CN104897907A - Kit for detecting glycosylated hemoglobin and detection method thereof - Google Patents
Kit for detecting glycosylated hemoglobin and detection method thereof Download PDFInfo
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- CN104897907A CN104897907A CN201510262100.6A CN201510262100A CN104897907A CN 104897907 A CN104897907 A CN 104897907A CN 201510262100 A CN201510262100 A CN 201510262100A CN 104897907 A CN104897907 A CN 104897907A
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- glycosylated hemoglobin
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- kit
- hemoglobin
- borate derivative
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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
- G01N33/72—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
- G01N33/726—Devices
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- 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
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
-
- 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
Abstract
The invention relates to the technical field of detection of glycosylated hemoglobin and in particular relates to a kit for detecting glycosylated hemoglobin and a detection method thereof. The kit comprises a first reagent, a second reagent and a chromatograph, wherein the first reagent comprises a borate derivative used for splitting red cells, releasing the glycosylated hemoglobin and precipitating total hemoglobin; the second reagent comprises a buffer solution used for washing the borate derivative which is not combined with the glycosylated hemoglobin; and the chromatograph comprises a reaction film used for remaining hemoglobin precipitation. According to the kit disclosed by the invention, only trace whole blood live peripheral blood sample is needed, namely the content of the glycosylated hemoglobin can be quantitatively detected within 2-3 minutes, the screening speed is greatly improved, and the kit has the advantages of high sensitivity, high specificity, simplicity and convenience in operation and the like; and moreover, the preparation method is simple, and large-scale production is easily realized.
Description
Technical field
The present invention relates to glycosylated hemoglobin detection technique field, be specifically related to a kind of kit and the detection method thereof that detect glycosylated hemoglobin.
Background technology
Diabetes are incretion metabolism diseases that one group of cause of disease and pathogenesis understand not yet completely, and the current incidence of disease is only second to angiocardiopathy and tumour.Recent years, the incidence of disease of diabetes was continuous ascendant trend, was the worldwide public health problem of serious threat human health.Traditional diabetes diagnosis and Treatment monitoring adopt fasting blood-glucose, postprandial blood sugar and oral glucose tolerance test etc., but moment blood sugar level when glycemic parameters only represents blood drawing.Nearly car comes, and the detection of glycosylated hemoglobin (HbA1C) is subject to clinical great attention day by day.That a part of haemoglobin that glycosylated hemoglobin (HbA1C) refers in blood and glucose combines.The amino of valine that hemoglobin β-chain N holds and the reversible condensation of free aldehyde radical of glucose are aldimine (schiff bases), and then Amadori rearrangement reaction occurs aldimine, forms comparatively stable N end fructosyl structure and syn diol structure unit.When the concentration of glucose in blood is higher, the saccharification hemoglobin content that human body is formed also can be higher.In human body, the erythrocytic life-span is generally 120 days, and before red blood cell death, in blood, glycosylated hemoglobin (HbA1C) content also keeps relatively constant.Glycosylated hemoglobin (HbA1C) horizontal reverse has answered the average blood glucose levels in first 120 days of detection, and with get blood time, patient whether on an empty stomach and whether use the factors such as insulin to have nothing to do, therefore, glycosylated hemoglobin (HbA1C) is the goldstandard of reaction long-term blood glucose level, is also the important indicator of diabetes auxiliary diagnosis, monitoring, treatment.
The assay method of glycosylated hemoglobin (HbA1C) is varied, can be divided into two large classes on the whole: a class is different based on the electric charge of HbA1C with Hb, as ion exchange chromatography, electrophoresis; Another kind of is design feature based on saccharification group on Hb, as affinity chromatography, immunization and enzyme process etc.
Multiple research shows that the difference in principle and methodology determines the quality detecting reagent performance, and diabetic therapy target call measured value is by the impact of assay method, therefore under lab apply the result comparison that different GHb assay method produces extremely important.
