nylon filter membrane.
The reagent II comprises 200mmol/LPH value of Tris-HCl buffer solution of 8.0, 3.5g/L ethylene glycol bis-aminoethyl ether tetraacetic acid, 0.5g/L LTriton X-100, 50g/L glycerol, 30g/L ethylene glycol, 1g/LADPS, 0.2g/L hydroxyl-2, 4, 6-triiodobenzoic acid, 10ku/L fructosamine amino acid oxidase, 15ku/L peroxidase, 30 mu mol/L potassium ferrocyanide and 0.5g/L ProClin 300; the preparation method comprises the following steps: adding purified water accounting for about 80% of the total amount required by the preparation of the reagent II into a mixing container, respectively and accurately weighing a full batch of Tris, ethylene glycol bisaminoethyl ether tetraacetic acid, Triton X-100, glycerol, ethylene glycol, ADPS, hydroxyl-2, 4, 6-triiodobenzoic acid, potassium ferrocyanide and ProClin300, sequentially adding into the mixing container, stirring one by one until the fructosyl amino acid oxidase and the peroxidase are completely dissolved, continuing stirring for 5 minutes, testing whether the pH value is 8.0 +/-0.05, otherwise adjusting the pH value to the range by using a 6mol/L hydrochloric acid solution or a 10mol/L sodium hydroxide solution, respectively accurately weighing the full batch of the fructosyl amino acid oxidase and the peroxidase, sequentially adding the fructosyl amino acid oxidase and the peroxidase into a mixing container, stirring one by one until the solution is completely dissolved, continuing stirring for 5 minutes, quantifying with purified water to a preparation amount, stirring uniformly, standing and balancing the solution for 15min, and filtering insoluble substances with a 0.8 micron-aperture nylon filter membrane.
Example 2: the first reagent component adopts 50mol/LPH value 7.8 HEPES buffer solution, 3g/L proteinase K, 0.5 g/L4-aminoantipyrine, 0.4g/LPTIO, 10KU/L catalase, 1g/LBRIJ 35, 0.5g/L sodium azide, and the preparation method is the same as that of the example 1;
the reagent II adopts Tricine buffer solution with 200mmol/LPH value of 8.2, 3g/L ethylene glycol bisaminoethyl ether tetraacetic acid, 0.5g/LBRIJ 35, 30g/L glycerol, 30g/L sorbitol, 1g/LTODB, 0.3g/L hydroxyl-2, 4, 6-triiodobenzoic acid, 15ku/L fructosamine oxidase, 20ku/L peroxidase, 30 mu mol/L potassium ferrocyanide and 0.5g/L sodium azide, and the preparation method is the same as the example 1.
Example 3: the reagent adopts MES buffer solution with 100mol/LPH value of 7.8, 2.5g/L proteinase K, 0.75 g/L4-aminoantipyrine, 0.1g/LPTIO, 0.2g/L nitroxide radical piperidinol, 15KU/L catalase, 1g/LTriton X-100, 0.5g/LBRIJ 35 and 1g/L sodium azide as components; the preparation method is the same as that of example 1;
the preparation method of the reagent II comprises the steps of adopting 200mmol/LPH value of Tris-HCl buffer solution with 8.5, 5g/L ethylene glycol bisaminoethyl ether tetraacetic acid, 1g/L LTriton X-100, 0.5g/LBRIJ 35, 50g/L glycerol, 20g/L mannitol, 0.5 g/LMOAS, 1g/L hydroxyl-2, 4, 6-triiodobenzoic acid, 12ku/L fructosamine oxidase, 20ku/L peroxidase, 25 mu mol/L potassium ferrocyanide and 1g/L sodium azide to prepare the same as the example 1.
Through determination, in a stability load experiment that the first reagent and the second reagent are placed at 37 ℃ for 7 days and a storage stability experiment that the first reagent and the second reagent are stored at 4-8 ℃ for 12 months, the appearance of the reagents has no obvious change, when the reagents are used for testing a sample with the glycated albumin concentration of 14.00g/L, the absorbance difference (delta A) is 0.0500-0.0540 which is far higher than the standard requirement of a standard YY/T1578-2018 glycated albumin testing kit (enzyme method) of not less than 0.0200, the upper limit of the GA concentration of the reagents can reach 30.0g/L, and if the samples contain the following interferents, the detection result is not influenced: ascorbic acid is less than 50mg/dL, bilirubin is less than 20mg/dL, hemoglobin is less than 200mg/dL, uric acid is less than 35mg/dL, heparin sodium is less than 100U/mL, fat emulsion (intralipd) is less than 600mg/dL, and glucose is less than 2400 mg/dL.
Interference test: referring to fig. 3, the anti-interference performance of the first reagent and the second reagent is tested by an addition method, items and concentrations marked on a table shown in fig. 3 are selected, an interfering substance concentrated solution is prepared according to 10 times of the marked amount of the interfering concentration, 1 part of the concentrated solution is sucked from the interfering substance concentrated solution, 9 parts of sample serum is added to the interfering substance concentrated solution and mixed uniformly, physiological saline with the same volume is used for replacing the interfering substance concentrated solution for a comparison sample, each sample is tested for 3 times, and the relative deviation of the average value is calculated; and (3) judging: no interference was found if the test results deviate by no more than ± 10% from the blank.
And (3) verifying clinical relevance: referring to fig. 4-6, the reagent prepared by the embodiment formula is compared with a GA assay kit sold in some companies approved by the national drug administration for comparison detection, 100 clinical sample test samples are simultaneously detected on a hitachi 7180 biochemical analyzer, and correlation and paired T-test analysis are performed on two groups of test results, the results show that the linear correlation coefficient R of two groups of data of the three embodiment formulas and the comparison reagent is greater than 0.9750 (R of the first embodiment is 0.9989; R of the second embodiment is 0.9971; R of the third embodiment is 0.9975), and P of the T-test analysis is greater than 0.05, which indicates that the reagent and the similar detection reagent have good correlation in clinical test results and no obvious difference in results.
And (3) verification of accuracy: referring to FIG. 7, three levels of International glycated Albumin Standard (JCCRM, Japan) JCCRM 611-1M, JCCRM611-1H, JCCRM611-1HH were tested using the formulated reagent of example 1, and the results met the accuracy requirement that the relative deviation as calibrated by the YY/T1578-2018 glycated Albumin assay kit (enzymatic method) was no more than. + -. 10%.
The invention relates to a high-sensitivity glycated albumin assay kit with high stability and high anti-interference capability, which realizes rapid enzymolysis of glycated albumin under a low metal ion interference system by adopting serine protease proteinase-proteinase K with high specificity and good stability and assisted by a specific nonionic surfactant, and masks a reducing endogenous substance which has serious interference on a trinder reaction in a sample by using a specific nitroxide oxide. The influence of an oxide on 4-aminoantipyrine in the reagent is eliminated through a certain amount of catalase, the long-time stability of the blank of the reagent test is realized, the glycated albumin is accurately measured through the high specificity effect of the relatively cheap and efficient lysine type fructosyl amino acid oxidase on the glycated amino acid under the condition that the ethylene glycol bisaminoethyl ether tetraacetic acid inhibits the activity of proteinase K, and meanwhile, a Trinder reaction color development system with high sensitivity and good stability is established by means of the combination of sodium salt of aniline sulfonate and hydroxy-2, 4, 6-triiodobenzoic acid.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A high-sensitivity glycated albumin assay kit with stability and strong anti-interference capability is characterized in that,
the reagent kit comprises a first reagent and a second reagent, wherein the ratio of the first reagent to the second reagent is 4: 1;
the reagent I comprises: 20-200 mmol/L buffer solution, 1-5 g/L proteinase K, 0.1-1 g/L4-aminoantipyrine, 0.05-0.1 g/L nitroxide free radical oxide, 5-50 KU/L catalase, 0.5-5 g/L nonionic surfactant and 0.1-1 g/L preservative;
the second reagent comprises: 20-200 mmol/L buffer solution, 1-10 g/L ethylene glycol bis-aminoethyl ether tetraacetic acid, 0.5-5 g/L nonionic surfactant, 10-100 g/L glycerol, 10-100 g/L polyalcohol, 0.5-5 g/L sodium anilinesulfonate, 0.1-0.5 g/L hydroxyl-2, 4, 6-triiodobenzoic acid, 5-20 ku/L fructosamine oxidase, 10-30 ku/L peroxidase, 20-50 mu mol/L potassium ferrocyanide and 0.1-1 g/L preservative.
2. The kit for assaying glycated albumin with high sensitivity and high stability and high anti-interference capability according to claim 1, wherein the first reagent is prepared by a method comprising:
mixing the buffer solution, the nonionic surfactant, the preservative, the 4-aminoantipyrine and the nitroxide free radical oxide into purified water to obtain a first mixed solution;
adding the proteinase K and the catalase into the first mixed solution, stirring until the proteinase K and the catalase are completely dissolved, adding purified water, uniformly mixing, standing, and filtering insoluble substances to obtain the reagent I.
3. The kit for assaying glycated albumin with high sensitivity, stability and high anti-interference ability according to claim 1, wherein the second reagent is prepared by a method comprising:
mixing the buffer solution, the ethylene glycol bis (aminoethyl ether) tetraacetic acid, the non-ionic surfactant, the glycerol, the polyol, the sodium salt of the aniline sulfonic acid, the hydroxy-2, 4, 6-triiodobenzoic acid, the potassium ferrocyanide and the preservative into purified water to obtain a second mixed solution;
and adding the fructosamine oxidase and the peroxidase into the second mixed solution, stirring until the fructosamine oxidase and the peroxidase are completely dissolved, adding purified water, uniformly mixing, standing, and filtering insoluble substances after standing to obtain the reagent II.
4. The kit according to claim 1, wherein the glycated albumin with high sensitivity is stable and resistant to interference,
the buffer solution in the reagent I is an alkyl sulfonic acid buffer solution in Good's biological buffer solution with the pH value of 7.5-8.5.
5. The kit according to claim 1, wherein the glycated albumin with high sensitivity is stable and resistant to interference,
the buffer solution in the reagent II is Good's biological buffer solution with the pH value of 7.5-8.5.
6. The kit for assaying glycated albumin with high sensitivity and high stability and anti-interference ability according to claim 1, wherein,
the nonionic surfactants in the reagent I and the reagent II are both one or two of polyoxyethylene lauryl ether and alkylphenol polyoxyethylene.
7. The kit according to claim 1, wherein the glycated albumin with high sensitivity is stable and resistant to interference,
the preservative in the first reagent and the second reagent is one or two of methyl chloro isothiazolinone and methyl isothiazolinone.
8. The kit for assaying glycated albumin with high sensitivity and high stability and anti-interference ability according to claim 1, wherein,
and the aniline sulfonic acid sodium salt in the reagent II is one of N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline sodium salt, N-N-bis (4-sulfobutyl) -3-methylaniline and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline sodium salt.
9. The kit according to claim 1, wherein the glycated albumin with high sensitivity is stable and resistant to interference,
and the polyhydric alcohol in the reagent II is one of hexahydric alcohol and dihydric alcohol.
10. The reagent kit for assaying glycated albumin with high sensitivity, stability and high anti-interference ability as claimed in claim 1, wherein the nitroxide oxide in the first reagent is one or two of nitroxide piperidinol and 3-oxo-2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxide.
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