CN112485453A

CN112485453A - Liquid chromatography reagent for measuring glycosylated hemoglobin and preparation method thereof

Info

Publication number: CN112485453A
Application number: CN202011295914.7A
Authority: CN
Inventors: 肖江涛; 秦川
Original assignee: Chongqing Zhongyuan Huiji Biotechnology Co Ltd
Current assignee: Chongqing Zhongyuan Huiji Biotechnology Co Ltd
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2021-03-12

Abstract

The invention discloses a liquid chromatography reagent for measuring glycosylated hemoglobin, which comprises: the immunochromatographic test strip comprises an immunochromatographic test strip, a reagent R1 and a reagent R2, wherein the immunochromatographic test strip comprises: the kit comprises a bottom lining, a sample pad, a nitrocellulose membrane and a water absorption pad, wherein one or more detection lines are fixed on the nitrocellulose membrane, the detection lines are anti-human glycosylated hemoglobin antibodies, the reagent R1 comprises 1-10g/L SDS, and the reagent R2 comprises tracer particles labeled anti-human hemoglobin antibodies. The invention is characterized in that a special reagent R2 is configured, a tracer particle labeled antibody is dissolved in a reagent R2, the tracer particle labeled antibody is combined with the tracer particle labeled antibody in the pretreatment stage of a sample to be tested, and meanwhile, the reagent R1 is configured to perform hemolysis treatment on the sample in the pretreatment stage. The invention simplifies the preparation process of the test strip on one hand, and improves the specificity and stability of the test strip on the other hand, so that the detection precision CV value is lower than 3%.

Description

Liquid chromatography reagent for measuring glycosylated hemoglobin and preparation method thereof

Technical Field

The invention relates to the field of medical examination, in particular to a liquid chromatography reagent for measuring glycosylated hemoglobin and a preparation method thereof

Background

Glycated hemoglobin HbA1c is a product of hemoglobin in red blood cells combined with saccharides in serum. It is formed by slow, continuous and irreversible glycation reaction, the content depends on the blood sugar concentration and the contact time of the blood sugar and the hemoglobin, is related to the survival of red blood cells, the synthesis rate is in direct function relation with the glycation amount of the red blood cells in the average life cycle of 120d, clinically, HbA1c represents the control of the average blood sugar level of the human body in the last 2-3 months, and is not related to factors such as blood drawing time, whether patients are fasting or not and whether insulin is used, therefore, HbA1c is the gold standard for measuring the blood sugar control, and is also an important means for diagnosing and managing diabetes.

The methods for measuring glycated hemoglobin known at present include an enzymatic method, an immunological method, a boric acid affinity chromatography, an ion exchange HPLC method, and a capillary electrophoresis method. The ion exchange HPLC method is an IFCC-guided standard reference method, has the advantages of good precision, accurate detection result and the like, and generally adopts the ion exchange HPLC method to detect that the precision CV value is less than 3 percent, but the required equipment is expensive, the detection cost is high, and the detection time is long. The immunochromatography method is relatively easy to develop in primary hospitals due to simple operation of immunochromatography, but the precision and accuracy of detection results are poor due to the heterogeneity of local chromatography materials, the precision CV value detected by the immunochromatography method is usually less than 15%, and the detection accuracy and precision cannot be guaranteed.

Disclosure of Invention

The invention provides a liquid chromatography reagent for measuring glycosylated hemoglobin and a preparation method thereof, wherein a special reagent R2 is configured, a tracer particle labeled antibody is dissolved in a reagent R2, the tracer particle labeled antibody is combined with the tracer particle labeled antibody in a pretreatment stage of a sample to be measured, and meanwhile, a reagent R1 is configured, hemolysis treatment is carried out on the sample in a pretreatment stage, so that the preparation process of a test strip is simplified, the specificity and the stability of the test strip are improved, and the CV value of the detection precision is lower than 3%.

In order to achieve the purpose, the invention adopts the following technical means: a liquid chromatography reagent for measuring glycated hemoglobin, comprising: an immunochromatographic strip, a reagent R1 and a reagent R2;

the reagent R1 contains 1-10g/L of a hemolytic agent, preferably SDS;

the reagent R2 contains 0.05-0.2g/L tracer particle labeled anti-human hemoglobin antibody.