Ion exchange chromatography: mainly contain high performance liquid chromatography (HPLC) and manual microtrabeculae method.The method sets up based on the electrically charged difference of institute after the saccharification of HBB N terminal Valine.But due to the expensive equipment that the method uses, be difficult to popularize in the hospital of relatively basic unit and laboratory; Microtrabeculae method manual steps is loaded down with trivial details, and the quality of chromatography time and microtrabeculae is wayward, easily produces operative technique error, and repeatability is not good enough; And disturbing factor is a lot, especially to the sensitive of pH value and temperature, HbF and variation haemoglobin (HbS, HbC, HbE etc.) are to result interference, and annoyance level is determined, so must examine collection of illustrative plates according to the separating power of post.
Electrophoresis: for agargel electrophoresis, Hb (pH6.0) electrophoretic migration on Ago-Gel under acidic buffer condition depend on the absorption situation of Hb on gel and with electric charge.The method sample consumption is few, and resolution is high, reproducible, studies have found that blood glucose value and HbAlc value have significant correlation, and result is not subject to the impact of temperature and fetal hemoglobin.In addition, because its measurable Hb range of linearity wider (13.0 ~ 39.0g/L), can note abnormalities Hb.The shortcoming of the method is that each mensuration all needs to carry out sample analysis in batch, speed is slow, individual in real time detection cannot be carried out, automaticity is poor, measured result scans with technician and judges relevant to the crest of electrophoresis, therefore and be not suitable for clinical labororatory's routine and use to affect by subjective factor, and somewhat expensive.
Affinity chromatography: boric acid has the character made Reversible binding with the cis-position glycol-based being incorporated into glucose on Hb molecule and react.Normally used is m-aminobenzene boric acid agarose, and after blood sample is added to chromatographic column, all GHb are combined with boric acid and stay in post, and non-GHb directly flows out chromatographic column; Add the polyhydroxy compound (as sorbierite) that high concentration also comprises cis-position glycol-based again, the combination of GHb and boric acid is replaced and is eluted, and measures two components respectively, and ratio calculated.Affinity chromatography is insensitive relative to additive method with the impact of pathology haemoglobin on variation haemoglobin, but mensuration is HbA1 and GHb total amount.In addition, Gold standard is closely related with boric acid affinity chromatography, operate all simple and easy to do, quick and precisely, stable reagent.It is reported, this method is by the interference of any haemoglobin variant except Hbs and Hbc and catabolite, and reliable results, compares and be applicable to clinically to detect at any time.
Immunoturbidimetry: utilize antigen, the principle of antibody response measures.The β chain N end of GHb provides one easily by the epitope of antibody recognition, can with monoclonal antibody or polyclonal antibody, the epitope of last 4 ~ 6 amino acid composition of β chain N end of specific recognition GHb, in conjunction with colorimetric or turbidimetry, take GHb as standard, measure the content of HbA1c, then measure the content of Hb, finally calculate the percentage composition that HbA1c accounts for total Hb.These class methods as the index judging diabetes glucose level, can only be not useable for the research of variation haemoglobin.In contrast, the method that immunoturbidimetry detects is more simple, does not need additionally to add instrument, provides a kind of quick, accurate, reliable, easy conventional method, have more wide prospect in clinical practice for clinical.
Enzyme process: whole blood is after haemolysis process, with differential protein restriction endonuclease, Hb enzymolysis, digestion is become fructosyl amino acid, hydrogen peroxide (hydrogen peroxide is produced again under fructosyl amino acid oxidase effect, H2O2), the concentration of H2O2 is directly proportional to the content of GHb in blood, H2O2 is coupled with corresponding chromogen under the effect of peroxidase, thus can obtain H2O2 concentration according to color intensity of variation, and then learns the content of GHb in sample; Total Hb concentration of Simultaneously test same pipe digestive juice, calculates the concentration proportion of GHb and Hb, is GHb result.This method provides a quick homogeneous reactive system (as glucose, alanine aminotransferase) as clinical biochemical reaction, has good precision, can detect GHb and Hb simultaneously, and have good correlativity with conventional H PLC method and immunoassay.
