CN113311171A - Method for detecting glycated serum albumin by using boric acid affinity principle - Google Patents
Method for detecting glycated serum albumin by using boric acid affinity principle Download PDFInfo
- Publication number
- CN113311171A CN113311171A CN202110745326.7A CN202110745326A CN113311171A CN 113311171 A CN113311171 A CN 113311171A CN 202110745326 A CN202110745326 A CN 202110745326A CN 113311171 A CN113311171 A CN 113311171A
- Authority
- CN
- China
- Prior art keywords
- serum albumin
- glycated
- albumin
- absorbance
- glycated serum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/765—Serum albumin, e.g. HSA
Abstract
The invention belongs to the technical field of glycated protein detection, and particularly relates to a method for detecting glycated serum albumin by using a boric acid affinity principle, which is characterized by comprising the following steps: putting a certain volume of bromocresol purple solution in a cuvette; adding a serum sample to be detected into a bromocresol purple solution; total protein concentration C was obtained by measuring the absorbance at 585nmT(ii) a Adding a phenylboronic acid adsorbent into a cuvette to react with BCP-GA; waiting for the adsorbent to be completely deposited at the bottom of the cuvette; measuring the absorbance at 585nm to obtain the concentration C of non-glycated serum albuminngAnd by the formula (C)T‑Cng)/CTThe proportion of glycated serum albumin was calculated at 100%. The invention uses bromcresol purple to test the total albumin content under the alkaline condition, the absorbance is reduced after the albumin and the bromcresol purple are combined, the albumin content and the absorbance reduction value are in a linear relation and are all in a full rangeThe test is completed by absorptiometry, the reagent is stable, the detection is rapid and simple, and the method is completely compatible with hospital general equipment.
Description
Technical Field
The invention relates to the technical field of glycated serum albumin detection, in particular to a method for detecting glycated serum albumin by using a boric acid affinity principle.
Background
The economy of China continues to develop rapidly for half a century, and the living standard of people's materials is continuously improved. The incidence of diabetes is rapidly increased in a large part of people in recent years due to unreasonable dietary habits and dietary structures and lack of sufficient exercise. At present, 1 hundred million diabetics exist in China, and the trend is not slowed, so that the situation cannot be effectively inhibited, and serious social problems can be caused after years.
The existing method for detecting the content of the glycated serum albumin has complicated steps, is time-consuming, or has poor stability of enzyme and antibody protein, so the method cannot be widely used clinically.
The retrieved Chinese patent No. 104198472 discloses a kit for assaying glycated serum albumin by an enzymatic method, which comprises reacting glycated albumin with a protease specific to albumin to obtain glycated amino acids, reacting the glycated albumin with glycated amino acids with glycated amino acid oxidase (KAOD) to obtain gluconal, amino acids and hydrogen peroxide, quantitatively converting the hydrogen peroxide with catalase to obtain bluish purple pigment under the coexistence of chromogen and 4-aminoantipyrine, determining glycated albumin by measuring bluish purple absorbance, and determining total albumin by bromocresol purple method to determine the ratio of the glycated albumin to the total albumin.
Further, chinese patent No. 104345150 discloses an immunochromatographic test strip for detecting glycated albumin, which determines the ratio of glycated albumin and goat anti-mouse IgG antibody by binding glycated albumin with metalloporphyrin-labeled anti-albumin antibody, separating glycated and non-glycated serum albumin by strip chromatography, and measuring fluorescence of total albumin and fluorescent label bound glycated hemoglobin.
The boric acid affinity method is widely applied to separation and detection of the glycosylated hemoglobin. U.S. Pat. Nos. 5631364 and 7374943 and International patent No. 2014-033258 disclose a method of determining the ratio of a boronic acid derivative bound to a dye and glycated hemoglobin by reacting the two substances, separating glycated and non-glycated serum albumin by strip chromatography, and measuring the reflectance (%) of total hemoglobin and dye-bound glycated hemoglobin.
