CN107490684A - A kind of Blood glycated haemoglobin collaurum detection method - Google Patents
A kind of Blood glycated haemoglobin collaurum detection method Download PDFInfo
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- CN107490684A CN107490684A CN201610404112.2A CN201610404112A CN107490684A CN 107490684 A CN107490684 A CN 107490684A CN 201610404112 A CN201610404112 A CN 201610404112A CN 107490684 A CN107490684 A CN 107490684A
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- hemoglobin
- glycosylated hemoglobin
- standard comparison
- comparison product
- immune detection
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention discloses a kind of Blood glycated haemoglobin collaurum detection method.This detection method, including：（1）, colloid gold immune detection kit preparation；（2）, blood sample to be detected is put into colloid gold immune detection kit, read standard comparison product immune detection result b corresponding to the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin；（3）, identical blood sample to be detected be put into colloid gold immune detection kit, read standard comparison product immune detection result d corresponding to the immune detection result c and hemoglobin of sample hemoglobin；（4）, ratio A=a/b, ratio B=c/d is calculated；（5）, calculate glycosylated hemoglobin content=A/B.The high sensitivity that the present invention detects, high specificity, and can quantify, solve the problem of traditional glycosylated hemoglobin quantitative detecting method is complex for operation step, and detection time is long, while solve the problems, such as that glycosylated hemoglobin qualitative detection accuracy is low.
Description
Technical field
The invention belongs to technical field of immune assay, more particularly to a kind of Blood glycated haemoglobin collaurum to detect
Method.
Background technology
Glycosylated hemoglobin（HbA1c）It is blood-glucose by non-enzyme effect, through blood red egg in cell membrane and red blood cell
The product that in vain-chain a word used in person's names propylhomoserin combines to form, its synthesis rate is directly proportional to concentration sugared in red blood cell local environment, is human body blood
The product that endoerythrocytic hemoglobin is combined with blood glucose in liquid.
The combination generation glycosylated hemoglobin of blood glucose and hemoglobin is irreversible reaction, and directly proportional to blood sugar concentration,
And kept for 120 days or so.Blood glucose is the monose in the blood come from the carbohydrate breakdown in food, generally only refers to grape
Sugar, what blood glucose test result reflected is blood sugar level at once.So glycosylated hemoglobin more has clinical meaning compared with blood sugar detection,
" goldstandard " of diabetic condition monitoring is described as, index of the routine testing as glycemic control is carried out in clinical detection.
The method that predominantly detects for clinically determining glycosylated hemoglobin at present has the affine layers of Gao Xiao Ye Xiang Se Pu ﹑ electricity Yong Fa ﹑
Analysis method etc..
Ion-exchange high-performance liquid chromatography analytic approach, it is on the basis of classical liquid chromatography, has introduced gas chromatography
Theory there are all advantages of gas chromatography.Needed before test using instrumentation to the hemoglobin in blood sample and saccharification blood
Lactoferrin is separated, and need to be detected using large-scale chromatogram analysis equipment during test, therefore detection must be entered in hospital by special messenger
OK, it is difficult to meet the needs of glycosylated hemoglobin detects immediately.
Electrophoresis, compared to both the non-glycated hemoglobin, the total electrical charge changed by saccharification and glycosylated hemoglobin etc. electricity
Point change is the basis of the isoelectrofocusing gel electrophoretic separation of Ago-Gel or pH gradient 5.0~6.5.Ago-Gel electricity
The hemoglobin subfraction resolution ratio very little of swimming, and isoelectric focusing preferably can separate subfraction.May be due to experiment
Automaticity deficiency, importance have declined.