Ion capture: application antigen-antibody reaction principle, in parallel with fluorescent marker, by connecting electronegative polyanionic compound, be adsorbed onto positively charged fiber surface, after the steps such as a series of thorough cleanings, measure fluorescence intensity change rate, calculate GHb concentration.Its detection system is easy to specification and repetition, can reduce operative technique error, and susceptibility and the specificity of detection are high, and in batch, interassay coefficient of variation is little.Have this method influence factor of bibliographical information few, accuracy is high, and the recovery reaches 98.85%, cross pollution rate <0.01%.The method is the new method grown up in recent years, adopts automatic analyzer, is applicable to the detection of batch sample.
Several method in sum, or reagent, instrument cost are high, complicated operation otherwise accuracy low, poor stability, and be all not suitable for community and basic hospital.Therefore, need to improve the method for existing mensuration glycosylated hemoglobin ratio, propose a kind of new method and solve these problems.
Summary of the invention
In order to overcome the shortcoming and defect existed in prior art, the object of the present invention is to provide a kind of kit detecting glycosylated hemoglobin, this kit only needs the whole blood of trace peripheral blood sample alive, the content quantitatively detecting glycosylated hemoglobin can be realized in 2-3 minute, greatly improve the speed of examination, have highly sensitive, specificity good and advantage easy and simple to handle; Preparation method is simple, is easy to large-scale production.
Another object of the present invention is to provide a kind of detection method detecting glycosylated hemoglobin, this detection method only needs the whole blood of trace peripheral blood sample alive, the content quantitatively detecting glycosylated hemoglobin can be realized in 2-3 minute, greatly improve the speed of examination, have highly sensitive, specificity good and advantage easy and simple to handle.
Object of the present invention is achieved through the following technical solutions: a kind of kit detecting glycosylated hemoglobin, and described kit comprises:
First reagent, this first reagent comprises the borate derivative for splitting erythrocyte, release glycosylated hemoglobin, precipitation total hemoglobin;
Second reagent, this second reagent comprises the damping fluid for washing away the borate derivative in conjunction with glycosylated hemoglobin;
And chromatography device, this chromatography device comprises the reaction film for retaining haemoglobin precipitation.
Preferably, described kit also comprises brown centrifuge tube, reagent bottle and aluminium foil bag, and brown centrifuge tube is equipped with the first reagent, and brown centrifuge tube is contained in aluminium foil bag, and reagent bottle is equipped with the second reagent.
More preferred, described kit also comprises packing box, liner and capillary heparin tube, and liner is arranged in packing box, is lining with in capillary heparin tube is arranged at.Chromatography device also comprises plastic casing, and reaction film is fixed on the bosom of plastic casing, and plastic casing and reaction film are assembled into chromatography device, and the aperture of reaction film is 0.5 μm.
First reagent is borate derivative, pH8.0, and total amount 200 μ L, is placed in the brown centrifuge tube of 1mL capacity; Second reagent is damping fluid, PH8.0, total amount 2mL, is placed in 5mL capacity plastic reagent bottle.
Kit of the present invention only needs the whole blood of trace to live peripheral blood sample, can realize the content quantitatively detecting glycosylated hemoglobin, greatly improve the speed of examination in 2-3 minute, have highly sensitive, specificity good and advantage easy and simple to handle; Preparation method is simple, is easy to large-scale production.
Preferably, often liter of described first reagent comprises following component:
Borate derivative 0.4-0.6g
Magnesium chloride 1.9-2.9g
Potassium chloride 22-26g
Barium chloride 6.2-8.2g
Glycyl amide hydrochloride 6.6-8.6g
Formamide 25-35g
Potassium azide 8-12g
NPE 15-25g
Water surplus.
