CN108254563A - Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of cTnI and preparation method thereof - Google Patents
Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of cTnI and preparation method thereof Download PDFInfo
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Classifications
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention discloses a kind of time resolution immuno-chromatographic test paper strips for detecting cTnI, kit and preparation method thereof, the test strips include bottom liner and the sample pad, bonding pad, coated film and the blotting paper that are sequentially arranged on the bottom liner, the cTnI monoclonals detection antibody of fluorescent microsphere label is coated on the bonding pad, the coated film includes being arranged in parallel and spaced detection zone and check plot, the detection zone is coated with the cTnI monoclonals 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 of conjugated polymer, and the polymer is to have less than 10% light transmittance less than 450nm wavelength, the material with more than 95% light transmittance more than 500nm wavelength.The test strips of the present invention, can be with Quantitative detection, and accurately and reliably, high sensitivity, preparation method is simple for result of the test, is suitble to large-scale production, positive effect has quantitatively been detected to cTnI.
Description
Technical field
The invention belongs to technical field of medical examination, specifically, the present invention relates to a kind of times for quantitatively detecting cTnI
Resolved fluorometric immuno-chromatographic test paper strip, kit and preparation method thereof.
Background technology
Troponin is the regulatory protein of contraction of muscle.Cardiac troponin (cTn) is by the subunit of three kinds of different genes
Composition：Serum cardiac troponin T (cTnT), cardiac muscle troponin I (cTnI) and troponin C (TnC).It is presently used for ACS experiments
Room diagnosis is cTnT and cTnI.Since cTnI and the heteroplasmon in skeletal muscle are encoded respectively by different genes, has different ammonia
Base acid sequence, has unique antigenicity, therefore its specificity will be substantially better than CK-MB isodynamic enzymes.Musculature other than cardiac muscle
Appearance damages or during disease, and CK and CK-MB may be increased, and cTnI does not exceed its critical value then.Since they are normal
Content is atomic in serum, significantly increases in AMI, and increases the variation that multiple is generally above total CK and CK-MB.CTnI due to
Molecular weight is small, after morbidity dissociate cTn from cardiac muscle cell slurry in discharge rapidly people's blood, blood level increases rapidly, the time and
CK-MB is quite or a little earlier.Although troponin half life, is very short (half life of free cTnI for 2h~5d it is reported that differ),
The process duration that it degrades from muscle fibril is very long, the raising that can be maintained for a long time in blood, therefore it has CK-MB concurrently
The advantages of raising is long compared with early and LD1 Diagnostic Time windows.Therefore cTn has the trend of gradually substitution Enzyme target at present.
The measure of troponin mainly uses the immunological method of double-antibody sandwich, and detection method then includes:
1st, double antibodies sandwich immunochemiluminescence method --- the method is easy to operate, high specificity, and sensibility is high.
2nd, Gold standard --- the method has the characteristics of fast and convenient, easily to observe, but sensitivity is not high.
3rd, immune turbidimetry is transmitted --- the assay method is easy, quick, can automate, be suitable for batch detection, still
The turbid methodology of immune transmittance and clinical practice are verified it is still necessary to further.
The troponin that measures of early stage be usually present in a free form very least a portion of Troponin I in screened stock and
Troponin T, as cardiac muscle cell is further downright bad, on muscle fibre, Troponin I and troponin T, which are constantly disintegrated, is discharged into periphery
Cycle, approximately 4 hours this period.
From physiological angle cardiac muscle cell also in continuous metabolism, usual aging and apoptosis are inevitable, same to opportunity
Body sub-health state such as prolonged exercise, fever, abnormal thyroid function, impaired renal function can result in the micro- damage of cardiac muscle
Evil, of short duration reversible myocardial ischemia, anoxic such as coronary spasm, coronary disease and angina pectoris these factors can cause flesh calcium in blood
The micro raising of protein I, T, excessively sensitive diagnosis index are unfavorable for heart infarction diagnosis.
Troponin needs delay a period of time just a large amount of after traditional troponin detection method can not change myocardial necrosis
The pathophysiological features of peripheral circulation are discharged into, so detection sensitivity is continuously improved, are not much practicability.Troponin without
Image of Buddha electrocardiogram can be in the information for capturing myocardial infarction more in early days.