Affinity chromatography, after the hemoglobin in blood sample is added into chromatographic column, all glycosylated hemoglobins（HbA1 and
The hemoglobin of side chain saccharification；Total glycosylated hemoglobin）Combined with boric acid rather than glycosylated hemoglobin can be tested by chromatographic column
Amount.The polyhydroxy base complex of cis-hydroxyl groups is also included in addition high concentration, such as after sorbierite, glycosylated hemoglobin and boric acid
Eluted with reference to being replaced from pillar.Affinity chromatography is blood red to the translated hemoglobin of modification and pathology later
The influence relative insensitivity of albumen.Using affinity chromatography, it is only capable of determining total glycosylated hemoglobin.Widely used affinity chromatography
Method, also only it is to calculate " the HbA1c " of standard, therefore exist quantitatively can not essence from total saccharification Hemoglobin Value with empirical algorithms
The problem of quasi-.
The content of the invention
In view of this, it is an object of the invention to propose a kind of Blood glycated haemoglobin collaurum detection method, including
Following steps：
（1）Colloid gold immune detection kit is prepared, including the preparation of standard comparison product, the standard comparison product is not
Immunity occurs with hemoglobin, anti-hemoglobin antibodies, glycosylated hemoglobin and anti-glycosylated hemoglobin antibody to be combined instead
The material answered；
（2) blood sample to be detected is put into colloid gold immune detection kit, using standard comparison product as reference product, with saccharification
Hemoglobin carries out immune detection reaction, reads the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin
Corresponding standard comparison product immune detection result b；
（3）Will be with step（2）In same blood sample to be detected be put into colloid gold immune detection kit, with step（2）
In same standard comparison product as reference product, carry out immune detection reaction with hemoglobin, read exempting from for sample hemoglobin
Standard comparison product immune detection result d corresponding to epidemic disease testing result c and hemoglobin；
（4）Ratio A=a/b, ratio B=c/d is calculated；
（5）Calculate content=A/B of glycosylated hemoglobin.
Further, the detection method is raw materials used including standard comparison product, anti-hemoglobin antibodies, anti-HbAle
Protein antibodies, sample pad, gold labeling antibody pad, carrier, adsorptive pads and PVC board.
Further, the standard comparison product be rabbit igg, donkey IgG, horse IgG, sheep IgG, mouse IgG one or two or
It is two or more.
Further, the anti-hemoglobin antibodies are anti-hemoglobin monoclonal antibody and anti-hemoglobin Anti-TNF-α
The one or two of body.
Further, the anti-glycosylated hemoglobin antibody is anti-glycosylated hemoglobin monoclonal antibody, anti-HbAle
Protein polyclone antibody, anti-hemoglobin monoclonal antibody, anti-hemoglobin polyclonal antibody one or two or two kinds with
On.
Further, the carrier is nitrocellulose filter.
Embodiment
The embodiments of the invention provide a kind of glycosylated hemoglobin gold-immunochromatographyreagent reagent for assay box, including：
Get stuck, Hemoglobin Reagent Strip and glycosylated hemoglobin reagent strip；
Get stuck including above get stuck and under get stuck, above get stuck on have observation window and well, under be fixed with blood in the groove that gets stuck that gets stuck
Lactoferrin reagent strip and glycosylated hemoglobin reagent strip, above get stuck and under get stuck and connect and fix；
Hemoglobin Reagent Strip includes sample pad, gold labeling antibody pad, nitrocellulose membrane, adsorptive pads and PVC board, PVC board position
In the bottom land layer that gets stuck, nitrocellulose membrane is located at the middle part on the PVC board upper strata, and gold labeling antibody pad is located at the PVC
The top of plate and overlapping with the nitrocellulose membrane upper part, adsorptive pads are located at the bottom of the PVC board and and cellulose nitrate
The end portion of film is overlapping, sample pad be located at the top of PVC board and with the non-close nitrocellulose membrane one end of gold labeling antibody pad
Partly overlap, nitrocellulose membrane close to sample pad, as initiating terminal, to be clearing end close to adsorptive pads, it is more to be coated with hemoglobin successively
Clonal antibody and standard comparison product, the hemoglobin monoclonal antibody of colloid gold label is coated with gold labeling antibody pad.