Preferably, the structural formula of described borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 1-6.
Preferably, often liter of described second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 23-33g
Potassium azide 4-6g
Sodium chloride 0.4-0.8g
Formamide 8-12g
Triton X-100 4-6g
Water surplus.
Another object of the present invention is achieved through the following technical solutions: a kind of detection method using kit detection glycosylated hemoglobin described above for non-treatment object, comprises the steps:
A, draw 5 μ L blood samples with capillary heparin tube, add in the brown centrifuge tube that 200 μ L first reagent are housed, turn upside down 8-12 time, fully mix, in left at room temperature 1-3min, obtain reactant liquor;
B, to draw the above-mentioned reactant liquor of 25 μ L with pipettor and drip on the reaction film in the middle of chromatography device;
After C, 8-12s, get 25 μ L second reagent dropwise on the reaction film in the middle of chromatography device;
After D, 8-12s, chromatography device is placed on glycolated hemoglobin analysis and detects.
Blood sample requires: the blood sample that anticoagulant blood-collecting pipe gathers, and can preserve 7 days at 2 ~ 8 DEG C; Or finger tip blood.
Cleaning Principle of the present invention is: the first reagent splitting erythrocyte 1, utilizing special formulation, release glycosylated hemoglobin, precipitation total hemoglobin; Meanwhile, " c/s-diol " structural specificity in the first reagent in the blue borate derivative of ad hoc structure and glycosylated hemoglobin reacts, and generates blue " borate-glycosylated hemoglobin compound ";
2, get a certain amount of reaction mixture and join chromatography device, the haemoglobin of all precipitations and to be combined with glycosylated hemoglobin or unconjugated boric acid conjugate remains on the reaction film in the middle of chromatography device;
3, a certain amount of second reagent dropwise is to chromatography device, and any borate derivative in conjunction with glycosylated hemoglobin is removed;
4, chromatography device is placed in glycolated hemoglobin analysis detects, by the color intensity of instrument quick determination and analysis blueness (glycosylated hemoglobin) and red (total hemoglobin) respectively, thus the number percent of glycosylated hemoglobin in mensuration blood sample.
Detection method of the present invention only needs the whole blood of trace to live peripheral blood sample, can realize the content quantitatively detecting glycosylated hemoglobin, greatly improve the speed of examination in 2-3 minute, have highly sensitive, specificity good and advantage easy and simple to handle.
Preferably, in described steps A, often liter of first reagent comprises following component:
Borate derivative 0.4-0.6g
Magnesium chloride 1.9-2.9g
Potassium chloride 22-26g
Barium chloride 6.2-8.2g
Glycyl amide hydrochloride 6.6-8.6g
Formamide 25-35g
Potassium azide 8-12g
NPE 15-25g
Water surplus.
Preferably, in described steps A, the structural formula of borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 1-6.
Preferably, in described step C, often liter of second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 23-33g
Potassium azide 4-6g
Sodium chloride 0.4-0.8g
Formamide 8-12g
Triton X-100 4-6g
Water surplus.
Beneficial effect of the present invention is: kit of the present invention only needs the whole blood of trace peripheral blood sample alive, the content quantitatively detecting glycosylated hemoglobin can be realized in 2-3 minute, greatly improve the speed of examination, have highly sensitive, specificity good and advantage easy and simple to handle; Preparation method is simple, is easy to large-scale production.
Detection method of the present invention only needs the whole blood of trace to live peripheral blood sample, can realize the content quantitatively detecting glycosylated hemoglobin, greatly improve the speed of examination in 2-3 minute, have highly sensitive, specificity good and advantage easy and simple to handle.
Tool of the present invention has the following advantages:
1, to adapt to instrument platform small and exquisite, be easy to carry;
2, instrument cost, reagent cost are far below the detection platform of other principles, are applicable to and community and township hospital;
3, easy and simple to handle, detect rapidly (each pattern detection time < 3 minutes);
4, result is accurate, stable, and disturbing factor is few;
5, required sample size is few, detection of bleeding (sample size 5 μ L).
