Detection method for directly measuring content of glycosylated hemoglobin
阅读说明：本技术 一种直接测定糖化血红蛋白含量的检测方法 (Detection method for directly measuring content of glycosylated hemoglobin ) 是由 戴瞻 于 2019-10-10 设计创作，主要内容包括：一种直接测定糖化血红蛋白含量的检测方法,包括以下步骤：A、取EDTA抗凝全血样本与溶血剂按照1:100进行稀释；B、先取4μL的溶血样本加入试剂R1中；所述试剂R1含有聚苯乙烯胶乳微球；C、经5min温预后再加入试剂R2；所述试剂R2是鼠抗人HbA1c单克隆抗体、羊抗鼠IgG抗体和甘氨酸缓冲液的混合液；D、免疫反应在600nm～700nm的波长光下,通过全自动生化分析仪读取反应后的吸光度；E、最终根据反应吸光度之间的差值,从校准曲线中直接读取糖化血红蛋白的百分比含量。本发明方法以免疫透射比浊法为检测原理,不需要测定EDTA抗凝全血样本中的总血红蛋白及糖化血红蛋白含量,即可直接确定其中的糖化血红蛋白百分比。(A detection method for directly measuring the content of glycosylated hemoglobin comprises the following steps: A. diluting an EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample with a hemolytic agent according to a ratio of 1: 100; B. adding 4 mu L of hemolysis sample into the reagent R1; the reagent R1 contains polystyrene latex microspheres; C. adding a reagent R2 after 5min of warm prognosis; the reagent R2 is a mixed solution of a mouse anti-human HbA1c monoclonal antibody, a sheep anti-mouse IgG antibody and a glycine buffer solution; D. the immunoreaction is under the wavelength light of 600 nm-700 nm, and the absorbance after the reaction is read by a full-automatic biochemical analyzer; E. and finally, directly reading the percentage content of the glycosylated hemoglobin from the calibration curve according to the difference between the reaction absorbances. The method takes an immunity transmission turbidimetry as a detection principle, and can directly determine the percentage of the glycosylated hemoglobin in the EDTA anticoagulated whole blood sample without determining the content of the total hemoglobin and the content of the glycosylated hemoglobin in the EDTA anticoagulated whole blood sample.)
1. A detection method for directly measuring the content of glycosylated hemoglobin is characterized in that: the reagent used for detecting the content of the glycosylated hemoglobin comprises a reagent R1, a reagent R2, a hemolytic agent, polystyrene latex microspheres, a mouse anti-human HbA1c monoclonal antibody, a goat anti-mouse IgG antibody and a glycine buffer solution, and the detection method comprises the following steps:
A. diluting an EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample with a hemolytic agent according to a ratio of 1: 100;
B. adding 4 mu L of hemolysis sample into the reagent R1; the reagent R1 contains polystyrene latex microspheres;
C. adding a reagent R2 after 5min of warm prognosis; the reagent R2 is a mixed solution of a mouse anti-human HbA1c monoclonal antibody, a sheep anti-mouse IgG antibody and a glycine buffer solution;
D. the immunoreaction is under the wavelength light of 600 nm-700 nm, and the absorbance after the reaction is read by a full-automatic biochemical analyzer;
E. and finally, directly reading the percentage content of the glycosylated hemoglobin from the calibration curve according to the difference between the reaction absorbances.
2. The method according to claim 1, wherein the measurement method for directly measuring the glycated hemoglobin content comprises: the hemolytic agents in the step A are TTAB and H2O, the polystyrene latex microspheres in the step B are 0.15%, the concentration of the mouse anti-human HbA1c monoclonal antibody in the step c is 0.01-0.06 mg/ml, and the concentration of the goat anti-mouse IgG antibody in the step c is 0.01-0.20 mg/ml; the concentration of glycine buffer was 50 mmol/l.
3. The method according to claim 2, wherein the measurement method for directly measuring the glycated hemoglobin content comprises: the concentration of the mouse anti-human HbA1c monoclonal antibody is 0.06mg/ml, and the concentration of the goat anti-mouse IgG is 0.12 mg/ml.
Technical Field
The invention belongs to the field of medical immunity, particularly belongs to the technical field of glycosylated hemoglobin detection, relates to a detection method for directly determining the content of glycosylated hemoglobin, and particularly relates to a detection method for directly determining the percentage content of the glycosylated hemoglobin by using a full-automatic biochemical analyzer.
Background
Glycated hemoglobin (HbA 1c) is a product of hemoglobin in red blood cells in human blood that binds to blood glucose. The glycosylated hemoglobin formed by the combination of blood glucose and hemoglobin is irreversible reaction, is in direct proportion to the blood glucose concentration, and is kept for about 120 days, so the blood glucose concentration before 120 days can be observed, the glycosylated hemoglobin can stably and reliably reflect the average blood glucose level in 120 days before the detection, and the glycosylated hemoglobin has little interference by factors such as blood drawing time, whether the blood glucose is empty and whether the insulin is used.
