A kit for measuring glycated hemoglobin

Abstract

The present application belongs to the technical field of biological detection kit of in-vitro diagnosis, and discloses a kit for determination of glycated hemoglobin, which comprises reagent 1, reagent 2 and hemolytic liquid. The pH value of the reagent 1 is 5.5-8.5, and the reagent 1 comprises latex microspheres, a first buffer, a first preservative and deionized water; the pH value of the reagent 2 is 5.5-8.5, and the reagent 2 comprises glycated hemoglobin monoclonal nanobody, goat anti-mouse IgG antibody, a second buffer, a second preservative and deionized water; and the hemolytic liquid comprises sodium chloride, Triton X-100 and deionized water. The kit has the beneficial effects that the latex microspheres make the glycated hemoglobin antibody not be adsorbed or connected on the latex, eliminate the adverse effects of the hemoglobin content in different blood samples on the accuracy of the detection results, and the combination of the monoclonal nanobody and the goat anti-mouse antibody is more easily solidified on the latex microspheres, so that the kit has better sensitivity and specificity, higher accuracy, and the detection process is simple, efficient and low in cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection kit, more particularly relates to a glycosylated hemoglobin determination kit with high detection sensitivity and low cost. BACKGROUND

[0002] Glycosylated hemoglobin (HbAlc) is the product of the combination of blood glucose and hemoglobin in red blood cells in human blood. Total hemoglobin can be divided into A, A2 and F three combinations, among which F is mainly in the fetal period, and A is mainly in adults, which is composed 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 accounts for 97%, which can be divided into HbA0 and HbA1, HbA0 is not glycosylated, and HbA1 is glycosylated. The control of glycosylated hemoglobin and blood glucose: 4% to 6%, blood glucose control is normal; 6% to 7%, blood glucose control is relatively ideal; 7% to 8%, blood glucose control is general; 8% to 9%, blood glucose control is not ideal, and blood glucose control needs to be strengthened; > 9%, blood glucose control is very poor, which is a risk factor for the development of chronic complications.

[0003] The combination of blood glucose and hemoglobin to form glycosylated hemoglobin is an irreversible reaction, which is proportional to the concentration of blood glucose, and can be maintained for about 120 days after formation. The content of glycosylated hemoglobin can basically reflect the blood glucose control of patients in the past 8 to 12 weeks, because glycosylated hemoglobin has the following characteristics:

[0004] (1) Parallel with blood glucose value: the higher the blood glucose, the higher the glycosylated hemoglobin, so it can reflect the blood glucose control level;

[0005] (2) Slow generation: since blood glucose is constantly fluctuating, each blood draw can only reflect the blood glucose level at that time, while glycosylated hemoglobin is gradually generated, and short-term blood glucose rise will not cause glycosylated hemoglobin to rise, and vice versa, short-term blood glucose drop will not cause glycosylated hemoglobin to drop;

[0006] (3) Not easy to decompose: glycosylated hemoglobin is quite stable and not easy to decompose, although it cannot reflect short-term blood glucose fluctuations, it can well reflect the degree of blood glucose control for a long time;

[0007] (4) Little affected by hemoglobin level: accordingly, the International Diabetes Federation has issued a new version of the Asia-Pacific Diabetes Prevention and Treatment Guidelines, which clearly stipulates that glycosylated hemoglobin is the internationally recognized "gold standard" for diabetes monitoring.