Glycosylated hemoglobin reagent strip includes sample pad, gold labeling antibody pad, nitrocellulose membrane, adsorptive pads and PVC board,
PVC board is located at the middle part on PVC board upper strata positioned at the bottom land layer that gets stuck, nitrocellulose membrane, and gold labeling antibody pad is located at PVC board
Top and overlapping with nitrocellulose membrane upper part, adsorptive pads be located at the bottom of PVC board and with nitrocellulose membrane cellulose nitrate
The end portion of film is overlapping, sample pad be located at the top of PVC board and with the non-close nitrocellulose membrane one end of gold labeling antibody pad
Partly overlap, nitrocellulose membrane close to sample pad, as initiating terminal, to be clearing end close to adsorptive pads, be coated with HbAle egg successively
White monoclonal antibody and standard comparison product, the hemoglobin monoclonal that colloid gold label is coated with gold labeling antibody pad resist
Body.
Nitrocellulose membrane coating hemoglobin polyclonal antibody on Hemoglobin Reagent Strip for detection line, be coated with standard
Comparison product to compare line, and detection line is parallel with comparing line.
Nitrocellulose membrane coating glycosylated hemoglobin monoclonal antibody on glycosylated hemoglobin test paper for detection line, bag
By standard comparison product to compare line, and detection line is parallel with comparing line.
The preparation method of the glycosylated hemoglobin gold-immunochromatographyreagent reagent for assay box of embodiment 1.
The preparation of the glycosylated hemoglobin gold-immunochromatographyreagent reagent for assay box is mainly the Hemoglobin Reagent Strip and HbAle
The preparation of protein reagent bar；
Wherein, the preparation of the Hemoglobin Reagent Strip includes colloid gold label, the preparation of gold-marking binding pad and nitrocellulose membrane
Coating.
The preparation of the collaurum includes：
(1) by 100ml 0.01% AuCl3It is heated to seething with excitement under solution magnetic agitation, accurately adds 1.5~3ml 1% lemon
Lemon three sodium solutions of acid.Continue heating 15 minutes after colour stable, after cooling plus pure water is supplemented to 100ml, is kept in dark place；
(2) K is used2CO3Adjust pH to 7~9；
(3) K is added2CO35 minutes are stood, every milliliter of gold solution adds 10~30ug hemoglobin monoclonal antibodies, is preferably
20ug, stand 30 minutes；
(4) 10% BSA (bovine serum albumin) solution is added, until ultimate density is 0.1%, stands 20 minutes；
(5) above-mentioned solution 12000rpm is centrifuged 50 minutes, abandons supernatant, redissolved in equal volume with liquid is redissolved.Now colloid gold label
Complete, followed by the preparation of gold-marking binding pad, i.e., the solution after above-mentioned redissolution be uniformly added drop-wise on the wide pads of 6mm,
37 DEG C of dryings 12 hours, gold-marking binding pad is finally made.
Here is the coating of nitrocellulose membrane, including：
Hemoglobin polyclonal antibody is coated with and standard comparison product coating.
Wherein standard comparison product choose sheep anti-mouse igg.
(1) hemoglobin polyclonal antibody and sheep anti-mouse igg antibody are diluted with pH7.4PBS buffer solutions, and hemoglobin is more anti-
Concentration is 1mg/ml, and sheep anti-mouse igg antibody concentration is 0.8mg/ml.
(2) detection line and control line are coated with according to 1ul/cm speed.To prevent from spreading, the PVC board being coated with is put immediately
Dried 12 hours to 37 DEG C.
The preparation of the glycosylated hemoglobin reagent strip is also to include colloid gold label, the preparation of gold-marking binding pad and nitric acid
The coating of tunica fibrosa.