In order to solve some problems of the existing methods, we propose a method for detecting glycated serum albumin by using the principle of boric acid affinity to solve the above problems.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a method for detecting glycated serum albumin by using a boric acid affinity principle.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting glycated serum albumin by using the principle of boric acid affinity, which is characterized by comprising the following steps:
s1, putting a certain volume of bromcresol purple solution into a cuvette;
s2, adding the serum sample to be detected into bromocresol purple solution to form BCP-BSA;
s3 determination of the Total serum Albumin concentration C by measuring the absorbance at 585nmT；
S4, adding the phenylboronic acid adsorbent into a cuvette to react with BCP-GA to form MP-GA-BCP;
s5, waiting for the adsorbent to be completely deposited at the bottom of the cuvette;
s6, measuring the absorbance at 585nm to obtain the concentration C of non-glycated serum albuminngAnd by the formula (C)T-Cng)/CTThe proportion of glycated serum albumin was calculated at 100%.
In the above method for assaying glycated serum albumin using the principle of boronic acid affinity, the steps S1, S2 and S3 are performed under alkaline conditions, i.e., pH>9, obtaining an absorbance A0 of bromocresol purple solution at 585nm, adding a serum sample, forming a complex of total albumin and BCP, determining the absorbance A1, and calculating the content C of the total serum albumin from A0-A1T。
In the above method for assaying glycated serum albumin using the principle of boronic acid affinity, in the steps S4, S5 and S6, glycated albumin is separated from non-glycated albumin by the phenylboronic acid reagent which can selectively bind to glycated albumin, the absorbance A2 at 585nm is measured again, and the content C of non-glycated albumin is calculated from A2 to A1ng。
In the above method for assaying glycated serum albumin using the principle of boronic acid affinity, the method for assaying glycated serum albumin comprises:
(a) adding the blood sample into a bromocresol purple detection solution, and fully reacting the total serum albumin with the bromocresol purple to form BCP-HSA;
(b) quantification of total serum albumin using a spectrophotometer at 585 nm;
(c) adding microspheres coupled with phenylboronic acid groups into a solution reacted with bromocresol purple, and specifically combining the phenylboronic acid microspheres with BCP-GA and fully reacting to form microspheres-GA-BCP;
(d) MP-GA-BCP settles at the bottom after one minute because of gravity;
(e) non-glycated serum albumin was quantified using a spectrophotometer at 585 nm;
(f) the ratio of glycated serum albumin is calculated from the total serum albumin and the non-glycated serum albumin thus determined.
In the above-mentioned method for assaying glycated serum albumin using the principle of boronic acid affinity, in the method for assaying non-glycated serum albumin ratio, the microspheres coupled with phenylboronic acid groups may be directly added to the solution for assaying.
In the method for measuring the proportion of non-glycated serum albumin in the above-mentioned method for measuring glycated serum albumin using the principle of boronic acid affinity, a phenylboronic acid group may be coupled to a magnetic bead and added to the solution to bind to the glycated serum albumin, and then the glycated serum albumin may be measured by magnetic separation.
In the method for measuring the proportion of non-glycated serum albumin in the method for measuring glycated serum albumin using the boric acid affinity principle described above, the measurement may be carried out by filling a narrow tube with microspheres and using a syringe.
Compared with the prior art, the method for detecting the glycated serum albumin by using the boric acid affinity principle has the advantages that:
1. the method is characterized in that bromocresol purple is used for testing the total albumin content under an alkaline condition, the absorbance is reduced after albumin and bromocresol purple are combined, and the albumin content and the absorbance reduction value are in a linear relation;
2. the invention is also characterized in that the phenylboronic acid derivative is combined with the glycated albumin through an ester bond under the alkaline condition, the absorbance is increased, and the content of the glycated albumin and the absorbance increase value are in a linear relationship;
3. the invention is also characterized in that all tests are completed by absorptiometry, the reagent is stable, the detection is rapid and simple, and the method is completely compatible with hospital general equipment.
Drawings
FIG. 1 is a block diagram of a method for detecting glycated serum albumin according to the present invention using the principle of boric acid affinity;
FIG. 2 is a diagram showing the steps of assaying glycated serum albumin in the method for assaying glycated serum albumin according to the principle of boronic acid affinity according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in 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.
