CN115774110A - Biosafety glycosylated hemoglobin dissociation liquid and glycosylated hemoglobin dissociation method - Google Patents

Abstract

The invention provides a biologically safe glycosylated hemoglobin dissociation solution and a glycosylated hemoglobin dissociation method, and relates to the field of biological detection. The biosafety glycosylated hemoglobin dissociation liquid comprises a reagent I, a reagent II and a hemolytic agent, wherein the reagent I comprises latex and a buffer solution, the reagent II comprises a primary antibody, a sensitizing protein and a stabilizing agent, the primary antibody is a mouse anti-human HbA1c monomer clone antibody, and the sensitizing protein is a directional binding protein capable of being in contact with an area outside a mouse IgG Fab area of the primary antibody. 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 reaction test operation steps, saves the reagent cost, has high result accuracy, good sensitivity, good precision and wide linear range, and is convenient for popularization and use.

Description

Biosafety glycosylated hemoglobin dissociation liquid and glycosylated hemoglobin dissociation method

Technical Field

The invention relates to the field of biological detection, in particular to a biosafety glycosylated hemoglobin dissociation liquid and a glycosylated hemoglobin dissociation method.

Background

Hemoglobin (Hb) is generally composed of HbA (97%), hbA2 (2.5%) and HbF (0.5%). Chromatographic analysis of HbA revealed minor haemoglobins HbA1a, hbA1b, hbA1c and HbA0.HbA1a, hbA1b, and HbA1c are collectively referred to as glycated hemoglobin (HbA 1), and their glycosylation site is a.beta.chain N-terminal valine residue. HbA0 refers to hemoglobin without a carbohydrate attached.

The prior glycated hemoglobin dissociation solution has the following several types, but all have corresponding technical defects: 1) Organic solvent extraction method: for example, the extraction using acetonitrile is performed by dissolving glycated hemoglobin in an organic phase by the principle of similar phase dissolution, and centrifuging the solution using a centrifuge to obtain an extract containing glycated hemoglobin. In the method, the glycated hemoglobin needs to be centrifuged after dissociation, so that the automatic operation is not convenient to realize; 2) Acid-base method: depending on the pH, which is higher or lower, glycated hemoglobin denatures or conformational changes are dissociated to release. The vitamin D solution obtained by the method is a solution with higher pH or lower pH, which is not beneficial to the next immunoreaction; 3) A combination method. The methods currently more commonly used are 1% dodecyltrimethylammonium bromide, 0.1% Tween-80, 0.1% Tween-20, 0.1% TritonX-100, 0.8% ammonium chloride in combination to dissociate the glycated haemoglobins. The method contains dodecyl trimethyl ammonium bromide and ammonium chloride which are highly toxic substances and have biosafety risks. The excessive use of TritonX-100 can cause environmental pollution.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides the biosafety glycosylated hemoglobin dissociation liquid and the glycosylated hemoglobin dissociation method.

(II) technical scheme

In order to realize the purpose, the invention is realized by the following technical scheme: the biosafety glycosylated hemoglobin dissociation liquid comprises a reagent I, a reagent II and a hemolytic agent, wherein the reagent I comprises latex and buffer solution, the reagent II comprises a primary antibody, a sensitizing protein and a stabilizing agent, the primary antibody is a mouse anti-human HbA1c monomer clone antibody, and the sensitizing protein is a directional binding protein capable of being in contact with the region outside the Fab region of a mouse IgG of the primary antibody.

Preferably, the latex has a particle size of 50-500nm and a concentration of 0.05-0.5% (mass/volume ratio), the buffer is glycine, the pH value is 5.0-9.0, and the concentration is 0.01-0.1mol/L.

Preferably, the sensitizing Protein is one of an anti-mouse IgG Fc terminal polyclonal antibody, protein A and Protein G.

Preferably, the reagent II comprises a mouse anti-human HbA1c monoclonal antibody and a rabbit anti-mouse IgG Fc end polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 50-100mg/L, and the concentration of the rabbit anti-mouse IgG Fc end polyclonal antibody is 10-20mg/L.

Preferably, the stabilizer is composed of one or more of micro urea, thiocyanate or guanine hydrochloride.