Preferably, the immunochromatographic test strip comprises: the detection line is an anti-human glycosylated hemoglobin antibody, the end, close to the water absorption pad, of the nitrocellulose membrane is provided with 1 or more detection lines, and the quality control line is a goat anti-mouse antibody.

Preferably, the reagent R1 further comprises 20-100mmol/L buffer solution, 0.1-0.8g/L preservative, 50-100mmol/L sodium chloride and 15-30ml/L surfactant.

Preferably, the buffer is selected from at least one of MOPSO buffer, HEPES buffer, Tris buffer or phosphate buffer, the preservative is selected from at least one of proclin-300 or sodium azide, and the surfactant is selected from at least one of Tween-20, Tween-80, Triton X100 and Triton X405.

Preferably, the reagent R2 further comprises a buffer solution of 5-50mmol/L, a protein stabilizing agent of 2-10g/L, a protective agent of 20-50g/L and a preservative of 0.1-0.8 g/L.

Preferably, the buffer solution in the reagent R2 is selected from at least one of MOPSO buffer solution, HEPES buffer solution, Tris buffer solution or phosphate buffer solution, the protective agent is selected from at least one of trehalose or sucrose, the preservative is selected from at least one of proclin-300 or sodium azide, and the protein stabilizer is selected from at least one of sodium caseinate and hydrolysate thereof, BSA, skimmed milk powder, fish gelatin or amino acid.

Preferably, the tracer particles are coloured microspheres, preferably red microspheres.

A preparation method of a liquid chromatography reagent for measuring glycated hemoglobin is characterized by comprising the following steps:

(1) preparation of reagent R1: mixing uniformly after configuring according to 1-10g/L of hemolytic agent, 20-100mmol/L of buffer solution, 0.1-0.8g/L of preservative, 50-100mmol/L of sodium chloride and 15-30ml/L of surfactant, wherein the hemolytic agent is preferably SDS, the buffer solution is selected from at least one of MOPSO buffer solution, HEPES buffer solution, Tris buffer solution or phosphate buffer solution, the preservative is selected from at least one of proclin-300 or sodium azide, and the surfactant is selected from at least one of Tween-20, Tween-80, Triton X100 and Triton X405;

(2) preparation of reagent R2: labeling an anti-human hemoglobin antibody according to 0.05-0.2g/L of tracer particles, 5-50mmol/L of buffer solution, 2-10g/L of protein stabilizer, 20-50g/L of protective agent and 0.1-0.8g/L of preservative, stirring and mixing uniformly after configuration, wherein the tracer particles are preferably red microspheres, the buffer solution is selected from at least one of MOPSO buffer solution, HEPES buffer solution, Tris buffer solution or phosphate buffer solution, the protective agent is selected from at least one of trehalose or sucrose, the preservative is selected from at least one of proclin-300 or sodium azide, and the protein stabilizer is selected from at least one of sodium caseinate and hydrolysate thereof, BSA, skim milk powder, fish gelatin or amino acid;

(3) treatment of nitrocellulose membrane: diluting the anti-human glycated hemoglobin monoclonal antibody with a PBS buffer solution, and marking a detection line on the end of the nitrocellulose membrane close to the sample pad; marking a quality control line on the end, close to the water absorption pad, of the nitrocellulose membrane, of the goat anti-mouse IgG antibody, wherein the line is parallel to the detection line, and drying;

(4) assembling the test strip: overlapping the nitrocellulose membrane on the bottom lining, overlapping the sample pad on one end of the nitrocellulose membrane, overlapping the water absorption pad on the other end of the nitrocellulose membrane, and cutting.

The application of the kit in detecting the glycosylated hemoglobin is characterized by comprising the following steps:

(1) adding a sample to be detected into the reagent R1, and uniformly stirring;

(2) mixing the mixed solution in the step (1) with a reagent R2, and uniformly stirring;

(3) dropwise adding the mixed solution into the sample adding hole of the test strip prepared in the embodiment 1;

(4) detecting with an immune quantitative analyzer;

preferably, the volume ratio of the sample to be tested to the reagent R1 in the step (1) is 1:50-1:100, and the volume ratio of the mixed solution to the reagent R2 in the step (2) is 10:1-50: 1.