The main features of time resolution immunochromatography POCT are to emphasize out that result is quick, substantially reduce experimental result turnover
Time.For the patient of emergency treatment and rescue, these patients often in critical condition and etiology unknown, and traditional clinical examination
Room time of measuring generally wants 15 minutes or more, and test generally can be completed in POCT within 5 minutes, and doctor provides according to POCT
Information makes patient tentative diagnosis and drafts rescue protocol, will reduce the hospital stays in time, reduces incidence/death rate,
Generate very big Social benefit and economic benefit.The chronic disease of long-term monitoring is needed simultaneously for some, such as the patient of diabetes
The monitoring of blood glucose and glucose in urine can be easily carried out by patient oneself or family members according to the requirement of doctor.
POCT is without reliable quality assurance at present.Each test cell of POCT is independent, therefore can not be ensured every
A test cell quality is just as.The instrument of wherein optical method detection can be interfered by haemolysis in sample and chyle, chemistry
Luminescence method can be influenced by exogenous nitric oxide reducing substances.Based on immunochromatography, the various test paper of chromatography and dry chemical technology
Item and instrument all can be because of temperature, the activity of micro protein in the Different Effects matrix of humidity and pH value, and then influence result.Part
The defects of POCT instrumental methods, sensitivity and repeatability is not good enough, and the range of linearity is narrow, is used to join when only emergency treatment or urgency are asked
It examines, also needs to be sent to clinical laboratory when necessary and be checked.
The immunofluorescence chromatographic apparatus used at present is using the fluorescence signal on bounce technique detection perforated membrane, fluorescence detector
What is captured is the specific antibody of porous film surface fluorescent dye modification, and can't detect the fluorescence signal inside perforated membrane,
Detection sensitivity is caused to decline.
Invention content
Based on this, the defects of in order to overcome the above-mentioned prior art, the present invention provides a kind of times for quantitatively detecting cTnI
Resolved immuno chromatograph test strip, kit and preparation method thereof, the immuno-chromatographic test paper strip and kit can not only provide compared with
High sensitivity and specificity, it is easy to operate, meet the needs of clinical rapid checking, and reduce cost, meet domestic city
The demand of field.
In order to achieve the above-mentioned object of the invention, this invention takes following technical schemes：
A kind of time resolution immuno-chromatographic test paper strip for detecting cTnI, including bottom liner and is sequentially arranged on the bottom liner
Sample pad, bonding pad, coated film and blotting paper, the cTnI monoclonals detection of fluorescent microsphere label is coated on the bonding pad
Antibody, the coated film includes being arranged in parallel and spaced detection zone and check plot, the detection zone are coated with identification list
The cTnI monoclonals capture antibody of one epitope, the check plot is coated with sheep anti-mouse igg antibody；The coated film is chemistry
Crosslinking is combined with the nitrocellulose membrane of polymer, and the polymer is to have less than 10% light transmittance less than 450nm wavelength,
Material more than 500nm wavelength with more than 95% light transmittance.
In wherein some embodiments, the polymer is a kind of or different for polystyrene acrylonitrile and makrolon
The mixture of ratio.This material can allow most of visible light-transmissive, and photodetector can capture multilayer porous film
Surface and internal fluorescence signal, make testing result more accurate.
In wherein some embodiments, the CTNI monoclonals capture antibody and sheep anti mouse of the single epitope of identification
The concentration of IgG antibody is respectively 1-1.5mg/ml and 0.3-0.5mg/ml, and the package amount on the coated film is 1-
1.5ul/cm。
In wherein some embodiments, the bonding pad is nitrocellulose membrane, can be loaded with enough fluorescent microspheres, and
Microballoon can be discharged rapidly after meeting sample again.
In wherein some embodiments, the fluorescent microsphere selects the Eu well known in the art for labelled antibody3+Group of the lanthanides
Element fluorescent microsphere, microsphere surface carry active group, can connect the biological substances such as albumen, carbohydrate, include fluorescein.
In wherein some embodiments, a diameter of 290nm-350nm of the fluorescent microsphere.
In wherein some embodiments, the detection zone close to the bonding pad, the check plot close to the blotting paper,
0.3-0.5cm is divided between the detection zone and the check plot.
The present invention also provides the preparation method of the time resolution immuno-chromatographic test paper strip of above-mentioned detection cTnI, including following
Step：
(1), the fixed cTnI monoclonals for identifying single epitope capture antibody and sheep anti-mouse igg respectively on coated film
Antibody forms detection zone and check plot；
(2), the cTnI monoclonals detection antibody of fluorescent microsphere label is prepared, and is sprayed on bonding pad；
(3), on bottom liner overlap joint paste sample pad, bonding pad, coated film and blotting paper to get.
In wherein some embodiments, the preparation side of the cTnI monoclonals detection antibody of fluorescent marker described in step (2)
Method includes the following steps：
(1), at 4 DEG C, cTnI monoclonals are examined using the phosphate buffer of the pH7.2-7.6 of 0.02-0.05mol/L
Antibody dialysed overnight is surveyed, then the cTnI monoclonals detection antibody after dialysis is adjusted to a concentration of 2-4mg/ml；
(2), microballoon is washed using the MES activation buffers of the pH6.0 of 0.01-0.05mol/L, adds in carbodiimide and N-
HOSu NHS makes the final concentration of 0.2mol/L of microballoon, reacts at room temperature 15-30 minutes, fully washs microballoon, use 0.02-
The borate buffer of 0.05mol/LpH7.4-7.6 redissolves；
(3), it is 1 by the mass ratio of cTnI monoclonals detection antibody and microballoon:The ratio of 5-6, in the microballoon after redissolution
CTnI monoclonals detection antibody is added in, reacts at room temperature 2 hours, adds in the boron of the pH7.4-7.6 of the 0.02mol/L containing 5%BSA
Acid buffer reacts at room temperature 30 minutes, washing, then with containing 0.5%BSA, the 0.02mol/L's of 0.05%Tween-20
The borate buffer of pH7.4-7.6 is redissolved to original volume, and glass fibre membrane is sprayed at the amount of 4ul/cm using quantitative spray film instrument
On, it is protected from light, drying.
The present invention also provides a kind of time-resolved fluoroimmunoassay for detecting cTnI to chromatograph kit, and the kit includes
Plastics get stuck, above-mentioned test strips and are set to plastics and get stuck interior buffer solution bag.
In wherein some embodiments, the buffer solution is contains 0.5% BSA, 0.05% polysorbas20,0.1-1%
The PBS buffer solution of reducing agent；Buffer solution is the reducing agent that 0.01-0.1% is added on the basis of common phosphate liquid, for restoring
The peroxidase to dissociate in sample.
In wherein some embodiments, the reducing agent is reduced glutathione or ascorbic acid.
When in use, blood sample to be measured is added for the time-resolved fluoroimmunoassay chromatography kit of the detection cTnI of the present invention
To after well, bumping bag is pierced by needle, the mixing of sample buffer blood sample is immersed in sample pad, when the sample in sample pad
Originally after reaching saturation state, sample is transported to by bonding pad by capillarity.When containing cTnI in blood sample, cTnI
Antigen-antibody complex is formed with the antibody on fluorescent microsphere, as chromatography acts on, compound moves forward, and reaches coating and knows
At the detection zone T of the cTnI monoclonals capture antibody of not single epitope, antibody-antigen-antibody sandwich complex is formed, is gathered
Collection is at detection zone T.Rare earth ion microballoon (the Eu of unbonded cTnI monoclonal antibodies3+Lanthanide series) continue to move ahead, arrival pair
During according to area C, sheep anti-mouse igg antibody is combined with the mouse monoclonal antibody (i.e. cTnI monoclonals detection antibody) on rare earth ion microballoon,
Occurs the aggregation of rare earth ion microballoon at C lines.Entire reaction was completed, and carry out machine-read card in 10 minutes.In exciting light
The fluorescence intensity generated under source is directly proportional to the conjugate content in test strips, when light source is irradiated to the detection zone of test strips and right
During according to area, the fluorescent material of attachment is excited, emission light gathering is simultaneously converted into electric signal, the power and fluorescent molecular quantity of electric signal
Correlation, detector calculate the content of determinand in sample.
Compared with prior art, the invention has the advantages that：
(1) test strips of the invention, using special translucent material, can both reach the quantitative analysis of chemoluminescence method,
The quick detection of Gold standard can be reached again, and ensure that result of the test accurately and reliably；
(2) time resolution immunochromatography technique is introduced into the quantitative detection of cTnI by test strips of the invention, binding time
Resolved fluorometric detector, the single part for realizing cTnI are quantitatively detected, and high sensitivity, batch in, difference between batch it is small, be Clinical practice
Provide great convenience；
(3) preparation method of test strips of the invention is simple, is suitble to large-scale production, has for the quantitative detection of cTnI
Positive meaning.
Specific embodiment
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 detects the time resolution immuno-chromatographic test paper strip of cTnI
The time resolution immuno-chromatographic test paper strip of detection cTnI of the present embodiment a kind of, including bottom liner and is sequentially arranged at
Sample pad, bonding pad, coated film and blotting paper on the bottom liner are coated with the cTnI of fluorescent microsphere label on the bonding pad
Monoclonal detection antibody (Raybiotech.), the coated film include be arranged in parallel and the detection zone of spaced 0.5cm and
Check plot, the detection zone are coated with identification close to the bonding pad, the check plot close to the blotting paper, the detection zone
The cTnI monoclonals capture antibody (self-produced, to be prepared using existing known technology) of single epitope, the check plot packet
There is sheep anti-mouse igg antibody.
In the present embodiment, the coated film is the nitric acid for being chemically crosslinked makrolon and polystyrene acrylonitrile (polymer)
Tunica fibrosa, the makrolon have less than 10% light transmittance with polystyrene acrylonitrile polymer less than 450nm wavelength,
More than 500nm wavelength there is more than 95% light transmittance.This material can allow most of visible light-transmissive, light detection
Device can capture multi-layer porous film surface and internal fluorescence signal, make testing result more accurate.
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 Eu well known in the art for labelled antibody3+Lanthanide series fluorescence
Microballoon, microsphere surface carry active group, can connect the biological substances such as albumen, carbohydrate, include fluorescein.The fluorescent microsphere
A diameter of 290nm.
The preparation method of the time-resolved fluoroimmunoassay chromatograph test strip of the detection cTnI of the present embodiment, including following step
Suddenly：
(1), the fixed cTnI monoclonals for identifying single epitope capture antibody and sheep anti-mouse igg respectively on coated film
Antibody forms detection zone and check plot；Specific method is：PH using the 0.02mol/L containing 5% sucrose is 7.2-7.6's
The cTnI monoclonals for identifying single epitope are captured antibody respectively and sheep anti-mouse igg antibody are diluted to by phosphate buffer
The two is sprayed on nitrocellulose filter by the concentration of 1.5mg/ml with the amount of 1ul/cm using quantitative spray film instrument with the interval of 0.5cm
On, 50 DEG C of drying 5h, addition drier is sealed up for safekeeping spare；
(2), the cTNI monoclonals detection antibody of fluorescent microsphere label is prepared, and is sprayed on bonding pad；Specific method is：
(a), at 4 DEG C, the phosphate buffer using the pH7.2-7.6 of 0.02-0.05mol/L is saturating by CTNI monoclonals detection antibody
Analysis overnight, then by the cTNI monoclonals detection antibody after dialysis is adjusted to a concentration of 2-4mg/ml；(b), using 0.01-
The MES activation buffers washing microballoon of the pH6.0 of 0.05mol/L, adds in carbodiimide (EDC) and n-hydroxysuccinimide
(NHS), the final concentration of 0.2mol/L of microballoon reacts at room temperature 15 minutes, microballoon is fully washed, with 0.02mol/LpH7.4-7.6's
Borate buffer redissolves；(c), it is 1 by the mass ratio of cTNI monoclonals detection antibody and microballoon:5 ratio, it is micro- after redissolution
CTNI monoclonals detection antibody is added in ball, reacts at room temperature 2 hours, adds in the pH7.4-7.6 of the 0.02mol/L containing 5%BSA
Borate buffer, react at room temperature 30 minutes, washing, then with containing 0.5%BSA, the 0.02mol/L's of 0.05%Tween-20
The borate buffer of pH7.4-7.6 is redissolved to original volume, and glass fibre membrane is sprayed at the amount of 4ul/cm using quantitative spray film instrument
On, it is protected from light, is dried 6 hours at 50 DEG C, addition drier is sealed up for safekeeping spare；
(3), overlap joint pastes sample pad, bonding pad, coated film and blotting paper on bottom liner, and it is big for 0.42cm to cut into width
It is small to get.
Embodiment 2 detects the time resolution immune chromatography reagent kit of cTnI
The time-resolved fluoroimmunoassay chromatography kit of the detection CTNI of the present embodiment, the kit include：Embodiment 1
The test strips, plastics get stuck, buffer solution bag；In the test strips get stuck loaded on the plastics, the buffer solution bag is located at
The edge that the plastics get stuck, close to the sample pad of the test strips, the surface indwelling circular hole of the buffer solution bag is used for needle
Thorn.
In the present embodiment, the reagent strip is used containing 0.5% BSA, 0.05% polysorbas20,0.1-1% reduction
The PBS buffer solution leaching of agent is molten.Buffer solution is the reducing agent that 0.01-0.1% is added on the basis of common phosphate liquid, for restoring
The peroxidase to dissociate in sample.The reducing agent is reduced glutathione or ascorbic acid.
