CN107490699A - A kind of Blood glycated haemoglobin fluorescence immunoassay detection method - Google Patents
A kind of Blood glycated haemoglobin fluorescence immunoassay detection method Download PDFInfo
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- 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
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- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention discloses a kind of Blood glycated haemoglobin fluorescence immunoassay detection method.This detection method, including：（1）The preparation of fluorescence immunoassay detection kit；（2）Blood sample to be detected is put into fluorescence immunoassay detection kit, reads 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 is put into fluorescence immunoassay detection kit, reads 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 content=A/B of glycosylated hemoglobin.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 present invention relates to technical field of immune assay, particularly relates to a kind of Blood glycated haemoglobin fluorescence immunoassay detection method.
Background technology
Glycosylated hemoglobin（HbA1c）It is that blood-glucose passes through non-enzyme effect, the product combined to form through hemoglobin-chain a word used in person's names propylhomoserin in cell membrane and red blood cell, its synthesis rate is directly proportional to concentration sugared in red blood cell local environment, is the product that endoerythrocytic hemoglobin is combined with blood glucose in blood of human body.
The combination generation glycosylated hemoglobin of blood glucose and hemoglobin is irreversible reaction, and directly proportional to blood sugar concentration, and is 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 glucose, and 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 Gao Xiao Ye Xiang Se Pu ﹑ electricity Yong Fa ﹑ affinity chromatographies etc..
Ion-exchange high-performance liquid chromatography analytic approach, it is on the basis of classical liquid chromatography, and having introduced the theory of gas chromatography has all advantages of gas chromatography.Need to separate the hemoglobin in blood sample and glycosylated hemoglobin using instrumentation before test, it need to be detected during test using large-scale chromatogram analysis equipment, therefore detection must be carried out in hospital by special messenger, it is difficult to meet the needs of glycosylated hemoglobin detects immediately.
Electrophoresis, compared to both the non-glycated hemoglobin, the isoelectric point change of the total electrical charge changed by saccharification and glycosylated hemoglobin is the basis of the isoelectrofocusing gel electrophoretic separation of Ago-Gel or pH gradient 5.0~6.5.The hemoglobin subfraction resolution ratio very little of agarose gel electrophoresis, and isoelectric focusing preferably can separate subfraction.It may decline due to the automaticity deficiency of experiment, importance.
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 measured by chromatographic column.The polyhydroxy base complex of cis-hydroxyl groups is also included in addition high concentration, such as after sorbierite, the combination of glycosylated hemoglobin and boric acid is replaced and eluted from pillar.Affinity chromatography is to the translated influence relative insensitivity of the hemoglobin of modification and pathology hemoglobin later.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 quantitatively can not be accurate the problem of be present from total saccharification Hemoglobin Value with empirical algorithms.
The content of the invention
The purpose of the present invention is to be directed to above-mentioned the deficiencies in the prior art, there is provided a kind of high sensitivity, accuracy fluorescence immunoassay detection method that is strong, easy to operate, being capable of quick diagnosis glycosylated hemoglobin.
In order to solve the above technical problems, the present invention uses fluorescence immune chromatography technology, specific technical scheme is：
（1）Fluorescence immunoassay detection kit is prepared, including the preparation of standard comparison product, the standard comparison product are the not material with hemoglobin, anti-hemoglobin antibodies, glycosylated hemoglobin and anti-glycosylated hemoglobin antibody generation immunity association reaction；
（2）Blood sample to be detected is put into fluorescence immunoassay detection kit, reference product is used as using standard comparison product, immune detection reaction is carried out with glycosylated hemoglobin, reads standard comparison product immune detection result b corresponding to the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin；
（3）Will be with step（2）In same blood sample to be detected be put into fluorescence immunoassay detection kit, with step（2）In same standard comparison product as reference product, carry out immune detection reaction with hemoglobin, 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 content=A/B of glycosylated hemoglobin.
Further, the detection method is raw materials used including standard comparison product, anti-hemoglobin antibodies, anti-glycosylated hemoglobin antibody, sample pad, label pad, chromatographic film, adsorptive pads and bottom plate.
Further, 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.
Further, the anti-hemoglobin antibodies are anti-hemoglobin monoclonal antibody and the one or two of anti-hemoglobin polyclonal antibody.
Further, the anti-glycosylated hemoglobin antibody be anti-glycosylated hemoglobin monoclonal antibody, anti-glycosylated hemoglobin polyclonal antibody, anti-hemoglobin monoclonal antibody, anti-hemoglobin polyclonal antibody it is one or two kinds of or two or more.
Further, the carrier is nitrocellulose filter.
Embodiment
An embodiment provides a kind of fluorescence immune chromatography kit for quantitatively detecting glycosylated hemoglobin, including buckle, fluorescence immune chromatography test paper bar and buffer solution.
Fluorescence immune chromatography test paper bar structure includes sample pad, label pad, chromatographic film, adsorptive pads and bottom plate.When carrying out whole blood sample detection, test strips also include blood separation membrane.Wherein, sample pad, label pad, blood separation membrane, chromatographic film and adsorptive pads are equipped on bottom plate, sample pad is located at the lower section of well, and it is connected to label pad, label pad is connected to chromatographic film, and chromatographic film is connected to adsorptive pads, is fixed with a quantitative detection line and a comparison line thereon, it is preferred that the two distance 3mm~8mm, and positioned at the lower section of observation window.In the case of including blood separation membrane, blood separation membrane is arranged between label pad and chromatographic film, now label pad is connected to blood separation membrane, blood separation membrane is connected to chromatographic film, or blood separation membrane is arranged between sample pad and label pad, now sample pad is connected to blood separation membrane, and blood separation membrane is connected to label pad, or blood separation membrane directly merges into same structure with sample pad.
The hemoglobin monoclonal antibody of fluorescent dye modification, wave-length coverage 300-1300nm are fixed with glycosylated hemoglobin test strips label pad and hemoglobin test strips label pad simultaneously.
Glycosylated hemoglobin monoclonal antibody is fixed with the quantitative detection line of glycosylated hemoglobin test strips, the antigenic determinant that the specific antibody is identified is different from the antigenic determinant that the hemoglobin monoclonal antibody for the fluorescent dye modification being fixed in label pad is identified；Hemoglobin polyclonal antibody is fixed with the quantitative detection line of hemoglobin test strips, the antigenic determinant that the specific antibody is identified is different from the antigenic determinant that the hemoglobin monoclonal antibody for the fluorescent dye modification being fixed in label pad is identified.
Compare the goat anti-mouse igg being fixed with line as standard comparison product.
Another aspect of the present invention provides the preparation method of the above-mentioned fluorescence immune chromatography kit for quantitatively detecting glycosylated hemoglobin, comprises the following steps：
Step (1)：With specific effect between chemical crosslinking or biomolecule respectively by conjugated fluorescent dyes to hemoglobin monoclonal antibody, the hemoglobin monoclonal antibody of fluorescent dye modification is obtained, wave-length coverage is 300~1300nm；
Step (2)：The hemoglobin monoclonal antibody that the fluorescent dye that step (1) is obtained is modified is fixed in label pad (2)；
Step (3)：One quantitative detection line and a comparison line are set respectively in chromatographic film, wherein, compare the goat anti-mouse igg being fixed with line as standard comparison product, glycosylated hemoglobin monoclonal antibody is fixed with the quantitative detection line of glycosylated hemoglobin reagent strip, and the antigenic determinant that the specific antibody is identified is different from the antigenic determinant that the hemoglobin monoclonal antibody for the fluorescent dye modification being fixed in label pad is identified；Hemoglobin polyclonal antibody is fixed with the quantitative detection line of Hemoglobin Reagent Strip, and the antigenic determinant that the specific antibody is identified is different from the antigenic determinant that the hemoglobin monoclonal antibody for the fluorescent dye modification being fixed in label pad is identified；
Step (4)：Sample pad, label pad, chromatographic film, adsorptive pads and bottom plate are built into fluorescence immune chromatography test paper bar, when carrying out whole blood sample detection, test strips also include blood separation membrane；
Step (5)：Fluorescence immune chromatography test paper bar is loaded.
The present invention by conjugated fluorescent dyes to the surface of hemoglobin monoclonal antibody, obtains the hemoglobin monoclonal antibody that fluorescent dye is modified using specific effect between chemical crosslinking or biomolecule.In the present invention, when fluorescent dye surface has active group, it can directly be reacted with specific antibody, is not required to use chemical cross-linking agent；Conversely, then need to be coupled by chemical cross-linking agent.Wherein, chemical cross-linking agent includes 1- ethyls -3- (3- DimethylAminopropyls) carbodiimides (EDC), n-hydroxysuccinimide (NHS) and glutaraldehyde etc..
In a preferred embodiment of the invention, it is using the fluorescent dye DCLHb monoclonal antibody of active group, step：Fluorescent dye after purification is dissolved, then a certain amount of hemoglobin monoclonal antibody is added, reaction medium is used as using buffer solution, reaction 2~4 hours, add hydroxylamine hydrochloride terminating reaction, purified with modes such as chromatogram, chromatographic column or ultrafiltration centrifugations, obtain the hemoglobin monoclonal antibody of fluorescent dye modification.
In order to improve the discrimination of signal and background, the wave-length coverage of fluorescent dye is 300~1300nm, preferably 550-800nm in the present invention.In addition, chromatographic film, bottom plate and buckle are typically extremely weak near infrared region (600~800nm) fluorescence intensity, it is therefore preferable that wave-length coverage be 550-800nm fluorescent dye further to improve sensitivity, more preferably fluorescent dye launch wavelength is 665nm.
The fluorescent dye of the present invention includes organic fluorescent dye and rare earth element fluorescent dye.It is the form for becoming fluorescent microsphere on latex microsphere that the fluorescent dye of the present invention, which can be coupled to,.The present invention fluorescent dye can be single compound fluorescent dye or by several compound groups into composite fluorescent dye, but preferably single compound fluorescent dye and preferably have stronger photostability fluorescent dye.The fluorescein of the fluorescent dye such as Alexa Fluro series of the present invention.
In order to reduce the influence to fluorescent dye fluorescence signal, the present invention use hypofluorescence chromatographic film, bottom plate and buckle, its fluorescent noise be more than 550nm can be very weak, so as to ensure to obtain high fluorescence signal-to-background ratio, energy good discrimination signal and background, and then improve detection sensitivity.It is preferred that bottom plate is white, surface has adhesive sticker, and buckle, chromatographic film, bottom plate and adhesive sticker do not contain fluorescer.
In the present invention, testing sample can be serum or blood plasma, and now fluorescence immune chromatography test paper bar does not include blood separation membrane.Testing sample can also be whole blood, and now fluorescence immune chromatography test paper bar also includes blood separation membrane, for solidifying, filtration cell.Blood separation membrane may be provided between label pad and chromatographic film, respectively with label pad and the direct capillary contact of chromatographic film, or it is arranged between sample pad and label pad, respectively with sample pad and the direct capillary contact of label pad, also same structure directly can be merged into sample pad, so as to which sample pad has the function that sample collection, release and filtering simultaneously.
The fluorescence immune chromatography kit of the present invention carries out quantitative detection to the glycosylated hemoglobin in testing sample.During detection, testing sample is added in sample pad by well, sample moves along chromatographic film to adsorptive pads direction chromatography.The sample chromatography time is usually 8~25 minutes, preferably 15 minutes.After chromatography, chromatographic film is cleaned with cleaning buffer solution, the time is 3-8 minutes, preferably 5 minutes, to reduce background, improves detection sensitivity.Include bovine serum albumin(BSA), sucrose and surfactant in the cleaning buffer solution of the present invention, pH value 7.0-8.0, wherein, the concentration of bovine serum albumin(BSA) is 0.05~2%, and the concentration of sucrose is 1~15%, and the concentration of surfactant is 0.05~2%.The preferred polysorbas20 of surfactant, triton x-100, the preferred Tris-HCl buffer solutions of buffer solution, phosphate buffer.
After chromatography terminates, judge to detect the detection line in window with fluorescent quantitation instrument and compare line, to obtain a result.
Embodiment
Embodiment 1：The preparation of glycosylated hemoglobin fluorescence immunoassay detection kit
1) coupling of fluorescent dye and antibody
The fluorescent dyes rhodamine that launch wavelength is 665nm is mixed with l mg/ml hemoglobin monoclonal antibody, react at room temperature 3h, add 1mol/L hydroxylamine hydrochloride terminating reactions, and with chromatographic column or chromatograph column separating purification, obtain the hemoglobin monoclonal antibody of fluorescent dye modification, fluorescence emission wavelengths 665nm；
2) structure of kit
Take fluorochrome label thing, add bovine serum albumin(BSA) (content 1%), sucrose (content 10%) and surfactant triton x-100 (content 0.8%), subsequent even application is sealed after 40 DEG C of dryings, preserved at room temperature in label pad；
Glycosylated hemoglobin fluorescence immunoassay detection reagent bar is assembled, is made up of, is sequentially pasted on white bottom plate sample pad, label pad, chromatographic film, adsorptive pads.Wherein, sample pad is poroid barrier film, selects glass fibre, is testing sample collecting region；Contain fluorescent dye DCLHb monoclonal antibody in pad；Quantitative detection line is fixed with chromatographic film and compares line, detection line and comparison line are at intervals of 5mm, and detection line is fixed with and is different from glycosylated hemoglobin monoclonal antibody in label pad, compare line and are fixed with goat anti-mouse igg.After assembling, required width is cut into as requested；
Hemoglobin fluorescence immunoassay detection reagent bar is assembled, is made up of, is sequentially pasted on white bottom plate sample pad, label pad, chromatographic film, adsorptive pads.Wherein, sample pad is poroid barrier film, selects glass fibre, is testing sample collecting region；Contain fluorescent dye DCLHb monoclonal antibody in pad；Quantitative detection line is fixed with chromatographic film and compares line, detection line and comparison line are at intervals of 5mm, and detection line is fixed with and is different from hemoglobin polyclonal antibody in label pad, compare line and are fixed with goat anti-mouse igg.After assembling, required width is cut into as requested；
Glycosylated hemoglobin reagent strip and Hemoglobin Reagent Strip are placed in buckle, drier encapsulation is added, is configured to glycosylated hemoglobin fluorescence immunoassay detection kit jointly with cleaning buffer solution.
Embodiment 2：The detection mode of glycosylated hemoglobin fluorescence immunoassay detection kit
（1）Blood sample to be measured is taken, 80ul, forward direction chromatography reaction 15min is added dropwise respectively in glycosylated hemoglobin fluorescence immunoassay detection kit well obtained above；
（2）Prepare cleaning buffer solution, pH value is 7.5, buffer system be 20mM phosphate, add quality solubility be 1% bovine serum albumin(BSA), quality solubility be 10% sucrose and quality solubility be 0.8% surfactant triton x-100,50 μ L are respectively added in 2 sample wells, stand 5min；
（3）It is placed in fluorescent quantitation instrument and obtains fluorescence signal intensity；
（4）Goat anti-mouse igg corresponding to reading sample glycosylated hemoglobin inspection side line signal value a, glycosylated hemoglobin compares line signal value b, sample hemoglobin inspection side line signal value c and goat anti-mouse igg corresponding to hemoglobin and compares line signal value d；
（5）Ratio A=a/b, ratio B=c/d is calculated；
（6）Calculate content=A/B of glycosylated hemoglobin.
Embodiment 3：Kit Performance Evaluation is tested
1. accuracy testing
Same patient's anticoagulated whole blood sample is taken, repeats detection 3 times with a batch of kit, it is 0.02% to calculate relative deviation B values, meets product design requirement；
2. replica test
Same patient's anticoagulated whole blood sample is taken, is detected 20 times simultaneously with a batch of kit, it is 11.87% to calculate coefficient of variation CV values, meets product design requirement.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, and within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection.
Claims (6)
1. a kind of glycosylated hemoglobin fluorescence immunoassay detection method, comprises the following steps：
（1）Fluorescence immunoassay detection kit is prepared, including the preparation of standard comparison product, the standard comparison product are the not material with hemoglobin, anti-hemoglobin antibodies, glycosylated hemoglobin and anti-glycosylated hemoglobin antibody generation immunity association reaction；
（2）Blood sample to be detected is put into fluorescence immunoassay detection kit, reference product is used as using standard comparison product, immune detection reaction is carried out with glycosylated hemoglobin, reads standard comparison product immune detection result b corresponding to the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin；
（3）Will be with step（2）In same blood sample to be detected be put into fluorescence immunoassay detection kit, with step（2）In same standard comparison product as reference product, carry out immune detection reaction with hemoglobin, 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 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, label pad, chromatographic film, adsorptive pads and bottom plate.
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 antibodies are 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-glycosylated hemoglobin antibody be anti-glycosylated hemoglobin monoclonal antibody, anti-glycosylated hemoglobin polyclonal antibody, anti-hemoglobin monoclonal antibody, 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109142758A (en) * | 2018-10-19 | 2019-01-04 | 泰普生物科学（中国）有限公司 | It is a kind of to detect the immuno-chromatographic test paper strip of glycosylated hemoglobin, kit and preparation method thereof |
| CN110873800A (en) * | 2019-12-04 | 2020-03-10 | 海卫特(广州)医疗科技有限公司 | Glycosylated hemoglobin immunochromatographic test strip and preparation method and kit thereof |
| CN111273001A (en) * | 2020-02-12 | 2020-06-12 | 北京大弘生物技术有限公司 | System for rapidly detecting new coronavirus 2019-nCoV in blood sample and preparation method thereof |
| CN115267170A (en) * | 2022-07-25 | 2022-11-01 | 湖南中科蓝海生物科技有限公司 | Method and device for measuring glycosylated hemoglobin based on immunofluorescence chromatography |
| CN117402239A (en) * | 2022-07-07 | 2024-01-16 | 东莞市朋志生物科技有限公司 | Anti-glycosylated hemoglobin antibody, reagent for detecting glycosylated hemoglobin and kit |
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| CN109142758A (en) * | 2018-10-19 | 2019-01-04 | 泰普生物科学（中国）有限公司 | It is a kind of to detect the immuno-chromatographic test paper strip of glycosylated hemoglobin, kit and preparation method thereof |
| CN110873800A (en) * | 2019-12-04 | 2020-03-10 | 海卫特(广州)医疗科技有限公司 | Glycosylated hemoglobin immunochromatographic test strip and preparation method and kit thereof |
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| CN117402239A (en) * | 2022-07-07 | 2024-01-16 | 东莞市朋志生物科技有限公司 | Anti-glycosylated hemoglobin antibody, reagent for detecting glycosylated hemoglobin and kit |
| CN117402239B (en) * | 2022-07-07 | 2024-05-10 | 东莞市朋志生物科技有限公司 | Anti-glycosylated hemoglobin antibody, reagent for detecting glycosylated hemoglobin and kit |
| CN115267170A (en) * | 2022-07-25 | 2022-11-01 | 湖南中科蓝海生物科技有限公司 | Method and device for measuring glycosylated hemoglobin based on immunofluorescence chromatography |
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