Embodiment
For the ease of the understanding of those skilled in the art, below in conjunction with embodiment, the present invention is further illustrated, and the content that embodiment is mentioned not is limitation of the invention.
Embodiment 1
Detect a kit for glycosylated hemoglobin, described kit comprises:
First reagent, this first reagent comprises the borate derivative for splitting erythrocyte, release glycosylated hemoglobin, precipitation total hemoglobin;
Second reagent, this second reagent comprises the damping fluid for washing away the borate derivative in conjunction with glycosylated hemoglobin;
And chromatography device, this chromatography device comprises the reaction film for retaining haemoglobin precipitation.
Often liter of described first reagent comprises following component:
Borate derivative 0.4g
Magnesium chloride 1.9g
Potassium chloride 22g
Barium chloride 6.2g
Glycyl amide hydrochloride 6.6g
Formamide 25g
Potassium azide 8g
NPE 15g
Water surplus.
The structural formula of described borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 1.
Often liter of described second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 23g
Potassium azide 4g
Sodium chloride 0.4g
Formamide 8g
Triton X-100 4g
Water surplus.
Use kit described above to detect a detection method for glycosylated hemoglobin for non-treatment object, comprise the steps:
A, draw 5 μ L blood samples with capillary heparin tube, add in the brown centrifuge tube that 200 μ L first reagent are housed, turn upside down 8 times, fully mix, in left at room temperature 1min, obtain reactant liquor;
B, to draw the above-mentioned reactant liquor of 25 μ L with pipettor and drip on the reaction film in the middle of chromatography device;
After C, 8s, get 25 μ L second reagent dropwise on the reaction film in the middle of chromatography device;
After D, 8s, chromatography device is placed on glycolated hemoglobin analysis and detects.
In described steps A, often liter of first reagent comprises following component:
Borate derivative 0.4g
Magnesium chloride 1.9g
Potassium chloride 22g
Barium chloride 6.2g
Glycyl amide hydrochloride 6.6g
Formamide 25g
Potassium azide 8g
NPE 15g
Water surplus.
In described steps A, the structural formula of borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 1.
In described step C, often liter of second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 23g
Potassium azide 4g
Sodium chloride 0.4g
Formamide 8g
Triton X-100 4g
Water surplus.
Embodiment 2
Detect a kit for glycosylated hemoglobin, described kit comprises:
First reagent, this first reagent comprises the borate derivative for splitting erythrocyte, release glycosylated hemoglobin, precipitation total hemoglobin;
Second reagent, this second reagent comprises the damping fluid for washing away the borate derivative in conjunction with glycosylated hemoglobin;
And chromatography device, this chromatography device comprises the reaction film for retaining haemoglobin precipitation.
Often liter of described first reagent comprises following component:
Borate derivative 0.45g
Magnesium chloride 2.2g
Potassium chloride 23g
Barium chloride 6.7g
Glycyl amide hydrochloride 7.2g
Formamide 28g
Potassium azide 9g
NPE 18g
Water surplus.
The structural formula of described borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 2.
Often liter of described second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 25g
Potassium azide 4.5g
Sodium chloride 0.5g
Formamide 9g
Triton X-100 4.5g
Water surplus.
Use kit described above to detect a detection method for glycosylated hemoglobin for non-treatment object, comprise the steps:
A, draw 5 μ L blood samples with capillary heparin tube, add in the brown centrifuge tube that 200 μ L first reagent are housed, turn upside down 9 times, fully mix, in left at room temperature 1.5min, obtain reactant liquor;
B, to draw the above-mentioned reactant liquor of 25 μ L with pipettor and drip on the reaction film in the middle of chromatography device;
After C, 9s, get 25 μ L second reagent dropwise on the reaction film in the middle of chromatography device;
After D, 9s, chromatography device is placed on glycolated hemoglobin analysis and detects.