[0008] Currently, there are various methods for determining glycated hemoglobin (HbAlc), which can be generally divided into two categories: one is based on the different charges of HbAlc and Hb, such as ion exchange chromatography and electrophoresis; the other is based on the structural characteristics of the glycosylation group on Hb, such as affinity chromatography, immunological method and enzyme method. Among them, ion exchange chromatography is based on the different charges of the N-terminal valine of the hemoglobin β chain after glycosylation, mainly including high performance liquid chromatography (HPLC) and manual microcolumn method;

[0009] 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 the charge carried by Hb. The disadvantages of electrophoresis are that each determination needs to be performed in batches, the speed is relatively slow, real-time individual detection cannot be performed, the degree of automation is poor, the measured results are related to the scanning and peak judgment of the technicians, are greatly affected by subjective factors, and are expensive, so it is not suitable for routine use in clinical laboratories;

[0010] Affinity chromatography: boronic acid has the property of reversible combination reaction with the vicinal diol group integrated on the Hb molecule. The commonly used is m-aminophenylboronic acid agarose. After adding the blood sample to the chromatography column, all GHb (glycated hemoglobin) is combined with boronic acid and remains in the column, and non-GHb directly flows out of the chromatography column; then a high concentration of a polyhydroxy compound (such as sorbitol) containing a vicinal diol group is added, the combination of GHb and boronic acid is replaced and eluted, and the two components are measured respectively, and the ratio is calculated. Affinity chromatography is relatively insensitive to variant hemoglobin and pathological hemoglobin compared to other methods, but it measures HbA1, i.e. the total amount of GHb;

[0011] Enzymatic method: after hemolysis, the Hb is enzymatically digested into fructose amino acids by a special endoprotease, and then hydrogen peroxide (H2O2) is generated under the action of fructose amino acid oxidase. The concentration of H2O2 is proportional to the content of GHb in the blood. H2O2 is coupled with the corresponding chromogen under the action of peroxidase, so that the concentration of H2O2 can be obtained according to the degree of color change, and then the content of GHb in the sample is obtained. The total Hb concentration of the same tube of digested solution is also measured, and the concentration ratio of GHb and Hb is calculated, which is the GHb result;

[0012] Ion capture method: based on the principle of antigen-antibody reaction, a fluorescent marker is connected to a negatively charged polyanion complex, which is adsorbed to a positively charged fiber surface. After a series of thorough cleaning steps, the fluorescence intensity change rate is measured to calculate the GHb concentration;

[0013] Immune nephelometry: using the principle of antigen-antibody reaction to determine. The N-terminal of the beta chain of GHb provides an antigen epitope easily recognized by antibodies, which can be specifically recognized by monoclonal antibodies or polyclonal antibodies, and the antigen epitope composed of the last 4-6 amino acids of the N-terminal of the beta chain of GHb is combined with colorimetric or nephelometry method, and the content of HbA1c is determined with GHb as a standard, then the content of Hb is determined, and finally the percentage of HbA1c in total Hb is calculated.

[0014] The detection methods of glycosylated hemoglobin widely used in the clinic at present, such as high performance liquid chromatography, affinity chromatography, manual microcolumn method, electrophoresis method, enzyme method, ion capture method, etc., not only have complex operation procedures, time-consuming, low efficiency, but also need to use some special detection instruments, and the cost is high, and some detection methods also have the defect of low detection accuracy. SUMMARY

[0015] In summary, the purpose of the present application is to solve the technical problems of the existing glycosylated hemoglobin detection, such as complex detection operation procedure, time-consuming, low efficiency, poor sensitivity, low accuracy, and the need to use professional detection equipment, and the detection cost is relatively high, and an improved glycosylated hemoglobin determination kit with simple detection operation procedure, high sensitivity, good specificity, high accuracy, good stability and low cost is provided.

[0016] In order to solve the technical problems of the present application, the technical scheme adopted is a glycosylated hemoglobin determination kit, comprising reagent 1, reagent 2 and hemolytic liquid, characterized in that:

[0017] The pH value of the reagent 1 is 5.5-8.5, which comprises latex microspheres, a first buffer, a first preservative and deionized water;

[0018] The pH value of the reagent 2 is 5.5-8.5, which comprises glycosylated hemoglobin monoclonal nanobody, goat anti-mouse IgG antibody, second buffer, second preservative and deionized water;

[0019] The hemolytic liquid comprises sodium chloride, Triton X-100 and deionized water.

[0020] Further, the latex microspheres in the reagent 1 are hydrophobic microspheres, which are synthesized by emulsifier-free emulsion polymerization method, have one or more groups of sulfonic acid group, carboxyl group, amide group or aldehyde group, and the average particle size is 80-120 nm, and the concentration is 0.1-10 g / L.

[0021] Preferably, the concentration of the latex microspheres is 0.1-5 g / L, and most preferably 0.5 g / L.

[0022] Further, the first buffer in the reagent 1 is selected from one or more of MOPSO-Na buffer, TES buffer, MES buffer, HEPES buffer, phosphate buffer, Tris buffer, borate buffer, and the concentration is 0.01 mol / L-0.5 mol / L.

[0023] Preferably, the concentration of the first buffer is 0.01 mol / L-0.05 mol / L.

[0024] Further, the first preservative in the reagent 1 is selected from one or more of sodium azide, sodium benzoate, potassium sorbate, proclin 300, phenol, and the concentration is 0.01 ml / L-5 ml / L.

[0025] Preferably, the concentration of the first preservative is 0.2 ml / L-2 ml / L, and most preferably 1 ml / L.

[0026] Further, the content of each component in the reagent 2 is: glycosylated hemoglobin monoclonal nanobody 0.01 mg / ml-0.5 mg / ml, goat anti-mouse IgG antibody 0.01 mg / ml-0.5 mg / ml, second buffer 0.01 mol / L-0.1 mol / L, and second preservative 0.01 ml / L-5 ml / L.

[0027] Preferably, the concentration of the glycosylated hemoglobin monoclonal nanobody is 0.1 mg / ml.

[0028] Preferably, the concentration of the goat anti-mouse IgG antibody is 0.2 mg / ml.

[0029] Further, the glycosylated hemoglobin monoclonal nanobody is a monoclonal nanobody in which a plurality of the same antibody genes or different antibody genes are connected in series by a flexible peptide gene, and a lysine-rich polypeptide gene is fused at the N-terminal or C-terminal of the sequence.

[0030] Further, the second buffer is selected from one or more of glycine buffer, carbonate buffer, TES buffer, MES buffer, HEPES buffer, phosphate buffer, Tris buffer, and borate buffer.

[0031] Preferably, the concentration of the second buffer is 0.01 mol / L-0.05 mol / L.

[0032] Further, the second preservative is selected from one or more of sodium azide, sodium benzoate, potassium sorbate, proclin 300, 2-methyl-4-isothiazolin-3-one, and phenol.

[0033] Preferably, the concentration of the second preservative is 0.2ml / L-2ml / L, and most preferably 1ml / L.

[0034] Further, the content of sodium chloride in the hemolytic solution is 0.05mol / L-0.5mol / L, and the content of Triton X-100 is 0.05wt%-0.5wt%.

[0035] Further, the reagent 1 further comprises a surfactant with a concentration of 0.05ml / L-0.1ml / L, and the reagent 2 further comprises a stabilizer with a concentration of 0.5mM-6mM; the surfactant is selected from one or more of N-acyl taurine, alkyl sulfobutyric acid, polyoxyethylene alkyl ether acetic acid, and N-acyl amino acid, and the stabilizer is selected from one or more of maleic acid, malonic acid, glutaric acid, and tartaric acid.

[0036] Preferably, the surfactant is preferably mixed by N-acyl taurine and polyoxyethylene alkyl ether acetic acid at a volume ratio of 1:1.

[0037] Preferably, the concentration of the stabilizer is 1.0mM-3.0mM; and the stabilizer is preferably mixed by maleic acid and glutaric acid at a weight ratio of 2:1.

[0038] When the latex microspheres of the present application are applied to the detection of glycated hemoglobin, the recognition site of human hemoglobin antibody is the general reaction site of hemoglobin, so it can specifically bind to both hemoglobin and glycated hemoglobin in the sample to be solidified. After adding the two commonly used antibodies based on the latex-enhanced immunoturbidimetry (glycated hemoglobin (HbA1c) monoclonal nanobody and goat anti-mouse IgG antibody), the glycated hemoglobin (HbA1c) monoclonal nanobody specifically binds to form a complex of latex-human hemoglobin antibody-HbA1c-glycated hemoglobin (HbA1c) monoclonal nanobody. This complex forms agglutination due to the goat anti-mouse IgG antibody, and the amount of agglutination is positively correlated with the amount of glycated hemoglobin solidified on the surface of the latex. By measuring the transmission light intensity and comparing it with the calibration curve of the percentage concentration of glycated hemoglobin, the percentage content of glycated hemoglobin in hemoglobin in the sample can be calculated.

[0039] The method for using the glycated hemoglobin assay kit of the present application is as follows:

[0040] Step (1): Prepare the required reagent 1, reagent 2 and hemolytic solution according to any one of the components and content ratios in the above scheme;

[0041] Step (2): take the collected whole blood sample and dilute 100 times in lysate, take 10 muL of the diluted sample and add 400 muL of reagent 1, incubate at 35-38 DEG C for about 30 seconds, so that the hemoglobin and glycated hemoglobin in the sample are non-specifically adsorbed with the latex microspheres in reagent 1 to be solid-phase;

[0042] Step (3): reagent 2 is added, and the reaction is carried out at 35-38 DEG C for about 2 minutes, so that the glycated hemoglobin monoclonal nanobody and goat anti-mouse IgG antibody in reagent 2 are specifically combined with the latex microspheres with glycated hemoglobin to form an antigen-antibody immune complex;

[0043] Step (4): the specific protein analyzer is used to measure the absorbance difference of the reaction system with the antigen-antibody immune complex at 630nm, and the percentage content of HbA1c in the sample is calculated according to the standard curve of HbA1c percentage concentration.

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] 1、The determination of glycated hemoglobin in the present application has high accuracy, good sensitivity, good precision, wide linear range, and is convenient to use.

[0046] 2、The reagent kit of the present application can eliminate the influence of the hemoglobin content in different blood samples on the detection of glycated hemoglobin, so that the accuracy of the reagent kit is higher.

[0047] 3、The reagent kit of the present application has good stability, long shelf life, and is convenient to use and store.

[0048] 4、The sensitivity and specificity of the improved HbA1c monoclonal nanobody are greatly improved.

[0049] 5. The hydrophobic latex microspheres used in the kit of this invention have at least one of the following groups: sulfonic acid group, carboxyl group, amide group, or aldehyde group. These groups bind to the carboxyl site of the protein, which is located in the constant region of the antibody. This region does not affect the reactivity of the protein, ensuring the normal titer of the antibody. When used to label antigens or antibodies in immunoassays to detect reactants in target samples, the reaction system exhibits signal changes at wavelengths of 200–800 nm, demonstrating better accuracy and specificity. These latex microspheres are polymerized in pure water using an oxidizing thermal initiator to form uniform nanoscale microspheres. The experimental process is safe and non-toxic, and the prepared detection reagents require no special treatment and will not harm the environment. Detailed Implementation

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below through specific embodiments. The specific implementation methods used in the following embodiments are only some preferred implementation methods of the technical solution of the present invention and are not intended to limit the present invention.

[0051] Prepare reagent 1, reagent 2, and hemolysed blood for the kits of Examples 1-8 as shown in Tables 1 and 2.

[0052] Table 1: Distribution ratio of each group in Examples 1-4

[0053]

[0054]

[0055] Table 2: Distribution ratio of each group in Examples 5-8

[0056]

[0057]

[0058] Using the kits prepared in Examples 1-8 above, under the same detection environment conditions, and in accordance with the aforementioned method of using the kits of the present invention, at least three glycated hemoglobin measurements were performed on quality control 1 (glycated hemoglobin concentration of 5.20%) and quality control 2 (glycated hemoglobin concentration of 13.0%). The measurement data and the relative standard deviation (RSD) of the results are shown in Tables 3 and 4, respectively, to verify the accuracy of the measurement results of each kit.