Example 1
Referring to fig. 1-2, a method for detecting glycated serum albumin using the principle of boronic acid affinity, comprising the steps of:
s1, putting a certain volume of bromcresol purple solution into a cuvette;
s2, adding the serum sample to be detected into the bromocresol purple solution;
s3 determination of the Absorbance at 585nm to obtain the Total protein concentration CT；
S4, adding the phenylboronic acid adsorbent into a cuvette to react with BCP-GA;
s5, waiting for the adsorbent to be completely deposited at the bottom of the cuvette;
s6, measuring the absorbance at 585nm to obtain the concentration C of non-glycated albuminngAnd by the formula (C)T-Cng)/CTThe proportion of serum albumin was calculated at 100%.
Wherein the steps S1, S2 and S3 are under alkaline conditions, i.e. pH>9, obtaining the absorbance A0 of bromocresol purple solution at 585nm, adding serum sample to form a complex between total albumin and BCP, determining the absorbance A1, and calculating the total albumin content C from A0-A1T。
Further, in steps S4, S5 and S6, the glycated albumin is separated from the non-glycated albumin by the phenylboronic acid reagent which can selectively bind the glycated albumin, the absorbance A2 is measured again, and the content C of the non-glycated albumin is calculated from A2 to A1ng。
The method for measuring the glycated serum albumin comprises the following steps:
(a) adding the blood sample into a bromocresol purple detection solution, and fully reacting the total serum albumin with the bromocresol purple to form BCP-HSA;
(b) quantification of total serum albumin using a spectrophotometer at 585 nm;
(c) adding microspheres coupled with phenylboronic acid groups into a solution reacted with bromocresol purple, and specifically combining the phenylboronic acid microspheres with BCP-GA and fully reacting to form microspheres-GA-BCP;
(d) the microspheres-GA-BCP will settle to the bottom after one minute due to gravity;
(e) non-glycated serum albumin was quantified using a spectrophotometer at 585 nm;
(f) the ratio of glycated serum albumin is calculated from the total serum albumin and the non-glycated serum albumin thus determined.
Specifically, in the method for determining the proportion of the non-glycated serum albumin, the microspheres coupled with the phenylboronic acid groups can be added into the solution to be combined with the glycated serum albumin, and then the magnetic separation is used for determination, more specifically, in the method for determining the proportion of the non-glycated serum albumin, the microspheres can be filled into a thin tube and then a needle cylinder is used for determination.
The specific implementation mode is as follows:
1-1 determination of total serum albumin:
2ml of the solution containing 80. mu. mol/L-1Adding a detection solution (pH 9.0) of bromocresol purple into a quartz cuvette, adding 50 μ l of serum sample into the cuvette, reacting for two minutes, measuring the absorbance with a wavelength of 585nm by using a spectrophotometer (Unike 7200 type), and quantifying total serum albumin by using a standard curve of a BCP method to obtain CT。
1-2 measurement of non-glycated serum Albumin:
adding 5 μ l (70%) phenylboronic acid microsphere into 1-1 solution, reacting for two minutes, determining absorbance with wavelength of 585nm with spectrophotometer (Unikov 7200 type) when microsphere is deposited on the bottom of cuvette, and quantifying non-saccharified serum albumin with standard curve of BCP method to obtain Cng。
Finally, using the formula (C)T-Cng)/CTThe proportion of glycated serum albumin was calculated at 100%.