In described steps A, often liter of first reagent comprises following component:
Borate derivative 0.45g
Magnesium chloride 2.2g
Potassium chloride 23g
Barium chloride 6.7g
Glycyl amide hydrochloride 7.2g
Formamide 28g
Potassium azide 9g
NPE 18g
Water surplus.
In described steps A, the structural formula of borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 2.
In described step C, often liter of second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 25g
Potassium azide 4.5g
Sodium chloride 0.5g
Formamide 9g
Triton X-100 4.5g
Water surplus.
Embodiment 3
Detect a kit for glycosylated hemoglobin, described kit comprises:
First reagent, this first reagent comprises the borate derivative for splitting erythrocyte, release glycosylated hemoglobin, precipitation total hemoglobin;
Second reagent, this second reagent comprises the damping fluid for washing away the borate derivative in conjunction with glycosylated hemoglobin;
And chromatography device, this chromatography device comprises the reaction film for retaining haemoglobin precipitation.
Often liter of described first reagent comprises following component:
Borate derivative 0.5g
Magnesium chloride 2.4g
Potassium chloride 24g
Barium chloride 7.2g
Glycyl amide hydrochloride 7.6g
Formamide 30g
Potassium azide 10g
NPE 20g
Water surplus.
The structural formula of described borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 3.
Often liter of described second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 28g
Potassium azide 5g
Sodium chloride 0.6g
Formamide 10g
Triton X-100 5g
Water surplus.
Use kit described above to detect a detection method for glycosylated hemoglobin for non-treatment object, comprise the steps:
A, draw 5 μ L blood samples with capillary heparin tube, add in the brown centrifuge tube that 200 μ L first reagent are housed, turn upside down 10 times, fully mix, in left at room temperature 1-3min, obtain reactant liquor;
B, to draw the above-mentioned reactant liquor of 25 μ L with pipettor and drip on the reaction film in the middle of chromatography device;
After C, 10s, get 25 μ L second reagent dropwise on the reaction film in the middle of chromatography device;
After D, 10s, chromatography device is placed on glycolated hemoglobin analysis and detects.
In described steps A, often liter of first reagent comprises following component:
Borate derivative 0.5g
Magnesium chloride 2.4g
Potassium chloride 24g
Barium chloride 7.2g
Glycyl amide hydrochloride 7.6g
Formamide 30g
Potassium azide 10g
NPE 20g
Water surplus.
In described steps A, the structural formula of borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 1-6.
In described step C, often liter of second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 28g
Potassium azide 5g
Sodium chloride 0.6g
Formamide 10g
Triton X-100 5g
Water surplus.
Embodiment 4
Detect a kit for glycosylated hemoglobin, described kit comprises:
First reagent, this first reagent comprises the borate derivative for splitting erythrocyte, release glycosylated hemoglobin, precipitation total hemoglobin;
Second reagent, this second reagent comprises the damping fluid for washing away the borate derivative in conjunction with glycosylated hemoglobin;
And chromatography device, this chromatography device comprises the reaction film for retaining haemoglobin precipitation.
Often liter of described first reagent comprises following component:
Borate derivative 0.55g
Magnesium chloride 2.6g
Potassium chloride 25g
Barium chloride 7.7g
Glycyl amide hydrochloride 8.1g
Formamide 32g
Potassium azide 11g
NPE 22g
Water surplus.
The structural formula of described borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 4.
Often liter of described second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 30g
Potassium azide 5.5g
Sodium chloride 0.7g
Formamide 11g
Triton X-100 5.5g
Water surplus.
