CN112034186A - Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof - Google Patents
Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof Download PDFInfo
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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
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
A glycosylated hemoglobin kit based on biotin-streptavidin amplification relates to the technical field of biomedical diagnosis and is provided based on the problem that the existing glycosylated hemoglobin kit is poor in freeze-thaw resistance. The kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises glycine buffer solution, latex microspheres, a surfactant, salt, a stabilizer, a preservative and an anti-freezing and thawing agent; reagent R2 included MES buffer, salts, stabilizers, biotin, streptavidin, HbA1c monoclonal antibody and preservatives. The invention also discloses a preparation method of the glycosylated hemoglobin kit based on biotin-streptavidin amplification. Compared with the prior art, the invention has the advantages that: the kit R1 of the invention contains polystyrene microspheres without any modification, can rapidly adsorb glycosylated hemoglobin in blood, is added with an anti-freezing and thawing agent, can improve the anti-freezing and thawing stability of the kit, and is added with the anti-freezing and thawing agent and then repeatedly frozen and thawed R1 test values obviously tend to be stable.
Description
Technical Field
The invention relates to the technical field of medicine and biochemistry, in particular to a glycosylated hemoglobin kit based on biotin-streptavidin amplification and a preparation method thereof.
Background
According to the International Diabetes Federation (IDF), there are about 4.63 million diabetics in 2019 worldwide, wherein the number of diabetics in china ranks first and is about 1.164 million. Glycated hemoglobin (HbA1c) was found to be a good indicator of disease control in diabetic patients, and it is reflected in the average blood glucose level of the patients over the 120 days prior to the test. HbA1c is used as a gold standard for glycemic control, and all diabetic patients, regardless of the way they react to changes in blood glucose, will eventually use changes in HbA1c as an indicator for the ultimate detection of a drug or a treatment regimen. Therefore, the clinical significance of the detection of HbA1c is very important.
The current detection methods of HbA1c mainly include chromatography, immunization and electrophoresis. Wherein the chromatography comprises high performance liquid cation exchange chromatography, high performance liquid boric acid affinity chromatography, etc. As the gold standard for HbA1c detection, high performance liquid cation exchange chromatography is the most clinically valuable and accurate method. However, in the study of measuring HbA1c by liquid phase ion exchange chromatography, when HbA1c is detected after a sample (for example, a whole blood sample, a whole blood diluted sample, a whole blood lyophilized sample, or the like) is stored, the measurement value fluctuates with the lapse of the storage time of the sample.
The invention patent of patent CN101595230A discloses a method for measuring HbA1c, and also indicates that when the Hb 1c is detected after the Hb-containing sample is stored, a value lower than the measured value obtained by using the Hb-containing sample just collected is obtained. This patent uses a method of adding an inhibitor to a sample to avoid the fluctuation of the measured value of HbA1c due to the storage of the sample, but this method requires the direct involvement of the measurer, complicates the measurement process, and greatly reduces the detection efficiency. Patent CN107991500A discloses a glycated hemoglobin detection kit, which comprises reagent R1 and reagent R2, wherein reagent R1 comprises latex, buffer a and surfactant; reagent R2 comprises an antibody, buffer B and a surfactant. The kit can eliminate the influence of the hemoglobin content difference of an individual on the detection of the glycosylated hemoglobin, and has the advantages of good stability, and convenient use and storage. The problems with the above technique are as follows: poor freeze-thaw resistance, low sensitivity and poor linear range.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a glycosylated hemoglobin kit based on biotin-streptavidin amplification, which has good freeze-thaw resistance and wide linear range of glycosylated hemoglobin concentration.
The invention is realized by the following technical scheme, the glycosylated hemoglobin kit based on biotin-streptavidin amplification comprises a reagent R1 and a reagent R2, and comprises the following components in parts by weight:
reagent R1:
reagent R2:
the kit R1 of the invention contains polystyrene microspheres without any modification, can rapidly adsorb glycosylated hemoglobin in blood, is added with an anti-freezing agent, can improve the anti-freezing and thawing stability of the kit, and is added with anti-freezingAfter the thawing agent is repeatedly frozen and thawed, the R1 test value obviously tends to be stable; adopts double reagents, has simple operation, high sensitivity and wide linear range R20.9898, it can be widely applied to various transmission or scattering analyzers, including general biochemical analyzers, specific protein analyzers, and the like.
Preferably, the pH of the glycine buffer is 8.0.
Preferably, the MES buffer has a pH of 7.5.
Preferably, the surfactant comprises one or a combination of two or more of NP40, Triton X-100 and sodium dodecyl sulfate.
Preferably, the salt comprises sodium chloride or potassium chloride.
Preferably, the preservative comprises sodium azide or Proclin-300.
Preferably, the freeze/thaw resistance agent comprises one or a combination of betaine and glycerol.
Preferably, the stabilizer comprises bovine serum albumin, sucrose or trehalose.
Preferably, the latex microspheres are polystyrene latex microspheres; the diameter of the latex microsphere is 100-400 nm.
A preparation method of a glycosylated hemoglobin kit based on biotin-streptavidin amplification comprises the following steps:
(1) preparation reagent R1
Diluting the latex microspheres in a glycine buffer solution until the concentration is 0.15%, stirring at room temperature, aging at 37 ℃ for 12 hours after stirring for 2 hours, and dissolving a surfactant, salt, a preservative and an anti-freezing and thawing agent in the mixture according to the proportion to obtain a reagent R1 solution;
(2) preparation reagent R2
Mixing biotin and HbA1c antibodies according to the proportion, labeling the biotin onto the antibodies, adding streptavidin according to the proportion, reacting for 2 hours at room temperature, and then placing the mixture into a homogenizer for dispersing for 10min to obtain a biotin-streptavidin-HbA 1c antibody compound; dissolving the biotin-streptavidin-HbA 1c antibody compound in MES buffer solution, and uniformly stirring; and dissolving the surfactant, the salt and the preservative in the mixture according to the proportion to obtain the reagent R2 liquid.
Compared with the prior art, the invention has the advantages that:
(1) the kit R1 contains polystyrene microspheres without any modification, glycosylated hemoglobin in blood can be rapidly adsorbed, the freeze-thaw resisting agent is added into the reagent, the freeze-thaw resisting stability of the kit can be improved, and the repeated freeze-thaw resisting agent added into the kit obviously tends to be stable in the R1 test value; adopts double reagents, has simple operation, high sensitivity and wide linear range R20.9898, it can be widely applied to various transmission or scattering analyzers, including general biochemical analyzers, specific protein analyzers, and the like.
(2) The invention utilizes the strong binding effect of avidin-streptavidin to combine with the anti-glycated hemoglobin monoclonal antibody to form a firm and stable biotin-streptavidin-HbA 1c antibody compound, and can effectively amplify the turbidity degree of latex.
Drawings
FIG. 1 is a graph showing the labeling results of HbA1c antibody in the example of the present invention;
FIG. 2 is a calibration curve established for the latex reagents of example 1 of the present invention;
FIG. 3 is a cross-linking reaction diagram of the kit of the present invention for use in latex-enhanced immunoturbidimetry;
FIG. 4 is a graph of linear range results for a kit according to an embodiment of the invention;
FIG. 5 is a graph of the results of clinical correlations of the kits of the embodiments of the invention;
FIG. 6 is a graph showing the results of the detection of freeze-thaw resistance by the kit of example 1 of the present invention;
FIG. 7 is a graph showing the results of the freeze-thaw resistance test of the kit of comparative example 1 of the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided with reference to the drawings, and the embodiments are implemented on the premise of the technical solution of the present invention, and the detailed embodiments and the specific operation procedures are provided, but the protection scope of the present invention is not limited to the following embodiments.
A glycosylated hemoglobin kit based on biotin-streptavidin amplification, the kit comprising a reagent R1 and a reagent R2, comprising the following components in respective amounts:
reagent R1:
reagent R2:
the pH of the glycine buffer was 8.0.
The MES buffer had a pH of 7.5.
The surfactant comprises one or the combination of two or more of NP40, triton X-100 and sodium dodecyl sulfate.
The salt comprises sodium chloride or potassium chloride.
The preservative comprises sodium azide or Proclin-300.
The anti-freezing and thawing agent comprises one or the combination of betaine and glycerol.
The stabilizer comprises bovine serum albumin, sucrose or trehalose.
The latex microspheres are polystyrene latex microspheres; the diameter of the latex microsphere is 100-400 nm.
The latex microspheres of the present invention are selected from P2116 from the manufacturer JSRifeSciences corporation.
Example 1
The glycosylated hemoglobin kit based on biotin-streptavidin amplification of the embodiment comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
reagent R2:
the surface of the latex microsphere is not modified by any modification, and the particle size of the latex microsphere is 125 nm.
Example 2
The glycosylated hemoglobin kit based on biotin-streptavidin amplification of the embodiment comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
reagent R2:
the surface of the latex microsphere is not modified by any modification, and the particle size of the latex microsphere is 125 nm.
Example 3
The glycosylated hemoglobin kit based on biotin-streptavidin amplification of the embodiment comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
reagent R2:
example 4
The method for preparing the glycosylated hemoglobin kit based on biotin-streptavidin amplification comprises the following steps:
(1) preparation reagent R1
Diluting the latex microspheres in a glycine buffer solution until the concentration is 0.15%, stirring at room temperature, aging at 37 ℃ for 12 hours after stirring for 2 hours, and dissolving a surfactant, salt, a preservative and an anti-freezing and thawing agent in the mixture according to the proportion to obtain a reagent R1 solution;
(2) preparation reagent R2
Mixing biotin and HbA1c antibodies according to the proportion, labeling the biotin onto the antibodies, adding streptavidin according to the proportion, reacting for 2 hours at room temperature, and then placing the mixture into a homogenizer for dispersing for 10min to obtain a biotin-streptavidin-HbA 1c antibody compound; dissolving the biotin-streptavidin-HbA 1c antibody compound in MES buffer solution, and uniformly stirring; and dissolving the surfactant, the salt and the preservative in the mixture according to the proportion to obtain the reagent R2 liquid.
Principle of HbA1c antibody labeling process: the streptavidin molecule consists of 4 identical peptide chains, wherein each peptide chain can be combined with one biotin and does not have any glycosyl group, so that one streptavidin molecule can be combined with 4 biotin-labeled antibodies like avidin, namely, the HbA1c antibody labeling process is completed, and the labeling result is shown in FIG. 1.
(3) Preparation of standards
A series of standard products are prepared by using commercially available HbA1c antigen and standard product diluent, wherein the concentration of the standard product diluent is respectively 0L, 4.3%, 5.6%, 8.3%, 11.5% and 15%, and the standard product diluent consists of 50mMPBSpH7.4, 0.1% Proclin300, 0.1% Triton X-100, 1.8% sodium chloride and 1% bovine serum albumin.
(4) Preparation of quality control product
A high concentration HbA1c sample (15%) was diluted to 5.0% and 10% with standard dilutions as low and high quality controls, respectively.
Calibration curve was established on a biochemical analyzer by placing 6 HbA1c standards, at concentrations from 0 to 15%, in a sample pan of a biochemical analyzer (Hitachi full-automatic biochemical analyzer 7180 was used in this example) and reacting with the prepared HbA1c reagent R1 and the reaction solution R2, respectively, and the sample: r1: and (3) detecting R2 (3 ul), 150ul and 50ul, recording the reactivity, and automatically completing reagent sucking and sampling steps by the biochemical analyzer after the reagent parameters are set and the reagent position is correctly placed, wherein the whole process takes about 10-12 min. The detection wavelength is 800/660nm, a two-point end point method is adopted, the reaction time is 5min, and the difference of the two-point reactivity is calculated. Calibration curves can be established based on the difference in concentration and reactivity. FIG. 2 is a calibration curve established for the latex reagents of example 1.
Example 6
The kit provided by the invention is used for a latex enhanced immunoturbidimetry method, glycated hemoglobin is adsorbed on latex microspheres in R1, and then the latex microspheres adsorbed by the glycated hemoglobin are captured by using a biotin-streptavidin-HbA 1c antibody compound to form cross-linking among particles, so that the scattering degree or transmission absorbance of the solution is changed, as shown in figure 3.
Example 7
This example is used to evaluate the biotin-streptavidin amplification based glycated hemoglobin kit of the above examples:
(1) sensitivity verification
Using the reagent of example 1, a zero-value standard was selected as a blank sample, and 3 samples having a concentration of approximately 5.5% were measured, and the measurement was repeated 10 times at 37 ℃ under the conditions of 800/660nm wavelength and 1cm optical path in a fully automatic biochemical analyzer (Hitachi 7180), and the difference (. DELTA.A) between the absorbance change of the sample and the absorbance change of the blank was calculated and converted into the absorbance difference (. DELTA.A) between glycated hemoglobin having a concentration of 5.5%. The average value and Standard Deviation (SD) of the 20 test results were calculated according to the standard curve established in fig. 3, and the test results in3 groups are shown in table 1, which show that the analytical sensitivities are 0.1350, 0.1376 and 0.1370, respectively, and thus, it is proved that the sensitivity of the reagent can be improved by the monoclonal antibody labeled biotin-streptavidin. The clinical reference value for detecting the glycosylated hemoglobin (HbA1c) is about 5.0-8.0%, and the sensitivity of the reagent is smaller than the reference value by one order of magnitude, thereby completely meeting the use requirement.
TABLE 1 assay sensitivity test
| Numbering | Sample 1 (5.3%) | Sample 2 (5.5%) | Sample 3 (5.4%) |
| 1 | 0.1346 | 0.1398 | 0.1385 |
| 2 | 0.1348 | 0.1385 | 0.1376 |
| 3 | 0.1362 | 0.1356 | 0.1375 |
| 4 | 0.1326 | 0.1398 | 0.1356 |
| 5 | 0.1362 | 0.1357 | 0.1368 |
| 6 | 0.1347 | 0.1365 | 0.1375 |
| 7 | 0.1364 | 0.1385 | 0.1367 |
| 8 | 0.1345 | 0.1365 | 0.1364 |
| 9 | 0.1348 | 0.1362 | 0.1362 |
| 10 | 0.1352 | 0.1385 | 0.1367 |
| AVE | 0.1350 | 0.1376 | 0.1370 |
| SD | 0.0011 | 0.0016 | 0.0008 |
(2) Linear range verification
A low-value calibrator 4.3% (L) and a high-concentration HbA1c standard 15% (H) were prepared as 6 samples at different dilution concentrations (5L, 4L +1H, 3L +2H, 2L +3H, L +4H, and 5H), respectively. These samples were tested 3 times for each dilution concentration using the reagents prepared in example 1, and the results of the measurements were averaged for each dilution concentration. And (3) calculating a linear regression equation by taking the dilution concentration as an independent variable and the mean value of the detection result as a dependent variable of the measurement result, and calculating a correlation coefficient (r) of linear regression. The linear correlation curve is shown in fig. 4, and the linear regression equation is that y is 1.0148x +0.2037, R2The linear relationship is shown to be good at 0.9898.
(3) Repeatability verification of the kit
The control substance having a HbA1c concentration of 5% at the normal level and the control substance having a HbA1c concentration of 9.5% at the abnormal level were tested using the reagents prepared in example 1, each test was repeated 10 times, the mean value and Standard Deviation (SD) of the measured values were calculated, the Coefficient of Variation (CV) was calculated according to equation (1), and the test results are shown in table 2. According to the measurement result, the coefficient of variation CV is calculated to be 0.96 percent and 0.88 percent, which meets the technical requirement that the reagent CV is less than or equal to 3 percent.
Table 2 shows the results of the reproducibility test of the kit
| Serial number | Level one | Level two |
| 1 | 5 | 9.5 |
| 2 | 5 | 9.4 |
| 3 | 5.1 | 9.4 |
| 4 | 5 | 9.4 |
| 5 | 5.1 | 9.2 |
| 6 | 5 | 9.3 |
| 7 | 5.1 | 9.3 |
| 8 | 5 | 9.4 |
| 9 | 5 | 9.4 |
| 10 | 5 | 9.4 |
| M | 5.03 | 9.37 |
| SD | 0.048 | 0.082 |
| CV | 0.96％ | 0.88％ |
(4) Clinical relevance validation
By collecting 64 clinical samples and performing detection by using the reagent device in example 1, correlation analysis is performed between the detection result and the detection result of the kit imported in the market. The analysis results are shown in FIG. 5. The results of the correlation straight line fit show the fitting equation as Y-0.9751 x +0.1493, where Y-intercept 0.1493, slope 0.9751, and correlation coefficient R-0.9855. The result shows that the kit has high correlation with imported and marketed kits and has very consistent accuracy.
Comparative example 1
This comparative example differs from example 1 in that: the agent R1 of this comparative example did not have added a freeze-thaw resistance agent.
And (3) detecting the freeze-thaw resistance: the control substance at the normal level with the HbA1c concentration of 5% and the control substance at the abnormal level with the HbA1c concentration of 9.5% were tested by repeating freezing and thawing 5 times in a refrigerator at-40 ℃ using the reagent R1 prepared in example 1 and the reagent R1 prepared in comparative example 1, and by testing the data before and after 5 times of freezing and thawing, respectively, together with the reagent R2 in example 1. The test results are shown in fig. 6 and 7: after the anti-freezing and thawing agent is added, the repeated freezing and thawing R1 test value obviously tends to be stable, and the anti-freezing and thawing agent is not used in comparative example 1, so that the anti-freezing and thawing value is obviously reduced after freezing and thawing.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The glycosylated hemoglobin kit based on biotin-streptavidin amplification is characterized in that: the kit comprises a reagent R1 and a reagent R2, and comprises the following components in parts by weight:
reagent R1:
reagent R2:
2. the glycated hemoglobin kit based on biotin-streptavidin amplification according to claim 1, wherein: the pH of the glycine buffer was 8.0.
3. The biotin-streptavidin amplification-based glycated hemoglobin kit of claim 2, wherein: the MES buffer had a pH of 7.5.
4. The glycated hemoglobin kit based on biotin-streptavidin amplification according to claim 1, wherein: the surfactant comprises one or the combination of two or more of NP40, triton X-100 and sodium dodecyl sulfate.
5. The glycated hemoglobin kit based on biotin-streptavidin amplification according to claim 1, wherein: the salt comprises sodium chloride or potassium chloride.
6. The glycated hemoglobin kit based on biotin-streptavidin amplification according to claim 1, wherein: the preservative comprises sodium azide or Proclin-300.
7. The glycated hemoglobin kit based on biotin-streptavidin amplification according to claim 1, wherein: the anti-freezing and thawing agent comprises one or the combination of betaine and glycerol.
8. The glycated hemoglobin kit based on biotin-streptavidin amplification according to claim 1, wherein: the stabilizer comprises bovine serum albumin, sucrose or trehalose.
9. The glycated hemoglobin kit based on biotin-streptavidin amplification according to claim 1, wherein: the latex microspheres are polystyrene latex microspheres; the diameter of the latex microsphere is 100-400 nm.
10. A method for preparing a biotin-streptavidin amplification-based glycated hemoglobin kit according to any one of claims 1 to 9, comprising the steps of:
(1) preparation reagent R1
Diluting the latex microspheres in a glycine buffer solution until the concentration is 0.15%, stirring at room temperature, aging at 37 ℃ for 12 hours after stirring for 2 hours, and dissolving a surfactant, salt, a preservative and an anti-freezing and thawing agent in the mixture according to the proportion to obtain a reagent R1 solution;
(2) preparation reagent R2
Mixing biotin and HbA1c antibodies according to the proportion, labeling the biotin onto the antibodies, adding streptavidin according to the proportion, reacting for 2 hours at room temperature, and then placing the mixture into a homogenizer for dispersing for 10min to obtain a biotin-streptavidin-HbA 1c antibody compound; dissolving the biotin-streptavidin-HbA 1c antibody compound in MES buffer solution, and uniformly stirring; and dissolving the surfactant, the salt and the preservative in the mixture according to the proportion to obtain the reagent R2 liquid.
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