Example 2
The difference from example 1 is that:
repetition rate of the method
In-batch repeatability test: the same serum sample was taken and repeated for 20 consecutive times to calculate the CV value. Batch to batch repeatability test: the same serum sample is divided into 20 portions, stored in a refrigerator, 1 portion is taken every day for detection, and CV values are calculated after 20 days. The intra-batch CV value and the inter-batch (daytime) CV value have an intra-batch and inter-batch imprecision level of less than 5% as required by the american clinical laboratory standards committee (NCCLS) documentation, and the method is satisfactory.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A method for detecting glycated serum albumin by using the principle of boric acid affinity, which is characterized by comprising the following steps:
s1, putting a certain volume of bromcresol purple solution into a cuvette;
s2, adding the serum sample to be detected into the bromocresol purple solution;
s3 determination of the Total Albumin concentration C by measuring the absorbance at 585nmT；
S4, adding the phenylboronic acid adsorbent into a cuvette to react with BCP-GA;
s5, waiting for the adsorbent to be completely deposited at the bottom of the cuvette;
s6, measuring the absorbance at 585nm to obtain the concentration C of non-glycated albuminngAnd by the formula (C)T-Cng)/CTThe proportion of glycated serum albumin was calculated at 100%.
2. The method of claim 1, wherein the steps S1, S2 and S3 are performed under alkaline condition (pH)>9, obtaining the absorbance A0 of bromocresol purple solution at 585nm, adding a serum sample to form a compound of total serum albumin and BCP, determining the absorbance A1, and calculating the content C of the total serum albumin from A0-A1T。
3. The method of claim 2, wherein the glycated serum albumin is separated from the non-glycated serum albumin by the phenylboronic acid reagent that selectively binds to the glycated serum albumin in the steps S4, S5 and S6, the absorbance A2 at 585nm is measured again, and the content C of the non-glycated serum albumin is calculated from A2-A1ng。
4. The method of claim 1, wherein the method for assaying glycated serum albumin using the principle of boronic acid affinity comprises:
(a) adding the blood sample into a bromocresol purple detection solution, and fully reacting the total serum albumin with the bromocresol purple to form BCP-HSA;
(b) quantification of total serum albumin using a spectrophotometer at 585 nm;
(c) adding microspheres coupled with phenylboronic acid groups into a solution reacted with bromocresol purple, and specifically combining the phenylboronic acid microspheres with BCP-GA and fully reacting to form microspheres-GA-BCP;
(d) the microspheres-GA-BCP will settle to the bottom after one minute due to gravity;
(e) non-glycated serum albumin was quantified using a spectrophotometer at 585 nm;
(f) the ratio of glycated serum albumin is calculated from the total serum albumin and the non-glycated serum albumin thus determined.
5. The method of claim 4, wherein the non-glycated serum albumin fraction is assayed by adding the microbeads coupled with phenylboronic acid groups directly to the solution.
6. The method of claim 4, wherein the non-glycated serum albumin is assayed by the boronic acid affinity assay, wherein the phenylboronic acid group is coupled to a magnetic bead and bound to glycated serum albumin, and the assay is performed by magnetic separation.
7. The method of claim 4, wherein the assay of glycated serum albumin is carried out by filling the fine tube with the microspheres and using a syringe, in the method for assaying non-glycated serum albumin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110745326.7A CN113311171A (en) | 2021-07-01 | 2021-07-01 | Method for detecting glycated serum albumin by using boric acid affinity principle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110745326.7A CN113311171A (en) | 2021-07-01 | 2021-07-01 | Method for detecting glycated serum albumin by using boric acid affinity principle |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113311171A true CN113311171A (en) | 2021-08-27 |
Family
ID=77381445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110745326.7A Pending CN113311171A (en) | 2021-07-01 | 2021-07-01 | Method for detecting glycated serum albumin by using boric acid affinity principle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113311171A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114460310A (en) * | 2022-04-12 | 2022-05-10 | 天津康博尔生物基因技术有限公司 | Colored latex microsphere and preparation method and application thereof |