The invention has the beneficial effects that:

(1) according to the invention, by configuring the reagent R1, the reagent R1 is utilized to carry out hemolysis treatment on the sample to be detected in the sample pretreatment stage, so that the background interference of the test strip is reduced, and the stability and precision of detection are ensured.

(2) According to the invention, the special reagent R2 is configured, the tracer particle labeled antibody is dissolved in the reagent R2, and the tracer particle labeled antibody is combined with the tracer particle labeled antibody in the pretreatment stage of a sample to be detected, so that the traditional combination pad structure is replaced, on one hand, the preparation process of the test strip is simplified, on the other hand, the defect of poor precision and accuracy of a detection result caused by non-uniform chromatography material is overcome, the specificity and stability of the test strip are improved, and the detection precision CV value is lower than 3%.

Drawings

FIG. 1 is a schematic structural view of a glycated hemoglobin test strip according to example 1 of the present invention;

FIG. 2 is a standard curve graph of the concentration of HbA1c standard substance and the measurement value provided in example 3 of the present invention;

FIG. 3 is a graph showing the correlation between the measurement value of HbA1c and the clinical value, provided in example 3 of the present invention.

The reference numbers in the figures denote:

detecting a line 1; a quality control line 2; a sample pad 3; a nitrocellulose membrane 4; a water absorbent pad 5; a bottom lining 6.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.

EXAMPLE 1 preparation of glycated hemoglobin test strip

1. Preparation of reagent 1

The preparation is carried out according to the following formula, and the mixture is fully stirred, evenly mixed and stored at the temperature of 2-8 ℃.

The rest of the solvent is purified water

2. Preparation of reagent 2

The rest of the solvent is purified water

3. Assembly of test strips

(1) Treatment of nitrocellulose membranes

a. Preparation of detection line

The anti-human glycated hemoglobin HbA1c monoclonal antibody was diluted to 2.0mg/ml with 20mmol/L PBS buffer pH7.2, and the test line was scribed at the left end of the nitrocellulose membrane in an amount of 0.6 ul/cm.

b. Preparation of quality control line

A quality control line is scribed at the right end of the nitrocellulose membrane according to the concentration of 2mg/ml and the amount of 0.6ul/cm, the line and the detection line are parallel to each other, and then the goat anti-IgG antibody is dried for 24 hours at 45 ℃ in a drying oven.

(2) Preparation of sample pad

The glass cellulose film was cut into 20 x 30cm pieces.

(3) Preparation of absorbent pad

The absorbent paper was cut into 30 x 2.7cm pieces.

(4) Assembly

The plastic bottom lining, the sample pad and the absorbent pad are materials commonly used in the field, and the sample pad, the nitrocellulose membrane and the absorbent pad are tightly overlapped on the plastic bottom lining once (as shown in figure 1). The pasted intermediate was cut into strips of 3.72mm wide by a cutter.

Example 2 method of Using test strips

(1) Adding 0.05ml of sample to be detected into 2.45ml of reagent R1, and stirring and mixing uniformly to complete quick hemolysis;

(2) mixing the mixed solution obtained in the step (1) with 0.1ml of reagent R2, and uniformly stirring to ensure that the hemoglobin antibody and the hemoglobin in the sample fully react with the anti-human hemoglobin antibody;

(3) 60ul of the mixed solution was dropped into the well of the test strip prepared in example 1;

(4) carrying out chromatography on the sample mixed solution until the test strip detection line is combined with the antihuman glycosylated hemoglobin antibody, and then carrying out chromatography until the quality control line is combined with the goat anti-mouse antibody;

(5) the gray value after the reaction was measured with an immune quantitative analyzer (Q8 pro, yohui biotechnology limited in Chongqing), and the proportion of glycated hemoglobin in the sample was calculated from the gray value.

Example 3 Performance testing

(1) Detection result of HbA1c standard substance

By adopting the test strip using method in embodiment 2, the HbA1c standard samples with different concentrations are selected as samples to be tested (seven different concentrations, 2%, 4%, 6%, 8%, 12%, 14%, 16% are taken, and 3 repetitions are set for each concentration), the samples to be tested pretreated by the reagents R1 and R2 are respectively dripped onto the test strip sample pads prepared in embodiment 1, and signals are read by an immune quantitative analyzer (Q8 pro, yohui ji biotechnology limited in Chongqing). The results are shown in the following table:

TABLE 1 HbA1c Standard test results

Taking the concentration of HbA1c antigen standard as abscissa, taking the measured average concentration as ordinate, establishing logarithmic equation andfitting a standard curve, the result is shown in FIG. 2, R²The correlation is better when the value is 0.9993, and the detection range is 2% -16%. The result proves that the test strip has good linearity of the detection result when used for detection, and the HbA1c protein concentration in the sample can be quantitatively analyzed through the standard curve.

(2) Determination of precision

Preparing HbA1c standard substance solutions of 2% of a low-value sample, 6% of a medium-value sample and 14% of a high-value sample, measuring by using the test strip prepared in example 1 and the method used in example 2, respectively repeating the measurement for 10 times for each concentration, respectively calculating a measurement mean value and a standard deviation, calculating a variation coefficient, and performing repeated investigation, wherein the detection results are shown in the following table:

TABLE 2 HbA1c test paper strip precision

The coefficient of variation is 1.83%, 1.21% and 1.53%, and meanwhile, the accuracy examination is carried out by calculating the relative deviation by (1-mean/standard value) × 100%, and the results show that the relative deviation is-0.50%, -0.98% and-1.19%, and the experimental results show that the test strip obtained by adopting the preparation scheme of the embodiment has better precision and accuracy.

(3) Determination of the difference between different times

Preparing a sample with the concentration of 5%, repeatedly measuring the sample after sample addition at 1min, 3min, 5min, 10min, 20min, 30min and 60min by using an immune quantitative analyzer (Q8 pro, Yuanhui Gi Biotechnology Co., Ltd., Chongqing), comparing the difference of the detection results at different times, and calculating the relative deviation (the concentration value measured at different time points/the concentration value measured at the first time), wherein the detection results are shown in the following table:

TABLE 3 HbA1c test strip variation over time

Time

1min

3min

5min

10min

20min

30min

60min

Concentration of

5.12％

5.16％

5.09％

5.07％

5.14％

5.26％

5.27％

Relative deviation of

-2.4％

-0.2％

-1.8％

-1.4％

-0.8％

-1.2％

-3.4％

The result shows that the test strip prepared in the embodiment 1 of the invention has small detection difference within 2-60min after sample addition, which shows that the test strip has good time stability.

(4) Clinical evaluation

200 parts of whole blood samples for detecting HbA1c in a hospital are collected, and the kit for detecting HbA1c by the Roche immunoturbidimetry are used for simultaneous detection and comparison. The results of the two methods were analyzed linearly, and the results are shown in FIG. 3, where R is²0.9876, therefore, the test strip for detecting HbA1c has good correlation with the Roche immunoturbidimetry detection kit, meets the requirements of clinical analysis, and is suitable for clinical detection.

Example 4

This example sets up six sets of experiments, each set of experiments using kits differing from example 1 only in the concentration of the surfactant triton X100 in the reagent R1, and the remaining preparation methods were the same as in example 1. The six kits are adopted for detection at the same time, and the detection results are shown in the following table:

TABLE 4

Note: in the embodiment, the content of triton X100 in the group A reagent R1 is 5 mL/L; the group B is 10 mL/L; group C is 15 mL/L; the group D is 20 mL/L; group E is 30 mL/L; the F group is 40mL/L, and the rest components and the preparation process are the same as the example 1.

The experimental result shows that the precision is higher when the content of the surfactant triton X100 in the reagent R1 is between 15mL/L and 30 mL/L.

Example 5

This example sets up 4 sets of experiments, each set of experiments using kits differing from example 1 only in the type of surfactant in reagent R1, and the rest of the preparation procedure was the same as in example 1. The four kits are adopted for detection at the same time, and the detection results are shown in the following table:

TABLE 5

Note: in the group A reagent R1 in the embodiment, the surfactant is 20ml/L Tween-20; group B is 20ml/L Brij58, group C is 20ml/L triton X450; group D is 20ml/L Tween-80; group E is 20ml/L triton X100, and the rest of the components and the preparation process are the same as those in the example 1.

Experimental results show that when the surfactants adopted in the reagent R1 are Tween-20, Triton X405, Tween-80 and Triton X100, the detection precision of the test strip is high.

Example 6

This example sets up a total of five experiments, wherein each experiment uses a kit differing from example 1 only in the concentration of the protective agent (trehalose) in the reagent R2, and the remaining preparation method is the same as in example 1. The five kits are simultaneously adopted for detection, and the detection results are shown in the following table:

TABLE 4

Note: in the embodiment, the content of trehalose in the group A reagent R2 is 10 g/L; the group B is 20 g/L; group C is 30 g/L; the group D is 50 g/L; the E group is 70g/L, and the rest components and the preparation process are the same as those of the example 1.

The experimental result shows that the precision is higher when the content of the protective agent trehalose in the reagent R2 is between 20 and 50 g/L.

In summary, the reagent R1 is provided in the invention, and the reagent R1 is used to perform hemolysis treatment on the sample to be tested in the sample pretreatment stage, so that the background interference of the test strip is reduced, and the stability and precision of the test are ensured. Meanwhile, the special reagent R2 is configured, the tracer particle labeled antibody is dissolved in the reagent R2, and the tracer particle labeled antibody is combined with the tracer particle labeled antibody in the pretreatment stage of the sample to be detected, so that the traditional combination pad structure is replaced, on one hand, the preparation process of the test strip is simplified, on the other hand, the defect that the precision and the accuracy of a detection result are poor due to non-uniform chromatography materials is overcome, the specificity and the stability of the test strip are improved, and the CV value of the detection precision is lower than 3%.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A liquid chromatography reagent for measuring glycated hemoglobin, comprising: an immunochromatographic strip, a reagent R1 and a reagent R2;

the reagent R1 contains 1-10g/L of a hemolytic agent, preferably SDS;

2. The liquid chromatography reagent for glycated hemoglobin as set forth in claim 1, wherein the immunochromatographic strip comprises: the detection line is an anti-human glycosylated hemoglobin antibody, the end, close to the water absorption pad, of the nitrocellulose membrane is provided with 1 or more detection lines, and the quality control line is a goat anti-mouse antibody.

3. The liquid chromatography reagent for measuring glycated hemoglobin of claim 1 or 2, wherein the reagent R1 further comprises 20-100mmol/L of a buffer, 0.1-0.8g/L of a preservative, 50-100mmol/L of sodium chloride, and 15-30ml/L of a surfactant.

4. The liquid chromatography reagent for glycated hemoglobin as set forth in claim 3, wherein the buffer is at least one selected from the group consisting of MOPSO buffer, HEPES buffer, Tris buffer and phosphate buffer, the preservative is at least one selected from the group consisting of proclin-300 and sodium azide, and the surfactant is at least one selected from the group consisting of Tween-20, Tween-80, Triton X100 and Triton X405.

5. The liquid chromatography reagent for measuring glycated hemoglobin of any one of claims 1-4, wherein the reagent R2 further comprises a buffer solution of 5-50mmol/L, a protein stabilizer of 2-10g/L, a protective agent of 20-50g/L, and a preservative of 0.1-0.8 g/L.

6. The liquid chromatography reagent for glycated hemoglobin as set forth in claim 5, wherein the buffer solution in the reagent R2 is selected from at least one of MOPSO buffer, HEPES buffer, Tris buffer or phosphate buffer, the protecting agent is selected from at least one of trehalose or sucrose, the preservative is selected from at least one of proclin-300 or sodium azide, and the protein stabilizing agent is selected from at least one of sodium caseinate and its hydrolysate, BSA, skim milk powder, fish gelatin or amino acids.

7. The liquid chromatography reagent for measuring glycated hemoglobin as set forth in any one of claims 1 to 6, wherein the labeled particles are colored microspheres, preferably red microspheres.

8. A preparation method of a liquid chromatography reagent for measuring glycated hemoglobin is characterized by comprising the following steps:

9. Use of the kit according to any one of claims 1 to 6 for the detection of glycated hemoglobin, comprising the steps of:

(1) adding a sample to be detected into the reagent R1, and uniformly stirring;

(4) and detecting by using an immune quantitative analyzer.

10. The use of the kit according to claim 9 for detecting glycated hemoglobin, wherein the volume ratio of the sample to be detected to the reagent R1 in step (1) is 1:50-1:100, and the volume ratio of the mixture to the reagent R2 in step (2) is 10:1-50: 1.