A biosafety glycated hemoglobin dissociation method comprising the steps of;

s1, taking an EDTA (ethylene diamine tetraacetic acid) anticoagulation whole blood sample and a hemolytic agent according to the ratio of 1:100, diluting;

s2, adding 4 mu L of hemolysis sample into the reagent I;

s3, adding a second reagent after 5min Wen Yuhou;

s4, reading the absorbance of the immunoreaction through a full-automatic biochemical analyzer under the wavelength light of 600nm to 700nm;

and S5, finally, directly reading the percentage content of the glycosylated hemoglobin from the calibration curve according to the difference between the reaction absorbances.

Preferably, the linear equation is as shown in the formula:

Y=A/（A+B）+b

wherein A is the peak area of the N-terminal valine glycosylation modified beta globin with the mass-to-charge ratio of 15868 m/z; b is the peak area where the mass-to-charge ratio of hemoglobin and protein beta globin is 16030 m/z; y is the glycated hemoglobin value of the blood sample to be measured, and b is a linear analysis constant.

The working principle is as follows: the invention adopts a full-automatic biochemical analyzer as a testing platform and an immune transmission turbidimetry as a detection principle, a soluble antigen reacts with a specific antibody to form an immune complex, when light passes through a reaction suspension, the absorbance changes and is detected by the full-automatic biochemical analyzer, and the amount of the absorbance change is in a certain proportion to the percentage content of the glycosylated hemoglobin in a test sample; firstly, taking an EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample and a hemolytic agent according to the ratio of 1:100, diluting, adding 4 mu L of hemolysis sample into the first reagent, adding the second reagent after 5min Wen Yuhou, reading the absorbance of the immunoreaction by a full-automatic biochemical analyzer under the wavelength light of 600nm to 700nm, and finally directly reading the percentage content of the glycosylated hemoglobin from a calibration curve according to the difference between the reaction absorbances.

(III) advantageous effects

The invention provides a biologically safe glycated hemoglobin dissociation solution and a glycated hemoglobin dissociation method. The method has the following beneficial effects:

1. the method for detecting the glycosylated hemoglobin has the advantages of high detection result accuracy, good sensitivity, good precision, wide linear range and convenient popularization and use, and can be widely applied to hospitals at all levels, health prevention departments and medical biological research and development units for measuring the content of the glycosylated hemoglobin in human whole blood.

2. The invention provides a biologically safe glycosylated hemoglobin dissociation solution and a glycosylated hemoglobin dissociation method, the determination method of the invention takes an immunity transmission turbidimetry as a detection principle, the percentage content of the glycosylated hemoglobin in a blood sample can be directly determined without independently determining the content of the total hemoglobin and the glycosylated hemoglobin in the blood sample, double reagents are directly used, the reaction test operation steps are simplified, the reagent cost is saved, and a full-automatic biochemical analyzer can be utilized to reduce the detection cost.

3. In the glycosylated hemoglobin detection reagent, the sensitizing protein is selected from sensitizing proteins capable of directionally combining with an IgG Fc region of an anti-mouse anti-human HbA1c monoclonal antibody, and the sensitizing protein is combined into directional combination, so that an IgG Fab region of the anti-mouse anti-human HbA1c monoclonal antibody cannot be blocked, the combination of primary antibody and HbA1c cannot be influenced, and the detection sensitivity and accuracy of the glycosylated hemoglobin are higher.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The embodiment of the invention provides a biologically safe glycated hemoglobin dissociation solution, which comprises a first reagent, a second reagent and a hemolytic agent, wherein the first reagent comprises latex and buffer solution, the second reagent comprises a first antibody, a sensitizing protein and a stabilizing agent, the first antibody is a mouse anti-human HbA1c monomer clone antibody, and the sensitizing protein is a directional binding protein capable of being combined with the first antibody outside the Fab region of mouse IgG.

The reagent I also comprises latex and buffer solution, wherein the particle size of the latex is 50-500nm, the concentration is 0.05-0.5% (mass/volume ratio), the buffer solution is glycine, the pH value is 5.0-9.0, the concentration is 0.01-0.1mol/L, the sensitizing Protein is one of immune anti-mouse IgG Fc end polyclonal antibody, protein A and Protein G, the reagent II comprises mouse anti-human HbA1c monoclonal antibody and rabbit anti-mouse IgG Fc end polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 50-100mg/L, the concentration of the rabbit anti-mouse IgG Fc end polyclonal antibody is 10-20mg/L, and the stabilizer is formed by mixing one or more than two of micro urea, thiocyanate or guanine hydrochloride.

A biosafety glycated hemoglobin dissociation method comprising the steps of;

s1, taking an EDTA (ethylene diamine tetraacetic acid) anticoagulation whole blood sample and a hemolytic agent according to the ratio of 1:100, diluting;

s2, adding 4 mu L of hemolysis sample into the reagent I;

s3, adding a second reagent after 5min Wen Yuhou;

s4, reading the absorbance of the immunoreaction through a full-automatic biochemical analyzer under the wavelength light of 600nm to 700nm;

and S5, finally, directly reading the percentage content of the glycosylated hemoglobin from the calibration curve according to the difference between the reaction absorbances.

The equation of the type is shown as the formula:

Y=A/（A+B）+b

wherein A is the peak area of the N-terminal valine glycosylation modified beta globin with the mass-to-charge ratio of 15868 m/z; b is the peak area of the hemoglobin and the protein beta globin with the mass-to-charge ratio of 16030 m/z; y is the glycated hemoglobin value of the blood sample to be measured, and b is a linear analysis constant.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (7)

1. The biosafety glycosylated hemoglobin dissociation liquid comprises a reagent I, a reagent II and a hemolytic agent, and is characterized in that: the reagent I comprises latex and buffer solution, the reagent II comprises primary antibody, sensitizing protein and stabilizer, the primary antibody is a mouse anti-human HbA1c monomer clone antibody, and the sensitizing protein is a directional binding protein capable of being in contact with the region outside the Fab region of the primary antibody of mouse IgG.

2. The biosafety glycated hemoglobin dissociation solution according to claim 1, wherein: the latex particle size is 50-500nm, the concentration is 0.05-0.5% (mass/volume ratio), the buffer solution is glycine, the pH value is 5.0-9.0, and the concentration is 0.01-0.1mol/L.

3. The biosafety glycated hemoglobin dissociation solution according to claim 1, wherein: the sensitization Protein is one of polyclonal antibody, protein A and Protein G of an Fc end of an immune anti-mouse IgG.

4. The biosafety glycated hemoglobin dissociation of claim 1, wherein: the reagent II comprises a mouse anti-human HbA1c monoclonal antibody and a rabbit anti-mouse IgG Fc end polyclonal antibody, the concentration of the mouse anti-human HbA1c monoclonal antibody is 50-100mg/L, and the concentration of the rabbit anti-mouse IgG Fc end polyclonal antibody is 10-20mg/L.

5. The biosafety glycated hemoglobin dissociation solution according to claim 1, wherein: the stabilizer is formed by mixing one or more than two of micro urea, thiocyanate or guanine hydrochloride.

6. A biosafety glycated hemoglobin dissociation method, comprising the steps of:

s1, taking an EDTA (ethylene diamine tetraacetic acid) anticoagulation whole blood sample and a hemolytic agent according to the ratio of 1:100, diluting;

s2, adding 4 mu L of hemolysis sample into the reagent I;

s3, adding a second reagent after 5min Wen Yuhou;

s4, reading the absorbance of the immunoreaction through a full-automatic biochemical analyzer under the wavelength light of 600nm to 700nm;

and S5, finally, directly reading the percentage content of the glycosylated hemoglobin from the calibration curve according to the difference between the reaction absorbances.

7. The biosafety glycated hemoglobin dissociation method according to claim 6, wherein: the linear equation is shown as follows:

Y=A/（A+B）+b

wherein A is the peak area of the N-terminal valine glycosylation modified beta globin with the mass-to-charge ratio of 15868 m/z; b is the peak area where the mass-to-charge ratio of hemoglobin and protein beta globin is 16030 m/z; y is the glycated hemoglobin value of the blood sample to be measured, and b is a linear analysis constant.