| CN115950868A (en) * | 2022-12-26 | 2023-04-11 | 深圳市雷诺华科技实业有限公司 | Testing device and testing method for determining glycated albumin by antibody-spot method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103823071A (en) * | 2012-11-16 | 2014-05-28 | 北京怡成生物电子技术有限公司 | Method for directly determining proportion of glycosylated hemoglobin |
| CN104345150A (en) * | 2013-07-26 | 2015-02-11 | 深圳市艾瑞生物科技有限公司 | Glycated albumin detection immunochromatography test trip and preparation method thereof |
| CN104345149A (en) * | 2013-07-26 | 2015-02-11 | 深圳市艾瑞生物科技有限公司 | Immunochromatography test strip for detecting glycosylated hemoglubin and preparation method thereof |
-
2021
- 2021-07-01 CN CN202110745326.7A patent/CN113311171A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103823071A (en) * | 2012-11-16 | 2014-05-28 | 北京怡成生物电子技术有限公司 | Method for directly determining proportion of glycosylated hemoglobin |
| CN104345150A (en) * | 2013-07-26 | 2015-02-11 | 深圳市艾瑞生物科技有限公司 | Glycated albumin detection immunochromatography test trip and preparation method thereof |
| CN104345149A (en) * | 2013-07-26 | 2015-02-11 | 深圳市艾瑞生物科技有限公司 | Immunochromatography test strip for detecting glycosylated hemoglubin and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 中国食品药品检定研究院: "体外诊断试剂检验技术", 中国医药科技出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114460310A (en) * | 2022-04-12 | 2022-05-10 | 天津康博尔生物基因技术有限公司 | Colored latex microsphere and preparation method and application thereof |
| CN115950868A (en) * | 2022-12-26 | 2023-04-11 | 深圳市雷诺华科技实业有限公司 | Testing device and testing method for determining glycated albumin by antibody-spot method |
| CN115950868B (en) * | 2022-12-26 | 2024-02-13 | 深圳市雷诺华科技实业有限公司 | Testing device and testing method for measuring glycosylated albumin by antibody-spot method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113311171A (en) | Method for detecting glycated serum albumin by using boric acid affinity principle | |
| EP2613146B1 (en) | Method for measuring fibroblast growth factor-23 and reagent therefor | |
| CN104345149A (en) | Immunochromatography test strip for detecting glycosylated hemoglubin and preparation method thereof | |
| GB2181840A (en) | Method for the immunoassay of a macromolecular analyte | |
| JP3646097B2 (en) | Reagents and solid phase components of specific binding assays that do not contain an advanced glycosylation end product | |
| US5284777A (en) | Combined glycated hemoglobin and immunoturbidometric glycated albumin assay from whole blood lysate | |
| CN117396759A (en) | Biomarker for detecting thrombus or coagulation related diseases and application thereof | |
| EP0455225A2 (en) | Method for measuring the percentage of glycation of a particular protein | |
| EP3818375A1 (en) | Direct immunoassay measurement of autoantibodies | |
| WO2005029092A1 (en) | Hemoglobin assay for neonatal screening | |
| JP2004532388A (en) | Methods and devices for quantification of glycosylated proteins | |
| CN1217193C (en) | Luminol chemiluminescence immunological analysis detecting method for cardiac muscle calcium protein | |
| KR100646305B1 (en) | Cobalamin Assay | |
| CN114112957B (en) | Adiponectin determination kit and application thereof | |
| Kessler et al. | Nonimmunological assay of urinary albumin based on laser-induced fluorescence | |
| CN113777324A (en) | Growth stimulation expression factor 2 instant detection kit, preparation method and application thereof | |
| CN112213490A (en) | Hyaluronic acid chemiluminescence immunoassay kit and preparation method thereof | |
| AU703940B2 (en) | Regenerable solid phase for carrying out specific binding reactions | |
| US7544513B2 (en) | Hemoglobin assay for neonatal screening | |
| JP3171681B2 (en) | Method for measuring glycated protein | |
| CN113009144B (en) | Antibody detection kit and detection method based on microfluidic technology | |
| CN113933497A (en) | I type collagen amino-terminal extension peptide magnetic particle chemiluminescence immunoassay kit and detection method thereof | |
| CN117110613A (en) | Diagnostic kit and application of LMWK-Fc in preparation of liver fibrosis diagnostic reagent | |
| CN116381252A (en) | Glycosylated hemoglobin determination kit | |
| Backer et al. | Autoantibodies to lactate dehydrogenase in serum identified by use of immobilized protein G and immobilized jacalin, a jackfruit lectin. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |