CN109142758A - It is a kind of to detect the immuno-chromatographic test paper strip of glycosylated hemoglobin, kit and preparation method thereof - Google Patents
It is a kind of to detect the immuno-chromatographic test paper strip of glycosylated hemoglobin, kit and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of immuno-chromatographic test paper strips of quantitative detection glycosylated hemoglobin, detection kit and preparation method thereof, wherein, the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin includes bottom liner, and it is sequentially arranged at the sample pad on the bottom liner, bonding pad, coated film and blotting paper, glycosylated hemoglobin monoclonal antibody is coated on the bonding pad, the coated film includes being arranged in parallel and the detection zone being spaced apart from each other and check plot, the detection zone is coated with the glycosylated hemoglobin monoclonal antibody for identifying single epitope, the check plot is coated with goat anti-rabbit igg antibody；The coated film is nitrocellulose membrane.Kit based on immunochromatographic method of the invention can not only provide the method for accurate measurement glycosylated hemoglobin, and the kit is easy to operate, the needs for meeting clinical rapid checking, reduce costs simultaneously, can meet the needs of quickly detecting market to glycosylated hemoglobin both at home and abroad.
Description
Technical field
The present invention relates to medical test technology, in particular to a kind of quantitative detection HbAle egg based on immunochromatographic method
White test strips, kit and preparation method thereof.
Background technique
In recent years, glycosylated hemoglobin clinically starts gradually to be paid much attention to.Glycosylated hemoglobin is hemoglobin A
Certain particular molecule positions of component and glucose are formed by slow and irreversible non-enzymatic reaction bonded.Therefore,
When the concentration of glucose in blood is higher, the saccharification hemoglobin content that human body is formed also can be relatively high.
Diabetes are the endocrine metabolism diseases that one group of cause of disease and pathogenesis are not yet completely understood, and disease incidence is only at present
Inferior to cardiovascular and cerebrovascular disease and tumour, in China, diabetes morbidity is 2-3%, and is increased with every millesimal speed.Mesh
Preceding clinical carry out extensively detects patient blood glucose's work.But blood sugar detection only represents blood glucose level at once, prompts patient at that time
Physical condition, can not as evaluation disease control degree index.
Glycated hemoglobin levels reflection is average blood glucose levels in 120 days before detection, and with blood drawing time, disease
On an empty stomach whether people, and if it is unrelated using factors such as insulin, it is the good index for determining diabetes and controlling for a long time.It reflects 4~8
The average level of the internal blood glucose in week, and may be a major reason for causing diabetic complication.
The measurement result of glycosylated hemoglobin is expressed as a percentage, refers to that the hemoglobin combined with glucose accounts for whole
The ratio of hemoglobin.The level of the glycosylated hemoglobin of non-diabetic patients is 4-6%；Many is the study found that patient of diabetes
If glycated hemoglobin levels can be reduced to 8% hereinafter, the complication of diabetes will substantially reduce by person.If HbAle
Albumen > 9% illustrates patient's Persistent hyperglycemia, it may occur that nephrosis, artery sclerosis, the complication such as cataract, and have
It is likely to occur the acute complication such as ketoacidosis.Therefore, expert advice, if blood sugar in diabetic patients controls standard up to standard,
And glycemic control state is more steady, should at least receive 2 glycosylated hemoglobin detections every year；Those are needed to change
The patient of therapeutic scheme or glycemic control state labile, and carrying out the patient of insulin therapy, it should every three months
Carry out a glycosylated hemoglobin measurement.
Currently, the method for clinically measuring glycosylated hemoglobin has very much, more common is that HPLC method, enzyme process, latex are exempted from
Epidemic disease turbidimetry, immunochromatographic method etc..Wherein, HPLC method is adopted extensively as the goldstandard method of detection glycosylated hemoglobin
With, but higher cost；The precision of enzyme process detection, accuracy are poor；Latex immunoturbidimetry accuracy is although relatively good, still
It is needed using lot of antibodies, and needs just to can be carried out detection using large-scale biochemical analyzer, cannot achieve miniaturization, convenient
The detection of change, and while dry type immunochromatographyassay assay reagent can guarantee result accuracy, the requirement of instrument is substantially reduced,
It can be more convenient, more efficiently realize the detection of glycosylated hemoglobin, it is easier to be promoted in places such as community hospital, hygienic outpatient services
The detection of the project.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of dry type immunochromatography of quantitative detection glycosylated hemoglobin
Test strips can solve the problems, such as to cannot achieve miniaturization, facilitation measurement glycosylated hemoglobin ratio in the prior art；
And provide a kind of preparation method of the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin；
Further, a kind of dry type immuno-chromatographic test paper strip including above-mentioned quantitative detection glycosylated hemoglobin, complete is provided
The kit of blood sample lysate,
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows:
A kind of dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin, including bottom liner and it is sequentially arranged at institute
Sample pad, bonding pad, coated film and the blotting paper on bottom liner are stated, coating is marked with the blood red of label indicant on the bonding pad
Protein monoclonal antibody and rabbit igg antibody, the coated film include being arranged in parallel and the detection zone being spaced apart from each other and check plot, institute
It states detection zone and is coated with the glycosylated hemoglobin monoclonal antibody for identifying single epitope, the check plot is coated with goat-anti rabbit
IgG antibody；The coated film is nitrocellulose membrane.
A kind of preparation method of the dry type immuno-chromatographic test paper strip of above-mentioned quantitative detection glycosylated hemoglobin, including it is following
Step:
(1) glycosylated hemoglobin monoclonal antibody and the goat-anti rabbit for identifying single epitope are fixed respectively on coated film
IgG antibody is respectively formed detection zone and check plot；
(2) the hemoglobin monoclonal antibody and rabbit igg antibody for being marked with time-resolved fluorescence microballoon are first prepared, is then sprayed
It is coated on bonding pad；
(3) sample pad, bonding pad, coated film and blotting paper are set gradually on bottom liner, obtain the quantitative detection saccharification
The time-resolved fluoroimmunoassay chromatograph test strip of hemoglobin.
A kind of dry type immune chromatography reagent kit of quantitative detection glycosylated hemoglobin, including above-mentioned quantitative detection saccharification blood
The dry type immuno-chromatographic test paper strip and whole blood sample lysate of Lactoferrin.
The beneficial effects of the present invention are:
(1) time resolution immunochromatography technique is introduced and is saccharified by time-resolved fluoroimmunoassay chromatograph test strip of the invention
In the quantitative detection of hemoglobin, binding time resolved fluorometric detector, single part for realizing glycosylated hemoglobin is quickly determined
Amount detection, and high sensitivity, in batch, difference between batch it is small, provide great convenience for clinical use；
(2) mark is marked using special hemoglobin antibodies in time-resolved fluoroimmunoassay chromatograph test strip of the invention
Remember indicant (such as fluorescent microsphere etc.), the blood red egg in sample is in 1.0-1.5mg/ml, in conjunction with hemoglobin monoclonal antibody
Amount reach saturation state；The content of the hemoglobin of different people is between 100mg/ml-150mg/ml, after 100 times of dilution just
The hemoglobin antibodies saturated reaction of label is enabled to well, i.e., different sample standard deviations can make the antibody of label all in conjunction with upper same
The hemoglobin of quantity；
(3) time-resolved fluoroimmunoassay chromatograph test strip of the invention, preparation method is simple, is suitble to large-scale production,
There is positive meaning for the quantitative detection of glycosylated hemoglobin.