The preparation of the collaurum includes：
(1) by 100ml 0.01% AuCl3It is heated to seething with excitement under solution magnetic agitation, accurately adds 1.5~3ml 1% lemon
Lemon three sodium solutions of acid.Continue heating 15 minutes after colour stable, after cooling plus pure water is supplemented to 100ml, is kept in dark place；
(2) K is used2CO3Adjust pH to 7~9；
(3) K is added2CO35 minutes are stood, every milliliter of gold solution adds 10~30ug hemoglobin monoclonal antibodies, is preferably
20ug, stand 30 minutes；
(4) 10% BSA (bovine serum albumin) solution is added, until ultimate density is 0.1%, stands 20 minutes；
(5) above-mentioned solution 12000rpm is centrifuged 50 minutes, abandons supernatant, redissolved in equal volume with liquid is redissolved.Now colloid gold label
Complete, followed by the preparation of gold-marking binding pad, i.e., the solution after above-mentioned redissolution be uniformly added drop-wise on the wide pads of 6mm,
37 DEG C of dryings 12 hours, gold-marking binding pad is finally made.
Here is the coating of nitrocellulose membrane, including：
Glycohemoglobin monoclonal antibody is coated with and standard comparison product coating.
Wherein standard comparison product choose sheep anti-mouse igg.
(1) glycosylated hemoglobin monoclonal antibody and sheep anti-mouse igg antibody are diluted with pH7.4PBS buffer solutions, HbAle
Protein monoclonal antibody concentration is that 1mg/ml sheep anti-mouse igg antibody concentration is 0.8mg/ml.
(2) detection line and control line are coated with according to 1ul/cm speed.To prevent from spreading, the PVC board being coated with is put immediately
Dried 12 hours to 37 DEG C.
After the completion of above-mentioned steps are equal, respectively by the Hemoglobin Reagent Strip and the sample of glycosylated hemoglobin reagent strip
Pad, pad, NC films and adsorptive pads are pasted in PVC board successively respectively, are cut into 4mm bars, and pack is sealed, and room temperature preservation is standby.
A kind of glycosylated hemoglobin gold-immunochromatographyreagent reagent for assay box provided by the invention, detected using gold-immunochromatographyreagent reagent for assay box
Glycosylated hemoglobin in sample, compared with traditional collaurum detector bar, the sensitivity of detection is higher, and specificity is stronger, and can
To quantify, solves the problem of traditional glycosylated hemoglobin quantitative detecting method is complex for operation step, and detection time is long, simultaneously
Solve the problems, such as that the quantitative detection accuracy of glycosylated hemoglobin is low.
The detection method of the glycosylated hemoglobin gold-immunochromatographyreagent reagent for assay bar of embodiment 2.
（1）, take blood sample to be measured, add 3 in glycosylated hemoglobin gold-immunochromatographyreagent reagent for assay box well obtained above
~4 drip, and after 20min, are put into collaurum detector；Using sheep anti-mouse igg as comparison product, immune inspection is carried out with glycosylated hemoglobin
Reaction is surveyed, sheep anti-mouse igg corresponding to the detection line signal value a and glycosylated hemoglobin of sample glycosylated hemoglobin is read and compares
Line signal value b；
（2）, take identical blood sample to be measured, with step（1）Middle identical glycosylated hemoglobin gold-immunochromatographyreagent reagent for assay box adds
3~4 are added in sample hole to drip, and after 20min, are put into collaurum detector；Using sheep anti-mouse igg as comparison product, enter with hemoglobin
Row immune detection is reacted, and is read sheep anti-mouse igg corresponding to the detection line signal value c and hemoglobin of sample hemoglobin and is compared
Line signal value d；
（3）, ratio A=a/b, ratio B=c/d is calculated；
（4）, calculate glycosylated hemoglobin content=A/B.