[0059] Table 3: Evaluation Table of Test Results for Quality Control Sample 1

[0060]

[0061]

[0062] Table 4: Quality control product 2 determination result evaluation table

[0063]

[0064] According to the data comparison and analysis of Table 3 and Table 4, the kit of each embodiment 1-8 of the present application meets the accuracy requirement of the determination of the content of glycosylated hemoglobin, the detection RSD deviation of the kit of each embodiment to quality control product 1 and quality control product 2 is small, and is within the allowable range of the detection deviation value of the kit product, which proves that the detection accuracy of the kit of the present application is high, and meets the technical improvement purpose of accurate determination of glycosylated hemoglobin. Moreover, the kit of the present application is detected by the foregoing method, the result accuracy is high, special detection instrument is not needed, and the detection process is simple, convenient, fast, efficient and low in cost, which meets the technical improvement purpose of simplifying the detection process of glycosylated hemoglobin and reducing the cost.

[0065] The kit product prepared by each of the foregoing embodiments is used to detect the same whole blood sample at least 10 times under the same detection environment conditions according to the foregoing use method of the kit of the present application, and the standard deviation (SD) and coefficient of variation (CV) of the determination data and results are shown in Table 5 for the actual application detection of the kit product of the present application.

[0066] Table 5: Sample actual determination result evaluation table

[0067]

[0068] Under the storage condition of 2-8℃, the same batch of glycosylated hemoglobin calibrator is determined by the kit product of each embodiment at 6 months and 12 months, respectively, the reaction degree deviation of the kit before and after storage is calculated, and the long-term stability of the kit is observed, and the results are shown in Table 6.

[0069] Table 6: Long-term stability test result evaluation table

[0070]

[0071]

[0072] From the above results, it can be seen that the data of the kit prepared by each embodiment of the present application for determining the calibrator stored at 2-8℃ for 6 months and 12 months has little difference compared with the kit of 0 days, and the deviation is within the quality control requirement range (±15%), which shows that the kit product of the present application meets the requirement of long-term stability.

[0073] In addition, in combination with Tables 1-6 above, it can be seen that, under the conditions of satisfying the main technical feature requirements of reagent 1, reagent 2 and hemolytic liquid, after respectively adding surfactants and stabilizers in reagent 1 and reagent 2, the detection result deviation and stability of the kit product are further reduced, the detection stability is further enhanced, and the beneficial effect is better compared with the kit products of Examples 1-4.

[0074] The kit of the present application uses a glycated hemoglobin (HbA1c) monoclonal nanobody, which innovatively connects multiple same antibody genes or different antibody genes through a flexible peptide gene, and simultaneously fuses a lysine-rich polypeptide gene at the N-terminal or C-terminal of the sequence, so as to facilitate labeling of a marker and avoid direct labeling of a marker on an epitope gene. The modified HbA1c monoclonal nanobody has greatly improved sensitivity and specificity. That is, the nanobody has the advantages of high affinity, high specificity, high thermal stability and large-scale expression in bacteria compared with traditional antibodies, and its application in the field of in vitro diagnosis will greatly improve the sensitivity and specificity of diagnostic reagents and greatly reduce the production cost of in vitro diagnostic reagent production enterprises.