Use kit described above to detect a detection method for glycosylated hemoglobin for non-treatment object, comprise the steps:
A, draw 5 μ L blood samples with capillary heparin tube, add in the brown centrifuge tube that 200 μ L first reagent are housed, turn upside down 8-12 time, fully mix, in left at room temperature 1-3min, obtain reactant liquor;
B, to draw the above-mentioned reactant liquor of 25 μ L with pipettor and drip on the reaction film in the middle of chromatography device;
After C, 8-12s, get 25 μ L second reagent dropwise on the reaction film in the middle of chromatography device;
After D, 8-12s, chromatography device is placed on glycolated hemoglobin analysis and detects.
In described steps A, often liter of first reagent comprises following component:
Borate derivative 0.55g
Magnesium chloride 2.6g
Potassium chloride 25g
Barium chloride 7.7g
Glycyl amide hydrochloride 8.1g
Formamide 32g
Potassium azide 11g
NPE 22g
Water surplus.
In described steps A, the structural formula of borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 4.
In described step C, often liter of second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 30g
Potassium azide 5.5g
Sodium chloride 0.7g
Formamide 11g
Triton X-100 5.5g
Water surplus.
Embodiment 5
Detect a kit for glycosylated hemoglobin, described kit comprises:
First reagent, this first reagent comprises the borate derivative for splitting erythrocyte, release glycosylated hemoglobin, precipitation total hemoglobin;
Second reagent, this second reagent comprises the damping fluid for washing away the borate derivative in conjunction with glycosylated hemoglobin;
And chromatography device, this chromatography device comprises the reaction film for retaining haemoglobin precipitation.
Often liter of described first reagent comprises following component:
Borate derivative 0.6g
Magnesium chloride 2.9g
Potassium chloride 26g
Barium chloride 8.2g
Glycyl amide hydrochloride 8.6g
Formamide 35g
Potassium azide 12g
NPE 25g
Water surplus.
The structural formula of described borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 5.
Often liter of described second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 33g
Potassium azide 6g
Sodium chloride 0.8g
Formamide 12g
Triton X-100 6g
Water surplus.
Use kit described above to detect a detection method for glycosylated hemoglobin for non-treatment object, comprise the steps:
A, draw 5 μ L blood samples with capillary heparin tube, add in the brown centrifuge tube that 200 μ L first reagent are housed, turn upside down 12 times, fully mix, in left at room temperature 1-3min, obtain reactant liquor;
B, to draw the above-mentioned reactant liquor of 25 μ L with pipettor and drip on the reaction film in the middle of chromatography device;
After C, 12s, get 25 μ L second reagent dropwise on the reaction film in the middle of chromatography device;
After D, 12s, chromatography device is placed on glycolated hemoglobin analysis and detects.
In described steps A, often liter of first reagent comprises following component:
Borate derivative 0.6g
Magnesium chloride 2.9g
Potassium chloride 26g
Barium chloride 8.2g
Glycyl amide hydrochloride 8.6g
Formamide 35g
Potassium azide 12g
NPE 25g
Water surplus.
In described steps A, the structural formula of borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 5.
In described step C, often liter of second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 33g
Potassium azide 6g
Sodium chloride 0.8g
Formamide 12g
Triton X-100 6g
Water surplus.
Use the kit of embodiment 1-5 and commercially available biochemical reagents, high performance liquid chromatography testing result as shown in table 1:
Table 1
| Testing result | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Kit of the present invention | 3.6 | 5.7 | 6.8 | 9.3 | 11.2 |
| HPLC | 3.6 | 5.8 | 6.7 | 9.3 | 11.1 |
| Biochemical reagents | 3.3 | 5.4 | 6.2 | 9.7 | 10.5 |
As can be seen from the above table, the testing result of testing result of the present invention and high performance liquid chromatography is close, and is better than the testing result of commercially available biochemical reagents, highly sensitive, specificity good.Kit of the present invention only needs the whole blood of trace to live peripheral blood sample, can realize the content quantitatively detecting glycosylated hemoglobin, greatly improve the speed of examination in 2-3 minute, have highly sensitive, specificity good and advantage easy and simple to handle; Preparation method is simple, is easy to large-scale production.