The reagent strip Performance Evaluation of embodiment 3. is tested
1st, accuracy testing
Take same blood samples of patients sample, with a batch of reagent strip repeat detection 3 times, calculate relative deviation B values be-
0.04%.Meet product design requirement；
2nd, replica test
Same blood samples of patients sample is taken, is detected 20 times simultaneously with a batch of reagent strip, calculating coefficient of variation CV values is
11.21%, meet product design requirement.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
1. a kind of glycosylated hemoglobin collaurum detection method, comprises the following steps：
（1）Colloid gold immune detection kit is prepared, including the preparation of standard comparison product, the standard comparison product is not
Immunity occurs with hemoglobin, anti-hemoglobin antibodies, glycosylated hemoglobin and anti-glycosylated hemoglobin antibody to be combined instead
The material answered；
（2）Blood sample to be detected is put into colloid gold immune detection kit, using standard comparison product as reference product, with saccharification
Hemoglobin carries out immune detection reaction, reads the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin
Corresponding standard comparison product immune detection result b；
（3）Will be with step（2）In same blood sample to be detected be put into colloid gold immune detection kit, with step（2）
In same standard comparison product as reference product, carry out immune detection reaction with hemoglobin, read exempting from for sample hemoglobin
Standard comparison product immune detection result d corresponding to epidemic disease testing result c and hemoglobin；
（4）Ratio A=a/b, ratio B=c/d is calculated；
（5）Calculate content=A/B of glycosylated hemoglobin.
2. detection method as claimed in claim 1, it is characterised in that：The detection method is raw materials used including standard comparison
Product, the antibody of anti-hemoglobin, the antibody of anti-glycosylated hemoglobin, sample pad, gold labeling antibody pad, carrier, adsorptive pads and
PVC board.
3. detection method as claimed in claim 1, it is characterised in that：The standard comparison product be rabbit igg, donkey IgG, horse IgG,
Sheep IgG, mouse IgG it is one or two kinds of or two or more.
4. the detection method of Blood glycated haemoglobin as claimed in claim 1, it is characterised in that：The anti-hemoglobin resists
Body is anti-hemoglobin monoclonal antibody and the one or two of anti-hemoglobin polyclonal antibody.
5. the detection method of Blood glycated haemoglobin as claimed in claim 1, it is characterised in that：The anti-HbAle egg
Bai Kangti is anti-glycosylated hemoglobin monoclonal antibody, anti-glycosylated hemoglobin polyclonal antibody, anti-hemoglobin monoclonal resist
Body, anti-hemoglobin polyclonal antibody it is one or two kinds of or two or more.
6. detection method as claimed in claim 1, it is characterised in that：The carrier is nitrocellulose filter.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004042364A2 (en) * | 2002-11-05 | 2004-05-21 | Therasense, Inc. | Assay device, system and method |
| CN1522369A (en) * | 2002-03-29 | 2004-08-18 | ���µ�����ҵ��ʽ���� | Blood processing method, blood processing device, method of measuring hemoglobins and device for measuring hemoglobins |
| CN104407135A (en) * | 2014-11-03 | 2015-03-11 | 清华大学深圳研究生院 | Method and kit for detecting A type influenza virus H5 and H9 subtypes |
| CN105866440A (en) * | 2016-05-31 | 2016-08-17 | 常州博闻迪医药科技有限公司 | Detection method of glycosylated hemoglobin of blood |
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2016
- 2016-06-09 CN CN201610404112.2A patent/CN107490684A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1522369A (en) * | 2002-03-29 | 2004-08-18 | ���µ�����ҵ��ʽ���� | Blood processing method, blood processing device, method of measuring hemoglobins and device for measuring hemoglobins |
| WO2004042364A2 (en) * | 2002-11-05 | 2004-05-21 | Therasense, Inc. | Assay device, system and method |
| CN104407135A (en) * | 2014-11-03 | 2015-03-11 | 清华大学深圳研究生院 | Method and kit for detecting A type influenza virus H5 and H9 subtypes |
| CN105866440A (en) * | 2016-05-31 | 2016-08-17 | 常州博闻迪医药科技有限公司 | Detection method of glycosylated hemoglobin of blood |
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Address after: 213025 Dongfang Road 167, Qishuyan District, Changzhou, Jiangsu. Applicant after: Changzhou Bo Wen Di medical Limited by Share Ltd Address before: 213025 Dongfang Road 167, Qishuyan District, Changzhou, Jiangsu. Applicant before: CHANGZHOU BIOWIN BIOPHARM CO., LTD. |
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Application publication date: 20171219 |
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