CN116381252A - Glycosylated hemoglobin determination kit - Google Patents
Glycosylated hemoglobin determination kit Download PDFInfo
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- CN116381252A CN116381252A CN202310208306.5A CN202310208306A CN116381252A CN 116381252 A CN116381252 A CN 116381252A CN 202310208306 A CN202310208306 A CN 202310208306A CN 116381252 A CN116381252 A CN 116381252A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biological detection kits for in-vitro diagnosis, and discloses a kit for glycosylated hemoglobin measurement, which comprises a reagent 1, a reagent 2 and hemolysis. Wherein the pH value of the reagent 1 is 5.5-8.5, and the reagent comprises latex microspheres, a first buffer solution, a first preservative and deionized water; the pH value of the reagent 2 is 5.5-8.5, and the reagent comprises glycosylated hemoglobin monoclonal nano-antibody, goat anti-mouse IgG antibody, a second buffer solution, a second preservative and deionized water; the hemolytic liquid comprises sodium chloride, tritonX-100 and deionized water. The invention has the beneficial effects that: the latex microspheres prevent the glycosylated hemoglobin antibodies from being adsorbed or connected to the latex, eliminate the adverse effect of the hemoglobin content in different blood samples on the accuracy of detection results, and the monoclonal nano antibodies and the goat anti-mouse antibodies are combined and are easier to solidify on the latex microspheres, so that the kit has better sensitivity and specificity, higher accuracy, simple detection flow, high efficiency and low cost.
Description
Technical Field
The invention belongs to the technical field of biological detection kits, and particularly relates to a glycosylated hemoglobin determination kit with high detection sensitivity and low cost.
Background
Glycosylated hemoglobin (glycosylated hemoglobin, hbAlc) is the product of the binding of hemoglobin in erythrocytes in human blood to blood glucose. Total hemoglobin can be divided into three combinations a, A2, F, where F is mainly in fetal stage and the human is mainly a, consisting of two alpha chains and two beta chains. A2 is composed of two alpha chains and two delta chains, and F is composed of two alpha chains and two gamma chains. Adult HbA is 97% and can be divided into HbA0 and HbA1, hbA0 being the portion that is not glycosylated and HbA1 being the portion that is glycosylated. Control of glycosylated hemoglobin and blood glucose: 4% -6%, and normal blood sugar control; 6 to 7 percent, and the blood sugar control is more ideal; 7% -8%, and blood sugar control is general; 8% -9% of blood sugar control is not ideal, and blood sugar control needs to be enhanced; > 9%, blood glucose control is poor, which is a risk factor for the development of chronic complications.
The blood sugar and the hemoglobin are combined to generate the glycosylated hemoglobin which is an irreversible reaction and is in direct proportion to the blood sugar concentration, the glycosylated hemoglobin can be kept for about 120 days after generation, and the blood sugar control condition of a patient in about 8-12 weeks can be basically reflected by measuring the content of the glycosylated hemoglobin, because the glycosylated hemoglobin has the following characteristics:
(1) Parallel to blood glucose values: the higher the blood sugar, the higher the glycosylated hemoglobin, so that the blood sugar control level can be reflected;
(2) Slowly generating: because blood sugar is constantly fluctuated, blood sugar level can only be reflected at the time when blood is drawn each time, and glycosylated hemoglobin is gradually generated, the transient blood sugar rise does not cause the rise of glycosylated hemoglobin, otherwise, the transient blood sugar drop does not cause the drop of glycosylated hemoglobin;
(3) Is not easy to decompose: the glycosylated hemoglobin is quite stable and not easy to decompose, and can well reflect the blood sugar control degree for a long time although the glycosylated hemoglobin cannot reflect the blood sugar fluctuation in a short time;
(4) Is less affected by hemoglobin levels: accordingly, the international diabetes consortium has introduced a new edition of guidelines for the prevention and treatment of hypotonic diabetes, which clearly states that glycosylated hemoglobin is an internationally recognized "gold standard" for monitoring diabetes.
Currently, there are various methods for measuring glycosylated hemoglobin (HbAlc), and these methods are generally classified into two types: one is based on the difference in charge between HbAlc and Hb, such as ion exchange chromatography, electrophoresis; the other is based on structural features of the glycosylated group on Hb, such as affinity chromatography, immunoassay, enzymatic method, etc. The ion exchange chromatography is established based on different charges after saccharification of N-terminal valine of a beta chain of hemoglobin, and mainly comprises High Performance Liquid Chromatography (HPLC) and manual microcolumn method;
electrophoresis: taking agarose gel electrophoresis as an example, the electrophoretic migration of Hb on agarose gel under acidic buffer salt conditions (pH 6.0) depends on the adsorption of Hb on the gel and its charge. The electrophoresis method has the defects that sample analysis is required to be carried out in batches in each measurement, the speed is relatively low, real-time individual detection cannot be carried out, the degree of automation is poor, the measured result is related to scanning by technicians and wave crest judgment of electrophoresis, and the influence of subjective factors is large, and the cost is high, so that the electrophoresis method is not suitable for routine use in clinical laboratories;
affinity chromatography: boric acid has the property of reversibly binding to the cis-diol group of glucose integrated in Hb molecule. Typically, m-aminophenylboronic acid agarose is used, and after the blood sample is applied to the column, all GHb (glycosylated hemoglobin) is bound to boric acid and left in the column, and non-GHb flows directly out of the column; then adding high concentration polyhydroxy compound (such as sorbitol) containing cis-diol, and eluting with the combination of GHb and boric acid, measuring the two components, and calculating the ratio. Affinity chromatography is relatively insensitive to the effects of variant and pathological hemoglobin, but measures HbA1, the total GHb;
enzymatic method: after whole blood is subjected to hemolysis treatment, hb is subjected to enzymolysis and digestion to form a sugar amino acid by using special endoprotease, hydrogen peroxide (hydrogen peroxide, H2O 2) is generated under the action of fructosyl amino acid oxidase, the concentration of H2O2 is in direct proportion to the content of GHb in the blood, and H2O2 is coupled with a corresponding chromogen under the action of peroxidase, so that the concentration of H2O2 can be obtained according to the color change degree, and the content of GHb in a sample is obtained; simultaneously measuring the total Hb concentration of the digestive juice of the same tube, and calculating the concentration ratio of GHb to Hb to obtain a GHb result;
ion capturing method: applying antigen-antibody reaction principle, connecting fluorescent markers in parallel, adsorbing the fluorescent markers on the surface of positively charged fibers through connecting negatively charged polyanion complexes, measuring the change rate of fluorescence intensity after a series of steps of thorough cleaning and the like, and calculating GHb concentration;
immunoturbidimetry: the measurement is performed by using the principle of antigen and antibody reaction. The N end of the beta chain of GHB provides an epitope which is easy to be recognized by an antibody, a monoclonal antibody or a polyclonal antibody can be used for specifically recognizing the epitope which is formed by the last 4-6 amino acids of the N end of the beta chain of GHB, the content of HbA1c is measured by combining colorimetry or turbidimetry, the content of Hb is measured by taking GHB as a standard, and the percentage of HbA1c in total Hb is calculated finally.
Currently, these methods for detecting glycosylated hemoglobin are widely used clinically, such as: high performance liquid chromatography, affinity chromatography, manual microcolumn method, electrophoresis method, enzyme method, ion capturing method, etc., not only has complex operation, time consumption and low efficiency, but also needs to use special detection instruments, has higher cost, and has the defect of lower detection accuracy in some detection methods.
Disclosure of Invention
In summary, the invention aims to solve the technical problems of complex detection operation procedures, time and labor consumption, low efficiency, poor sensitivity, low accuracy and relatively high detection cost of the existing glycosylated hemoglobin detection, and provides an improved glycosylated hemoglobin detection kit with simple detection operation procedures, high sensitivity, good specificity, high accuracy, good stability and low cost.
In order to solve the technical defects provided by the invention, the adopted technical scheme is that the glycosylated hemoglobin determination kit comprises a reagent 1, a reagent 2 and hemolysis, and is characterized in that:
the pH value of the reagent 1 is 5.5-8.5, and the reagent comprises latex microspheres, a first buffer solution, a first preservative and deionized water;
the pH value of the reagent 2 is 5.5-8.5, and the reagent comprises glycosylated hemoglobin monoclonal nano-antibody, goat anti-mouse IgG antibody, a second buffer solution, a second preservative and deionized water;
the hemolytic liquid comprises sodium chloride, tritonX-100 and deionized water.
Further, the latex microsphere in the reagent 1 is a hydrophobic microsphere, is synthesized by adopting an emulsifier-free emulsion polymerization method, and has one or more groups of sulfonic group, carboxyl group, amido group or aldehyde group, wherein the average particle size is 80-120 nm, and the concentration is 0.1-10 g/L.
Preferably, the latex microspheres are present in a concentration of 0.1g/L to 5g/L, most preferably 0.5g/L.
Further, the first buffer solution in the reagent 1 is one or more selected from MOPSO-Na buffer solution, TES buffer solution, MES buffer solution, HEPES buffer solution, phosphate buffer solution, tris buffer solution and borate buffer solution, and the concentration of the first buffer solution is 0.01mol/L to 0.5mol/L.
Preferably, the concentration of the first buffer solution is 0.01mol/L to 0.05mol/L.
Further, the first preservative in the reagent 1 is one or more selected from sodium azide, sodium benzoate, potassium sorbate, proclin300 and phenol, and the concentration of the first preservative is 0.01 ml/L-5 ml/L.
Preferably, the concentration of the first preservative is 0.2ml/L to 2ml/L, and most preferably 1ml/L.
Further, the contents of each component in the reagent 2 are as follows: 0.01mg/ml to 0.5mg/ml of glycosylated hemoglobin monoclonal antibody, 0.01mg/ml to 0.5mg/ml of goat anti-mouse IgG antibody, 0.01mol/L to 0.1mol/L of second buffer solution and 0.01ml/L to 5ml/L of second preservative.
Preferably, the concentration of the glycosylated hemoglobin monoclonal antibody is 0.1mg/ml.
Preferably, the concentration of the goat anti-mouse IgG antibody is 0.2mg/ml.
Furthermore, the glycosylated hemoglobin monoclonal antibody is formed by connecting a plurality of identical antibody genes or different antibody genes in series through a flexible peptide gene, and simultaneously fusing a section of polypeptide genes rich in lysine at the N end or the C end of the sequence.
Further, the second buffer is one or more selected from glycine buffer, carbonate buffer, TES buffer, MES buffer, HEPES buffer, phosphate buffer, tris buffer and borate buffer.
Preferably, the concentration of the second buffer solution is 0.01mol/L to 0.05mol/L.
Further, the second preservative is selected from one or more of sodium azide, sodium benzoate, potassium sorbate, proclin300, 2-methyl-4-isothiazolin-3-one and phenol.
Preferably, the concentration of the second preservative is 0.2ml/L to 2ml/L, and most preferably 1ml/L.
Further, the content of sodium chloride in the blood is 0.05mol/L to 0.5mol/L, and the content of Triton X-100 is 0.05wt percent to 0.5wt percent.
Further, the reagent 1 also comprises a surfactant with the concentration of 0.05ml/L to 0.1ml/L, and the reagent 2 also comprises a stabilizer with the concentration of 0.5mM to 6 mM; the surfactant is one or more selected from N-acyl taurine, alkyl sulfoacetic acid, polyoxyethylene alkyl ether acetic acid and N-acyl amino acid, and the stabilizer is one or more selected from maleic acid, malonic acid, glutaric acid and tartaric acid.
Preferably, the surfactant is preferably formed by mixing N-acyl taurine and polyoxyethylene alkyl ether acetic acid in a volume ratio of 1:1.
Preferably, the concentration of the stabilizer is 1.0 mM-3.0 mM; the stabilizer is preferably formed by mixing maleic acid and glutaric acid in a weight ratio of 2:1.
The latex microsphere is applied to glycosylated hemoglobin detection, and the recognition site used by the human hemoglobin antibody is a general reaction site of hemoglobin, so that the latex microsphere can be specifically combined with both hemoglobin and glycosylated hemoglobin in a sample to be immobilized. When two antibodies (glycosylated hemoglobin (HbA 1 c) monoclonal antibody and goat anti-mouse IgG antibody) commonly used based on latex-enhanced immunoturbidimetry are added, the glycosylated hemoglobin (HbA 1 c) monoclonal antibody specifically binds to form a complex of latex-human hemoglobin antibody-HbA 1 c-glycosylated hemoglobin (HbA 1 c) monoclonal antibody. The complex forms aggregation due to goat anti-mouse IgG antibody, and the aggregation amount is positively correlated with the amount of glycosylated hemoglobin immobilized on the surface of the latex. The percentage of glycosylated hemoglobin in the sample can be calculated by measuring the intensity of the transmitted light and comparing it with a calibration curve of the percentage concentration of glycosylated hemoglobin.
The use method of the glycosylated hemoglobin determination kit comprises the following steps:
step (1): preparing a required reagent 1, a reagent 2 and hemolysis according to any one of the components and the content proportion in the scheme;
step (2): diluting the collected whole blood sample in blood for 100 times, adding 400 mu L of reagent 1 into 10 mu L of diluted sample, and incubating for about 30 seconds at the temperature of 35-38 ℃ to ensure that the hemoglobin and glycosylated hemoglobin in the sample have the same nonspecific adsorption with latex microspheres in the reagent 1 so as to be immobilized;
step (3): then adding a reagent 2, and reacting for about 2 minutes at the temperature of 35-38 ℃ to ensure that the glycosylated hemoglobin monoclonal nano-antibody and the goat anti-mouse IgG antibody in the reagent 2 are specifically combined with the latex microsphere attached with the glycosylated hemoglobin to form an antigen-antibody immune complex;
step (4): and measuring the absorbance difference value at 630nm of a reaction system formed with the antigen-antibody immune complex by adopting a specific protein analyzer, and calculating the percentage content of HbA1c in the sample according to a standard curve of the percentage concentration of HbA1 c.
Compared with the prior art, the invention has the following beneficial effects:
1. the method for measuring the glycosylated hemoglobin has the advantages of high accuracy, good sensitivity, good precision, wide linear range and convenient popularization and use.
2. According to the kit disclosed by the invention, the latex microspheres are added to ensure that the glycosylated hemoglobin antibodies are not adsorbed or connected to the latex, so that the influence of the hemoglobin content in different blood samples in glycosylated hemoglobin detection can be eliminated, and the accuracy of the kit is higher.
3. The kit has the advantages of good stability, long storage period and convenient use and storage.
4. The sensitivity and the specificity of the modified HbA1c monoclonal nano antibody are greatly improved. Compared with the traditional antibody, the nano antibody has the advantages of high affinity, high specificity, high thermal stability, capability of being expressed in a large amount in bacteria and the like, and the application of the nano antibody in the field of in-vitro diagnosis greatly improves the sensitivity and the specificity of the diagnostic reagent and greatly reduces the production cost of in-vitro diagnostic reagent production enterprises.
5. The hydrophobic latex microsphere used in the kit disclosed by the invention has at least one group selected from a sulfonic group, a carboxyl group, an amido group or an aldehyde group, and is combined with a carboxyl site of a protein, wherein the site is positioned in a constant region of an antibody, the reactivity of the protein is not influenced by the region, and the normal titer of the antibody can be ensured. When the method is used for labeling antigens or antibodies in an immunoassay to detect reactants to be detected in a target sample, a reaction system can generate signal change under the wavelength of 200-800 nm, so that better accuracy and specificity are reflected. The latex microsphere is prepared by initiating monomer polymerization by an oxidative thermal initiator in pure water to form nanoscale microspheres with uniform particle size. The experimental process is safe and nontoxic, and the prepared detection reagent does not need special treatment and cannot influence the environment.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the following description will further illustrate the present invention by way of specific examples, and the specific embodiments adopted in the following examples are only some preferred embodiments in the technical solution of the present invention, and are not limiting the present invention.
Reagent 1, reagent 2 and hemolysis of the kits of examples 1 to 8 were prepared as shown in tables 1 and 2, respectively.
Table 1: component proportion Table of examples 1 to 4
Table 2: component proportion Table of examples 5 to 8
The respective quality control product 1 (glycosylated hemoglobin concentration of 5.20%) and quality control product 2 (glycosylated hemoglobin concentration of 13.0%) were subjected to glycosylated hemoglobin measurement at least three times under the same detection environmental conditions by the aforementioned methods of using the respective kits of the present invention, and the Relative Standard Deviation (RSD) of the measurement data and results thereof were shown in tables 3 and 4, respectively, for checking the measurement result accuracy of each kit.
Table 3: evaluation table for quality control product 1 measurement result
Table 4: evaluation table for quality control product 2 measurement result
As can be seen from the data comparison analysis of tables 3 and 4, the kits of specific embodiments 1-8 of the present invention all meet the measurement accuracy requirement of glycosylated hemoglobin, and the detection RSD deviation of the kits of each embodiment on the quality control product 1 and the quality control product 2 is smaller, which is within the allowable range of the detection deviation value of the kit product, so that the detection accuracy of the kit of the present invention is high, and the technical improvement purpose of accurate glycosylated hemoglobin measurement is satisfied. In addition, the kit adopts the method to detect, has high result accuracy, does not need a special detection instrument, has simple detection flow, convenient operation, rapidness, high efficiency and low cost, and meets the technical improvement aims of simplified glycosylated hemoglobin detection flow and reduced cost.
The following kit products prepared by the above specific examples are subjected to at least 10 determinations of the same whole blood sample to be tested under the same detection environmental conditions according to the method of using the kit of the present invention, and Standard Deviation (SD) and Coefficient of Variation (CV) of the determination data and results are shown in table 5, and are used for practical application detection of the kit products of the present invention.
Table 5: sample actual measurement result evaluation table
The same batch of glycosylated hemoglobin calibrator was measured at 6 months and 12 months by the kit products of each specific example under the storage condition of 2-8 ℃, the reactivity deviation of the kit before and after storage was calculated, and the long-term stability of the kit was observed, and the results thereof are shown in table 6.
Table 6: evaluation table for long-term stability test result
From the results, the data of the calibration standard measured at 2-8 ℃ for 6 months and 12 months in the kit prepared by the specific examples of the invention are very small in measured value difference compared with the 0-day kit, and the deviation is within the quality control requirement range (+ -15%), which indicates that the kit product of the invention meets the requirement of long-term stability.
In addition, as can be seen from the above tables 1 to 6, under the condition that the main technical characteristics of the reagent 1, the reagent 2 and the hemolytic liquid are satisfied, after the surfactant and the stabilizer are respectively added in the reagent 1 and the reagent 2, the detection result deviation and the stability of the kit product are further reduced compared with those of the kit products of examples 1 to 4, the detection stability is further enhanced, and the beneficial effects are better.
The kit disclosed by the invention uses a glycosylated hemoglobin (HbA 1C) monoclonal nano antibody, creatively connects a plurality of identical antibody genes or different antibody genes in series through flexible peptide genes, and fuses a section of polypeptide gene rich in lysine at the N end or the C end of the sequence, so that the marker is conveniently marked, and the marker is prevented from being directly marked on an epitope gene. The sensitivity and the specificity of the modified HbA1c monoclonal nano antibody are greatly improved. Compared with the traditional antibody, the nano antibody has the advantages of high affinity, high specificity, high thermal stability, capability of being expressed in a large amount in bacteria and the like, and the application of the nano antibody in the field of in-vitro diagnosis greatly improves the sensitivity and the specificity of the diagnostic reagent and greatly reduces the production cost of in-vitro diagnostic reagent production enterprises.
In addition, the latex microsphere used in the kit is a hydrophobic microsphere with at least one of an amido group, a sulfonic group, a carboxyl group and an aldehyde group, and is combined with a carboxyl site of a protein, wherein the site is positioned in a constant region of an antibody, the region has no influence on the reactivity of the protein, and the normal titer of the antibody can be ensured. When the method is used for labeling antigens or antibodies in an immunoassay to detect reactants to be detected in a target sample, a reaction system can generate signal change under the wavelength of 200-800 nm, so that better accuracy and specificity are reflected. And the latex microsphere in the reagent 1 of the invention is polymerized by initiating the monomer by an oxidative thermal initiator in pure water (deionized water) to form the nanoscale microsphere with uniform particle size. The experimental process is safe and nontoxic, and the prepared detection reagent does not need special treatment and cannot influence the environment.
The foregoing examples are merely for the purpose of illustrating the technical solution of the present invention and are not intended to limit the embodiments of the present invention. Various modifications and alterations of this invention will be apparent to those skilled in the art without departing from the spirit and substance of this invention, and it is intended to cover all such modifications and alterations as fall within the true scope of this invention.
Claims (10)
1. The glycosylated hemoglobin determination kit comprises a reagent 1, a reagent 2 and hemolysis, and is characterized in that:
the pH value of the reagent 1 is 5.5-8.5, and the reagent comprises latex microspheres, a first buffer solution, a first preservative and deionized water;
the pH value of the reagent 2 is 5.5-8.5, and the reagent comprises glycosylated hemoglobin monoclonal nano-antibody, goat anti-mouse IgG antibody, a second buffer solution, a second preservative and deionized water;
the hemolytic liquid comprises sodium chloride, tritonX-100 and deionized water.
2. A glycosylated hemoglobin measurement kit according to claim 1, characterized in that: the latex microsphere in the reagent 1 is hydrophobic microsphere, is synthesized by adopting an emulsifier-free emulsion polymerization method, and has one or more groups of sulfonic group, carboxyl group, amido group or aldehyde group, wherein the average particle diameter is 80-120 nm, and the concentration is 0.1-10 g/L.
3. A glycosylated hemoglobin measurement kit according to claim 1, characterized in that: the first buffer solution in the reagent 1 is one or more selected from MOPSO-Na buffer solution, TES buffer solution, MES buffer solution, HEPES buffer solution, phosphate buffer solution, tris buffer solution and borate buffer solution, and the concentration of the first buffer solution is 0.01 mol/L-0.5 mol/L.
4. A glycosylated hemoglobin measurement kit according to claim 1, characterized in that: the first preservative in the reagent 1 is one or more selected from sodium azide, sodium benzoate, potassium sorbate, proclin300 and phenol, and the concentration of the first preservative is 0.01 ml/L-5 ml/L.
5. A glycosylated hemoglobin measurement kit according to claim 1, characterized in that: the content of each component in the reagent 2 is as follows: glycosylated hemoglobin monoclonal antibody 0.01 mg/ml-0.5 mg/ml, goat anti-mouse IgG antibody 0.01 mg/ml-0.5 mg/ml, second buffer solution 0.01 mol/L-0.1 mol/L, and second preservative 0.01 ml/L-5 ml/L.
6. The glycosylated hemoglobin measurement kit according to claim 5, wherein: the glycosylated hemoglobin monoclonal antibody is formed by connecting a plurality of identical antibody genes or different antibody genes in series through flexible peptide genes, and simultaneously fusing a section of polypeptide genes rich in lysine at the N end or the C end of the sequence.
7. The glycosylated hemoglobin measurement kit according to claim 5, wherein: the second buffer solution is one or more selected from glycine buffer solution, carbonate buffer solution, TES buffer solution, MES buffer solution, HEPES buffer solution, phosphate buffer solution, tris buffer solution and borate buffer solution.
8. The glycosylated hemoglobin measurement kit according to claim 5, wherein: the second preservative is selected from one or more of sodium azide, sodium benzoate, potassium sorbate, proclin300, 2-methyl-4-isothiazolin-3-ketone and phenol.
9. A glycosylated hemoglobin measurement kit according to claim 1, characterized in that: the content of sodium chloride in the dissolved blood is 0.05mol/L to 0.5mol/L, and the content of TritonX-100 is 0.05wt percent to 0.5wt percent.
10. A glycosylated hemoglobin measurement kit according to claim 1, characterized in that: the reagent 1 also comprises a surfactant with the concentration of 0.05ml/L to 0.1ml/L, and the reagent 2 also comprises a stabilizer with the concentration of 0.5mM to 6 mM; the surfactant is one or more selected from N-acyl taurine, alkyl sulfoacetic acid, polyoxyethylene alkyl ether acetic acid and N-acyl amino acid, and the stabilizer is one or more selected from maleic acid, malonic acid, glutaric acid and tartaric acid.
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| CN202310208306.5A CN116381252A (en) | 2023-03-07 | 2023-03-07 | Glycosylated hemoglobin determination kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310208306.5A CN116381252A (en) | 2023-03-07 | 2023-03-07 | Glycosylated hemoglobin determination kit |
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| CN202310208306.5A Pending CN116381252A (en) | 2023-03-07 | 2023-03-07 | Glycosylated hemoglobin determination kit |
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