Detection method of the present invention only needs the whole blood of trace to live peripheral blood sample, can realize the content quantitatively detecting glycosylated hemoglobin, greatly improve the speed of examination in 2-3 minute, have highly sensitive, specificity good and advantage easy and simple to handle.
Above-described embodiment is the present invention's preferably implementation, and in addition, the present invention can also realize by alternate manner, and any apparent replacement is all within protection scope of the present invention without departing from the inventive concept of the premise.
Claims (8)
1. detect a kit for glycosylated hemoglobin, it is characterized in that: described kit comprises:
First reagent, this first reagent comprises the borate derivative for splitting erythrocyte, release glycosylated hemoglobin, precipitation total hemoglobin;
Second reagent, this second reagent comprises the damping fluid for washing away the borate derivative in conjunction with glycosylated hemoglobin;
And chromatography device, this chromatography device comprises the reaction film for retaining haemoglobin precipitation.
2. a kind of kit detecting glycosylated hemoglobin according to claim 1, is characterized in that: often liter of described first reagent comprises following component:
Borate derivative 0.4-0.6g
Magnesium chloride 1.9-2.9g
Potassium chloride 22-26g
Barium chloride 6.2-8.2g
Glycyl amide hydrochloride 6.6-8.6g
Formamide 25-35g
Potassium azide 8-12g
NPE 15-25g
Water surplus.
3. a kind of kit detecting glycosylated hemoglobin according to claim 2, is characterized in that: the structural formula of described borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 1-6.
4. a kind of kit detecting glycosylated hemoglobin according to claim 1, is characterized in that: often liter of described second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 23-33g
Potassium azide 4-6g
Sodium chloride 0.4-0.8g
Formamide 8-12g
Triton X-100 4-6g
Water surplus.
5. use the kit described in any one of claim 1-4 to detect a detection method for glycosylated hemoglobin for non-treatment object, it is characterized in that: comprise the steps:
A, draw 5 μ L blood samples with capillary heparin tube, add in the brown centrifuge tube that 200 μ L first reagent are housed, turn upside down 8-12 time, fully mix, in left at room temperature 1-3min, obtain reactant liquor;
B, to draw the above-mentioned reactant liquor of 25 μ L with pipettor and drip on the reaction film in the middle of chromatography device;
After C, 8-12s, get 25 μ L second reagent dropwise on the reaction film in the middle of chromatography device;
After D, 8-12s, chromatography device is placed on glycolated hemoglobin analysis and detects.
6. a kind of detection method detecting glycosylated hemoglobin for non-treatment object according to claim 5, it is characterized in that: in described steps A, often liter of first reagent comprises following component:
Borate derivative 0.4-0.6g
Magnesium chloride 1.9-2.9g
Potassium chloride 22-26g
Barium chloride 6.2-8.2g
Glycyl amide hydrochloride 6.6-8.6g
Formamide 25-35g
Potassium azide 8-12g
NPE 15-25g
Water surplus.
7. a kind of detection method detecting glycosylated hemoglobin for non-treatment object according to claim 6, it is characterized in that: in described steps A, the structural formula of borate derivative is:
，
Wherein, R
1for azine, R
2for carbon number is the alkyl of 1-6.
8. a kind of detection method detecting glycosylated hemoglobin for non-treatment object according to claim 5, it is characterized in that: in described step C, often liter of second reagent comprises following component:
4-hydroxyethyl piperazine ethanesulfonic acid 23-33g
Potassium azide 4-6g
Sodium chloride 0.4-0.8g
Formamide 8-12g
Triton X-100 4-6g
Water surplus.
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| CN109923420A (en) * | 2016-09-29 | 2019-06-21 | 株式会社绿十字Ms | For measuring the separable box of glycosylated hemoglobin |
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