[0075] In addition, the latex microspheres used in the kit of the present application are hydrophobic microspheres with at least one of an amide group, a sulfonic acid group, a carboxyl group and an aldehyde group, which bind to the carboxyl site of a protein, which is located in the constant region of an antibody, and this region has no influence on the reactivity of the protein, and can ensure the normal titer of the antibody. When used for labeling antigens or antibodies in an immune experiment to detect the reactants in the target sample, the reaction system can cause signal changes at a wavelength of 200-800 nm, which reflects better accuracy and specificity. Moreover, the latex microspheres in reagent 1 of the present application are polymerized by an oxidative thermal initiator in pure water (deionized water) to form nanoscale microspheres with uniform particle size. The experimental process is safe and non-toxic, and the prepared detection reagent does not need special treatment and will not affect the environment.

[0076] The above embodiments are only for the purpose of clearly describing the technical solutions of the present application, and are not a limitation on the embodiments of the present application. Those skilled in the art can make various corresponding changes and modifications according to the present application without departing from the spirit and essence of the present application, and these corresponding changes and modifications should all belong to the protection scope of the present application.

Claims

1.A glycated hemoglobin assay kit, comprising reagent 1, reagent 2 and hemolytic solution, characterized in that: the pH value of the reagent 1 is 5.5-8.5, and the reagent 1 comprises latex microspheres, a first buffer, a first preservative and deionized water; the pH value of the reagent 2 is 5.5-8.5, and the reagent 2 comprises glycated hemoglobin monoclonal nanobodies, goat anti-mouse IgG antibodies, a second buffer, a second preservative and deionized water; the content of each component is as follows: 0.01 mg / ml-0.5 mg / ml of glycated hemoglobin monoclonal nanobodies, 0.01 mg / ml-0.5 mg / ml of goat anti-mouse IgG antibodies, 0.01 mol / L-0.1 mol / L of the second buffer and 0.01 ml / L-5 ml / L of the second preservative; the hemolytic solution comprises sodium chloride, Triton X-100 and deionized water; the latex microspheres in the reagent 1 are hydrophobic microspheres, which are synthesized by emulsifier-free emulsion polymerization, have one or more of sulfonic groups, carboxyl groups, amide groups or aldehyde groups, and have an average particle size of 80 nm-120 nm and a concentration of 0.1 g / L-10 g / L; the first buffer in the reagent 1 is selected from one or more of MOPSO-Na buffer, TES buffer, MES buffer, HEPES buffer, phosphate buffer, Tris buffer and borate buffer, and has a concentration of 0.01 mol / L-0.5 mol / L; the first preservative in the reagent 1 is selected from one or more of sodium azide, sodium benzoate, potassium sorbate, proclin 300 and phenol, and has a concentration of 0.01 ml / L-5 ml / L; the second buffer is selected from one or more of glycine buffer, carbonate buffer, TES buffer, MES buffer, HEPES buffer, phosphate buffer, Tris buffer and borate buffer; the second preservative is selected from one or more of sodium azide, sodium benzoate, potassium sorbate, proclin 300, 2-methyl-4-isothiazolin-3-one and phenol; the glycated hemoglobin monoclonal nanobodies are monoclonal nanobodies obtained by connecting multiple identical antibody genes or different antibody genes in series through a flexible peptide gene and simultaneously fusing a polypeptide gene rich in lysine at the N terminal or C terminal of the sequence; the content of sodium chloride in the hemolytic solution is 0.05 mol / L-0.5 mol / L, and the content of Triton X-100 is 0.05 wt%-0.5 wt%; the reagent 1 further comprises a surfactant with a concentration of 0.05 ml / L-0.1 ml / L, and the reagent 2 further comprises a stabilizer with a concentration of 0.5 mM-6 mM; the surfactant is selected from one or more of N-acyl taurine, alkyl sulfobutyric acid, polyoxyethylene alkyl ether acetic acid and N-acyl amino acid; and the stabilizer is selected from one or more of maleic acid, malonic acid, glutaric acid and tartaric acid. ​ ​ ​ ​ ​ ​ ​ ​ ​ 2. The reagent kit for the determination of glycated hemoglobin according to claim 1, characterized by: ​ 3. The reagent kit for the determination of glycated hemoglobin according to claim 1, characterized by: ​

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