Glycated hemoglobin was first separated from other types of hemoglobin by chromatography in 1958 and was classified as a glycoprotein in 1968. In 1975, researchers obtained a reaction formula for producing glycated hemoglobin. The total glycated hemoglobin is divided into several subfractions according to each glycated site and reaction participant. Glycated hemoglobin the native (non-glycated) hemoglobin is a0(2 α, 2 β chain). Subfractions (HbA1a1, HbA1a2, HbA1b and HbA1c) are formed by glycosylation of the free amino group of the beta chain-N-terminal valine of hemoglobin with different carbohydrates; these subfractions are collectively referred to as
Common methods for measuring the percentage of glycated hemoglobin include ion exchange chromatography, high performance liquid chromatography, immunological methods, enzymatic methods, and the like. Wherein, the ion exchange chromatography and the high performance liquid chromatography need specific instruments, are expensive and are not suitable for small and medium hospitals. The enzymatic method for detecting glycated hemoglobin utilizes redox reaction, and requires the participation of various enzymes. There are two clinically commonly used immunological methods, one is an immune competitive inhibition method which requires separate measurement of the concentration of total hemoglobin and glycated hemoglobin in a blood sample and subsequent calculation of the ratio of glycated hemoglobin to total hemoglobin, and the other is a latex agglutination method which can directly measure the percentage of glycated hemoglobin. The detection methods are complex and time-consuming to operate, special instruments are needed, the cost is high, other detection methods exist in the market, but the defect of low measurement accuracy is generally existed.
Therefore, how to solve the above problems is an important research content for those skilled in the art.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a method for directly measuring the content of glycosylated hemoglobin;
in order to achieve the above and other related objects, the present invention provides a method for directly measuring glycated hemoglobin content, wherein the reagents used for measuring glycated hemoglobin content include reagent R1, reagent R2, a hemolytic agent, polystyrene latex microspheres, a mouse anti-human HbA1c monoclonal antibody, a goat anti-mouse IgG antibody, and a glycine buffer solution, the method comprising the steps of:
A. diluting an EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample with a hemolytic agent according to a ratio of 1: 100;
B. adding 4 mu L of hemolysis sample into the reagent R1; the reagent R1 contains polystyrene latex microspheres;
C. adding a reagent R2 after 5min of warm prognosis; the reagent R2 is a mixed solution of a mouse anti-human HbA1c monoclonal antibody, a sheep anti-mouse IgG antibody and a glycine buffer solution;
D. the immunoreaction is under the wavelength light of 600 nm-700 nm, and the absorbance after the reaction is read by a full-automatic biochemical analyzer;
E. and finally, directly reading the percentage content of the glycosylated hemoglobin from the calibration curve according to the difference between the reaction absorbances.
In the above scheme, the following is explained:
1. in the scheme, the hemolytic agents in the step A are TTAB and H2O, the polystyrene latex microspheres in the step B are 0.15%, the concentration of the mouse anti-human HbA1c monoclonal antibody in the step c is 0.01-0.06 mg/ml, and the concentration of the goat anti-mouse IgG antibody is 0.01-0.20 mg/ml; the concentration of glycine buffer was 50 mmol/l.
2. In the scheme, the concentration of the mouse anti-human HbA1c monoclonal antibody is 0.06mg/ml, and the concentration of the goat anti-mouse IgG is 0.12 mg/ml.
The detection principle of the invention is as follows: the invention adopts a full-automatic biochemical analyzer as a testing platform and an immune transmission turbidimetry as a detection principle, soluble antigens react with specific antibodies to form immune complexes, when light passes through a reaction suspension, the absorbance changes and is detected by the full-automatic biochemical analyzer, and the degree of the absorbance change is in a certain proportion to the percentage content of the glycosylated hemoglobin in a test sample. In the invention, the percentage content of glycosylated hemoglobin in total hemoglobin is directly measured by utilizing an antigen-antibody reaction, the total hemoglobin and HbA1c in a sample have the same non-specific adsorption and solid phase with polystyrene latex microspheres, when a specific monoclonal antibody of HbA1c is added, a compound of the polystyrene latex microspheres-HbA 1 c-mouse anti-human HbA1c monoclonal antibody is formed, the compound is formed into agglutination due to goat anti-mouse IgG antibody, and the agglutination amount is in a certain proportion relation with the solid phase HbA1c amount on the surfaces of the polystyrene latex microspheres.
Due to the application of the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the determination method of the invention takes an immune transmission turbidimetry as a detection principle, can directly determine the percentage content of the glycosylated hemoglobin in the blood sample without independently determining the content of the total hemoglobin and the glycosylated hemoglobin in the blood sample, directly uses double reagents, simplifies the operation steps of reaction test and saves the reagent cost. And moreover, a full-automatic biochemical analyzer can be utilized, so that the detection cost is reduced.
Drawings
FIG. 1 is a calibration curve for different percentages of glycated hemoglobin calibrators (each point representing a percentage of the calibrator wherein the X-axis represents the percentage of glycated hemoglobin and the Y-axis represents absorbance);
FIG. 2 is a graph showing the correlation between a glycated hemoglobin reagent according to an embodiment of the present invention and a measurement sample of a glycated hemoglobin reagent according to the prior art (wherein the X-axis shows the measurement result of the reagent and the Y-axis shows the measurement result of a reagent according to the prior art).
Detailed Description
Other advantages and capabilities of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification by describing the embodiments of the present invention with reference to the specific embodiments thereof.