Detailed description of the invention
Fig. 1 is that the dry type immune chromatography reagent kit of the quantitative detection glycosylated hemoglobin of the embodiment of the present invention is fitted to mark
Directrix curve；
Fig. 2 is that the dry type immune chromatography reagent kit of the quantitative detection glycosylated hemoglobin of the embodiment of the present invention and Roche are immunized
Turbidimetry detection glycosylated hemoglobin kit is used to detect the correlation of the testing result of glycosylated hemoglobin.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, below in conjunction with embodiment and cooperate attached
Figure is explained.
The most critical design of the present invention is: the special label hemoglobin monoclonal antibody of selection, blood in the sample
When hemoglobin concentration is 1mg-1.5mg/ml range, the hemoglobin total amount in conjunction with the antibody is remained unchanged, according to conjugate
In hemoglobin contained by the amount of glycosylated hemoglobin react the ratio of glycosylated hemoglobin and hemoglobin.
A kind of dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin, including bottom liner and it is sequentially arranged at institute
Sample pad, bonding pad, coated film and the blotting paper on bottom liner are stated, coating is marked with the blood red of label indicant on the bonding pad
Protein monoclonal detects antibody and rabbit igg, and the coated film includes being arranged in parallel and the detection zone being spaced apart from each other and check plot, institute
It states detection zone and is coated with the glycosylated hemoglobin monoclonal antibody for identifying single epitope, the check plot is coated with goat-anti rabbit
IgG antibody；The coated film is nitrocellulose membrane.
A kind of preparation method of the dry type immuno-chromatographic test paper strip of above-mentioned quantitative detection glycosylated hemoglobin, including it is following
Step:
(1) glycosylated hemoglobin monoclonal antibody and the goat-anti rabbit for identifying single epitope are fixed respectively on coated film
IgG antibody is respectively formed detection zone and check plot；
(2) the hemoglobin monoclonal detection antibody and rabbit igg antibody of first preparation time resolved fluorometric microballoon label, then
It is sprayed on bonding pad；
(3) sample pad, bonding pad, coated film and blotting paper are set gradually on bottom liner, obtain the quantitative detection saccharification
The time-resolved fluoroimmunoassay chromatograph test strip of hemoglobin.
A kind of dry type immune chromatography reagent kit of quantitative detection glycosylated hemoglobin, including above-mentioned quantitative detection saccharification blood
The dry type immuno-chromatographic test paper strip and whole blood sample lysate of Lactoferrin.
As can be seen from the above description, the beneficial effects of the present invention are:
(1) time resolution immunochromatography technique is introduced and is saccharified by time-resolved fluoroimmunoassay chromatograph test strip of the invention
In the quantitative detection of hemoglobin, binding time resolved fluorometric detector, single part for realizing glycosylated hemoglobin is quantitatively examined
Survey, and high sensitivity, in batch, difference between batch it is small, provide great convenience for clinical use；
(2) time-resolved fluoroimmunoassay chromatograph test strip of the invention is carried out using a kind of hemoglobin monoclonal antibody
Time-resolved fluorescence microballoon is marked, the blood red egg in sample is in 1.0-1.5mg/ml, with hemoglobin monoclonal antibody knot
The amount of conjunction reaches saturation state；The content of the hemoglobin of different people is between 100mg/ml-150mg/ml, after 100 times of dilution
It can just make the hemoglobin antibodies saturated reaction of label；
(3) time-resolved fluoroimmunoassay chromatograph test strip of the invention, preparation method is simple, is suitble to large-scale production,
There is positive meaning for the quantitative detection of glycosylated hemoglobin.
Further, the bonding pad is nitrocellulose membrane, can be loaded with enough fluorescent microspheres, and after chance sample again
Microballoon can be discharged rapidly.
Further, colloidal gold, fluorescein, magnetic-particle etc. can be selected in the label indicant.
Further, the detection zone is close to the bonding pad, and the check plot is close to the blotting paper.The detection zone
0.3-0.5cm is divided between the check plot.
Further, kit further includes that plastics get stuck and the whole blood sample lysate of independent packaging.
Further, the whole blood sample lysate is the PBS of BSA, 0.1-1%TritonX-100 containing 0.5%
Buffer.Buffer is used for erythrocyte splitting, for discharging the hemoglobin in whole blood.
Further, decomposition agent can be added, decomposition agent is ammonium chloride, tetradecyltrimethylammonium bromide, TritonX-
100 etc..
Further, the glycosylated hemoglobin monoclonal antibody and goat anti-rabbit igg antibody of the single epitope of identification
Concentration be respectively 1-1.5mg/ml, the package amount on the coated film is 1-1.5ul/cm.
In preparation method, further, label has the hemoglobin Dan Ke of time-resolved fluorescence microballoon in step (2)
The preparation method of grand detection antibody and rabbit igg antibody includes the following steps:
(1) at 4 DEG C, using 0.02-0.05mol/L pH7.2-7.6 phosphate buffer by glycosylated hemoglobin
Monoclonal detects antibody dialysed overnight, then it is 1mg/ml that the antibody after dialysis, which is adjusted to concentration,；
(2) microballoon is washed using the MES activation buffer of the pH6.0 of 0.01-0.05mol/L, carbodiimide and N- is added
HOSu NHS makes the final concentration of 0.2g/L of microballoon, reacts at room temperature 15-30 minutes, then sufficiently washs microballoon, uses
The borate buffer solution of 0.02-0.05mol/LpH7.4-7.6 redissolves；
(3) in the mass ratio of hemoglobin monoclonal antibody and microballoon for the ratio of 1:5-15, in the microballoon after redissolution
Hemoglobin monoclonal is added and detects antibody, reacts at room temperature 2 hours, the pH7.4- of the 0.02mol/L containing 5%BSA is added
7.6 borate buffer reacts at room temperature 30 minutes, washing, then with containing 0.5%BSA, the 0.02mol/ of 0.05%Tween-20
The borate buffer of the pH7.4-7.6 of L is redissolved to original volume.
(4) labeling method of the labeling method of rabbit igg antibody with hemoglobin antibodies.
(5) after the hemoglobin antibodies and rabbit igg marked are mixed in a certain ratio, using quantitative spray film instrument with 4ul/cm
Amount be sprayed on glass fibre membrane, be protected from light, it is obtained by drying.
The dry type immune chromatography reagent kit of quantitative detection glycosylated hemoglobin of the invention, when in use, by blood to be measured
Sample is mixed well with whole blood sample lysate, is added in sample pad after mixing according to the volume of 75-100ul, is worked as sample pad
On sample reach saturation state after, sample is transported to by bonding pad by capillarity.It is blood red when containing in blood sample
When albumen, the antibody on hemoglobin and fluorescent microsphere forms anti-antigen-antibody complex, and as chromatography acts on, compound is forward
It is mobile, it reaches coating and identifies at the detection zone T of glycosylated hemoglobin monoclonal capture antibody of single epitope, formed blood red
Protein antibodies-antigen-glycosylated hemoglobin antibody sandwich compound, are gathered at detection zone T.Fluorescent marker rabbit igg antibody after
Continuous to move ahead, when reaching check plot C, there is the aggregation of fluorescent microsphere in conjunction with rabbit igg antibody at C line in goat anti-rabbit igg antibody.
Entire reaction is completed in 5 minutes, and carries out machine-read card.The knot in fluorescence intensity and test strips generated under excitation light source
It is directly proportional to close object content, when light source is irradiated to detection zone and the check plot of test strips, excites the fluorescent material of attachment, emits light
Electric signal is collected and is converted into, the power of electric signal is related to fluorescent molecule quantity, and detector is according to the fluorescence signal detected
The content of determinand in Strength co-mputation sample.
Below by way of specific embodiment, the present invention will be described in detail.
Raw material used in following embodiment unless otherwise specified, derives from commercially available.
Embodiment 1
The dry type immuno-chromatographic test paper strip of the quantitative detection glycosylated hemoglobin of the present embodiment, including bottom liner and successively
Sample pad, bonding pad, coated film and the blotting paper being located on the bottom liner are coated with fluorescent microsphere label on the bonding pad
Hemoglobin monoclonal detects antibody, and the coated film includes detection zone and the control for being arranged in parallel and being spaced apart from each other 0.5cm
Area, the detection zone is close to the bonding pad, and close to the blotting paper, it is single that the detection zone is coated with identification for the check plot
The glycosylated hemoglobin monoclonal antibody of epitope, the check plot are coated with goat anti-rabbit igg antibody.
In the present embodiment, the bonding pad is nitrocellulose membrane, can be loaded with enough fluorescent microspheres, and after chance sample
Microballoon can be discharged rapidly again.
In the present embodiment, the fluorescent microsphere selects the time-resolved fluorescence well known in the art for labelled antibody micro-
Ball, microsphere surface have active group, can connect the biological substances such as albumen, carbohydrate, include fluorescein.The fluorescent microsphere
Diameter is 100nm-300nm.
The preparation method of the time-resolved fluoroimmunoassay chromatograph test strip of the detection glycosylated hemoglobin of the present embodiment, including
Following steps:
(1) glycosylated hemoglobin monoclonal antibody and the goat-anti rabbit for identifying single epitope are fixed respectively on coated film
IgG antibody forms detection zone and check plot；Method particularly includes: the pH using the 0.02mol/L containing 5% sucrose is 7.2
Glycosylated hemoglobin monoclonal antibody and goat anti-rabbit igg antibody are diluted to the concentration of 1.0mg/ml by phosphate buffer respectively,
The two is sprayed on nitrocellulose filter with the interval of 0.5cm with the amount of 1ul/cm using quantitative spray film instrument, 50 DEG C of drying 12h,
Addition desiccant is sealed up for safekeeping spare；
(2) the hemoglobin monoclonal detection antibody of fluorescent microsphere label is prepared, and is sprayed on bonding pad；Specific method
Are as follows: (a), at 4 DEG C, glycosylated hemoglobin monoclonal is detected by antibody using the phosphate buffer of the pH7.2 of 0.02mol/L
Dialysed overnight, then it is 1mg/ml that the glycosylated hemoglobin monoclonal detection antibody after dialysis, which is adjusted to concentration,；(b), it uses
The MES activation buffer of the pH6.0 of 0.01mol/L washs microballoon, and carbodiimide (EDC) and n-hydroxysuccinimide is added
(NHS), the final concentration of 0.2g/L of microballoon reacts at room temperature 15 minutes, sufficiently washing microballoon, slow with the boric acid of 0.02mol/LpH7.4
Fliud flushing is redissolved；It (c), is the ratio of 1:5 in the mass ratio that hemoglobin monoclonal detects antibody and microballoon, the microballoon after redissolution
Middle addition hemoglobin monoclonal detects antibody, reacts at room temperature 2 hours, and the pH7.4 of the 0.02mol/L containing 5%BSA is added
Borate buffer, react at room temperature 30 minutes, washing, then with containing 0.5%BSA, the 0.02mol/L of 0.05% Tween-20
The borate buffer of pH7.4 redissolve the labeling method to the labeling method of original volume rabbit igg antibody with hemoglobin antibodies.Mark
After the hemoglobin antibodies and rabbit igg remembered are mixed in a certain ratio, glass is sprayed at the amount of 4ul/cm using quantitative spray film instrument
It on glass tunica fibrosa, is protected from light, is dried 6 hours at 50 DEG C, addition desiccant is sealed up for safekeeping spare；
(3) overlap joint pastes sample pad, bonding pad, coated film and blotting paper on bottom liner, and being cut into width is that 0.4cm is big
It is small to get.
Embodiment 2
The dry type immune chromatography reagent kit of the quantitative detection glycosylated hemoglobin of the present embodiment, comprising: described in embodiment 1
Time-resolved fluoroimmunoassay chromatograph test strip, plastics get stuck, whole blood sample lysate；The test strips are loaded on the plastic clip
In shell, whole blood sample lysate centrifuge tube or other containers.
In the present embodiment, the whole blood sample lysate use containing 0.5% BSA, 0.05% polysorbas20,
0.1-1%TritonX-100.
The dry type immune chromatography reagent kit of the quantitative detection glycosylated hemoglobin of the present embodiment when in use, by blood to be measured
It after sample is added to Sample dilution, turns upside down and mixes 1min, then mixture is added in sample pad, when the sample in sample pad
After reaching saturation state, sample is transported to by bonding pad by capillarity.When containing glycosylated hemoglobin in blood sample
When, glycosylated hemoglobin forms anti-antigen-antibody complex with the antibody on fluorescent microsphere, and as chromatography acts on, compound is forward
It is mobile, it reaches coating and identifies at the detection zone T of glycosylated hemoglobin monoclonal capture antibody of single epitope, formed anti-
Body-Ag-Ab sandwich complex, is gathered at detection zone T.The rabbit igg of fluorescent microsphere label continues to move ahead, and reaches check plot
When C, there is fluorescence at C line in conjunction with the mouse hemoglobin monoclonal of fluorescent marker detection antibody in goat anti-rabbit igg antibody
Signal.Entire reaction is completed in 5 minutes, and carries out machine-read card.The fluorescence intensity and test strips generated under excitation light source
On conjugate content it is directly proportional, when light source is irradiated to detection zone and the check plot of test strips, excite the fluorescent material of attachment,
Emission light gathering is simultaneously converted into electric signal, and the power of electric signal is related to fluorescent molecule quantity, and detector is glimmering according to what is detected
Light signal strength calculates the content of determinand in sample.
Effete test embodiment
It is detected in people's whole blood using the dry type immune chromatography reagent kit of the quantitative detection glycosylated hemoglobin of embodiment 41
Glycosylated hemoglobin.
A, fit standard curve
The glycosylated hemoglobin mark of various concentration is added in the sample application zone of the time resolution immuno-chromatographic test paper strip of embodiment 1
Quasi- product (take 6 different concentration, respectively 4.0%, 6.0%, 8.0%, 10.0%, 12.0%, 14.0%, each concentration is done
5 Duplicate Samples), after ten minutes, instrument reads nature controlling line C, detection line T signal, with the detection of the sample of detection for film layer analysis reaction
The ratio of the fluorescent value of line fluorescent value and nature controlling line is abscissa, and glycosylated hemoglobin standard concentration is ordinate, foundation side
Journey is simultaneously fitted to standard curve.Standard curve initial data such as the following table 1, standard curve are shown in Fig. 1.
Table 1
In Fig. 1, standard curve R2> 0.99, it is linear preferable, it can be by the standard curve to saccharification blood contained in sample
Hemoglobin concentration carries out quantitative analysis.
B, sample detection
Sample to be tested, film layer analysis is added in the sample application zone of the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin
Reaction 5 minutes.Fluorescence detection device is opened, the standard curve in IC card is read, and will test item insertion fluorescence detection device
Card inserting mouth runs instrument, and instrument calculates the glycosylated hemoglobin concentration in sample to be tested by analysis software accordingly automatically,
Actually detected value is brought into according to the information on calibration card and calculates quantitative result in preset standard curve.
The performance measurement of the time resolution immuno-chromatographic test paper strip of embodiment 1
1, the range of linearity: take the time resolution immune chromatography reagent kit of same lot number respectively to the HbAle of six concentration
Albumen reference material (4.0%, 6.0%, 8.0%, 10.0%, 12.0%, 14.0%, each concentration does 5 Duplicate Samples) is examined
It surveys, detection range is 4%~14%, calculates correlation coefficient r, wherein r value answers >=0.99.For details, reference can be made to the following table 2.
Table 2
| Theoretical concentration | 1 | 2 | 3 | 4 | 5 | Average value |
| 4.0% | 3.8% | 4.2% | 4.0% | 3.7% | 3.8% | 3.9% |
| 6.0% | 6.2% | 6.0% | 5.6% | 5.8% | 6.2% | 6.0% |
| 8.0% | 8.2% | 7.6% | 8.2% | 8.3% | 8.0% | 8.1% |
| 10.0% | 10.4% | 10.2% | 9.8% | 10.2% | 10.3% | 10.2% |
| 12.0% | 11.5% | 11.8% | 12.4% | 12.6% | 12.0% | 12.1% |
| 14.0% | 13.8% | 14.2% | 13.8% | 14.0% | 14.1% | 14.0% |
It is found that the linear equation of test value and theoretical ident value are as follows: Y=0.9879x+0.0009, linearly dependent coefficient are
0.9995。
2, precision: made time-resolved fluorescence detection glycosylated hemoglobin kit in three batches of embodiments 4 is taken, respectively
The repeated quality-control product of detection 4.0%, 8.0%, every batch of kit use repeated quality-control product Parallel testing 10 times, 4.0% concentration three
CV is respectively 4.64%, 4.20%, 4.00% in criticizing, and CV is 4.20% between three batches batches, and 8.0% concentration three batches criticizes interior CV points
Not Wei 4.53%, 4.27%, 4.00%, CV is 4.25% between three batches batches, within 5%.
200 parts of whole blood sample of hospital's detection glycosylated hemoglobin are acquired, with the immune ratio of kit and Roche of the invention
Two methods of turbid method detection glycosylated hemoglobin kit carry out detection comparison.In kit of the invention, 5 μ of whole blood sample is taken
Sample dilution is added in l, and 75 μ l is taken to be added in detection card well after mixing, passes through Thymopetidum Injection object science after chromatographing 5min
The TIB-FIA-01 type dry type fluorescence immunity analyzer of (China) Co., Ltd production reads concentration, and same sample is using comparison
System Roche immunoturbidimetry detects glycosylated hemoglobin kit and carries out Concentration Testing.Two methods testing result carries out line
Property analysis, as shown in Fig. 2, its
4, clinical sample detects
200 parts of whole blood sample of hospital's detection glycosylated hemoglobin are acquired, with the immune ratio of kit and Roche of the invention
Two methods of turbid method detection glycosylated hemoglobin kit carry out detection comparison.In kit of the invention, 5 μ of whole blood sample is taken
Sample dilution is added in l, and 75 μ l is taken to be added in detection card well after mixing, passes through Thymopetidum Injection object science after chromatographing 5min
The TIB-FIA-01 type dry type fluorescence immunity analyzer of (China) Co., Ltd production reads concentration, and same sample is using comparison
System Roche immunoturbidimetry detects glycosylated hemoglobin kit and carries out Concentration Testing.Two methods testing result carries out line
Property analysis, as shown in Fig. 2, its correlation is fine, i.e. r=0.9812, P > 0.05, mean relative deviation 5.4% is less than
10%, as a result meet clinical analysis requirement, is suitable for clinical detection.
In conclusion the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin provided by the invention, preparation side
Method and kit have the single part quantitative detection for realizing glycosylated hemoglobin, and high sensitivity, and in batch and difference between batch is small
Advantage.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright specification and accompanying drawing content are applied directly or indirectly in relevant technical field, similarly include
In scope of patent protection of the invention.
Claims (9)
1. a kind of dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin, which is characterized in that including bottom liner, Yi Jiyi
The secondary sample pad being located on the bottom liner, bonding pad, coated film and blotting paper, coating is marked with label instruction on the bonding pad
The hemoglobin monoclonal of object detects antibody, and the coated film includes being arranged in parallel and the detection zone being spaced apart from each other and check plot,
The detection zone is coated with the glycosylated hemoglobin monoclonal capture antibody for identifying single epitope, and the check plot is coated with
Sheep anti-mouse igg antibody；The coated film is the nitrocellulose membrane that chemical crosslinking is combined with polymer.
2. the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin according to claim 1, which is characterized in that
The label indicant is fluorescent microsphere, colloidal gold or magnetic particle.
3. the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin according to claim 1, which is characterized in that
Hemoglobin maximum combined concentration in the hemoglobin antibodies and detection sample of label used is 1.0-1.5mg/ml.
4. the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin according to claim 1, which is characterized in that
The glycosylated hemoglobin monoclonal capture antibody of the single epitope of identification and the concentration of sheep anti-mouse igg antibody are respectively 1-
1.5mg/ml, the package amount on the coated film are 1-1.5ul/cm.
5. the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin according to claim 4, which is characterized in that
The bonding pad is nitrocellulose membrane, and the marker material is time-resolved fluorescence microballoon.
6. the dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin according to claim 4, which is characterized in that
The detection zone is close to the bonding pad, and the check plot is between the blotting paper, the detection zone and the check plot
It is divided into 0.3-0.5cm.
7. a kind of dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin as claimed in any one of claims 1 to 6
Preparation method, which comprises the following steps:
(1) the glycosylated hemoglobin monoclonal antibody and goat anti-rabbit igg for identifying single epitope are fixed respectively on coated film
Antibody forms detection zone and check plot；
(2) the hemoglobin monoclonal antibody and rabbit igg antibody for being marked with label indicant are first prepared, bonding pad is then sprayed on
On；
(3) sample pad, bonding pad, coated film and blotting paper are set gradually on bottom liner, obtain the quantitative detection HbAle
The time-resolved fluoroimmunoassay chromatograph test strip of albumen.
8. a kind of dry type immune chromatography reagent kit of quantitative detection glycosylated hemoglobin, which is characterized in that including claim 1-6
The dry type immuno-chromatographic test paper strip of quantitative detection glycosylated hemoglobin described in any one.
9. the dry type immune chromatography reagent kit of quantitative detection glycosylated hemoglobin according to claim 8, which is characterized in that
It further include that plastics get stuck and whole blood sample lysate, the whole blood sample lysate is PBS buffer solution, the PBS buffer solution
Tween 20 and 0.1-1%TritonX-100 including 0.5%BSA, 0.05%.
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| CN110208544A (en) * | 2019-06-20 | 2019-09-06 | 南方医科大学深圳医院 | A kind of excrement Test paper and kit |
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| 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 |
| CN112485453A (en) * | 2020-11-18 | 2021-03-12 | 重庆中元汇吉生物技术有限公司 | Liquid chromatography reagent for measuring glycosylated hemoglobin and preparation method thereof |
| CN113740543A (en) * | 2021-09-28 | 2021-12-03 | 河南沃迈生物科技有限公司 | Kit for rapidly detecting proportion of glycosylated hemoglobin and preparation method thereof |
| CN115951072A (en) * | 2022-12-06 | 2023-04-11 | 北京鸿宇泰生物科技有限公司 | Glycosylated hemoglobin-C peptide combined detection kit |
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| CN112485453A (en) * | 2020-11-18 | 2021-03-12 | 重庆中元汇吉生物技术有限公司 | Liquid chromatography reagent for measuring glycosylated hemoglobin and preparation method thereof |
| CN113740543A (en) * | 2021-09-28 | 2021-12-03 | 河南沃迈生物科技有限公司 | Kit for rapidly detecting proportion of glycosylated hemoglobin and preparation method thereof |
| CN115951072A (en) * | 2022-12-06 | 2023-04-11 | 北京鸿宇泰生物科技有限公司 | Glycosylated hemoglobin-C peptide combined detection kit |
| CN115951072B (en) * | 2022-12-06 | 2024-05-14 | 北京鸿宇泰生物科技有限公司 | Glycosylated hemoglobin-C peptide joint detection kit |
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