When in use, blood sample to be measured is added for the time-resolved fluoroimmunoassay chromatography kit of the detection cTnI of the present invention
To after well, bumping bag is pierced by needle, sample buffer is mixed with blood sample and is immersed in sample pad, when in sample pad
After sample reaches saturation state, sample is transported to by bonding pad by capillarity.When containing cTnI in blood sample,
CTnI forms antigen-antibody complex with the antibody on fluorescent microsphere, and as chromatography acts on, compound moves forward, and reaches packet
It is identified at the detection zone T of cTnI monoclonals capture antibody of single epitope, it is sandwich compound to form antibody-antigen-antibody
Object is gathered at detection zone T.Rare earth ion microballoon (the Eu of unbonded cTnI monoclonal antibodies3+Lanthanide series) continue to move ahead, it arrives
During up to check plot C, sheep anti-mouse igg antibody and mouse monoclonal antibody (i.e. cTnI monoclonals detection antibody) knot on rare earth ion microballoon
It closes, occurs the aggregation of rare earth ion microballoon at C lines.Entire reaction was completed, and carry out machine-read card in 10 minutes.It is exciting
The fluorescence intensity generated under light source is directly proportional to the conjugate content in test strips, when light source be irradiated to test strips detection zone and
During check plot, the fluorescent material of attachment is excited, emission light gathering is simultaneously converted into electric signal, the power and fluorescent molecular number of electric signal
Amount is related, and detector calculates the content of determinand in sample.
Test example 1 detects cTnI using the time resolution immune chromatography reagent kit of embodiment 2
A, fit standard curve
The cTnI standard items that various concentration is added in the sample application zone of the time resolution immuno-chromatographic test paper strip of embodiment 1 (take 8
A different concentration, respectively 0ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 30ng/mL,
50ng/mL, each concentration do 5 Duplicate Samples), after ten minutes, instrument reads nature controlling line C, detection line T signal for film layer analysis reaction,
Using the ratio of the detection line fluorescent value of the sample of detection and the fluorescent value of nature controlling line as abscissa, cTnI standard concentrations are sat to be vertical
Mark, establishes equation and is fitted to standard curve y=0.9333x+0.0831.
Standard curve R2=0.9919, it is linear preferably, can by the standard curve to cTnI concentration contained in sample into
Row quantitative analysis.
B, sample detection
Sample to be tested, film layer analysis reaction 10 minutes are added in the sample application zone of the fluorescence immune chromatography test paper bar of cTnI.It opens
Fluorescence detection device reads the standard curve in IC card, and detector bar is inserted into the card inserting mouth of fluorescence detection device, running instrument
Device, instrument calculate the cTnI concentration in sample to be tested by corresponding analysis software automatically, will according to the information on calibration card
Actually detected value, which is brought into, calculates quantitative result in preset standard curve.
The performance measurement of the kit of 2 embodiment 2 of test example
The measure of aspect of performance is carried out to kit, including minimum detection limit, precision, sensitivity, specificity etc..
1st, the range of linearity：The time resolution immune chromatography reagent kit of same lot number is taken respectively to the myocardium myo calcium of six concentration
Albumen reference material (0ng/mL, 0.5mg/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, each concentration do 5 Duplicate Samples)
It is detected, detection range is 0ng/mL~50ng/mL, calculates correlation coefficient r, and wherein r values answer >=0.99.
2nd, withinrun precision：10 parts of the time resolution immune chromatography reagent kit of same lot number is randomly selected, respectively to same
The cardiac troponin reference material of concentration is detected, coefficient of variation CV (%) value≤15%.
3rd, betweenrun precision：The time resolution immune chromatography reagent kit of continuous three lot numbers is randomly selected, each lot number takes 3
Part is respectively detected the cardiac troponin reference material of same concentration, coefficient of variation CV (%) value≤15%.
4th, accuracy：The heart of three level concentrations is measured respectively with the time resolution immune chromatography reagent kit of same lot number
Flesh Troponin I reference material calculates sample measures result mean value and relative deviation, and wherein relative deviation (B%) is in ± 15%.
5th, minimum detection limit：10 parts of the time resolution immune chromatography reagent kit of same lot number is taken, to preparing reference material matrix
It is detected, calculates sample measures result mean valueWith standard deviation SD, wherein
6th, analysis specificity：Selection same concentration cardiac troponin reference material be separately added into cholesterol, triglycerides,
Bilirubin makes chaff interferent ultimate density cholesterol 60mg/ml, triglycerides 40mg/ml, bilirubin 2mg/ml, respectively interferes sample
Detection 3 times is repeated, calculates the mean value and relative deviation of pattern detection result, wherein relative deviation (B%) is in ± 15%.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that those of ordinary skill in the art are come
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of time resolution immuno-chromatographic test paper strip for detecting cTnI, which is characterized in that including bottom liner and be sequentially arranged at institute
Sample pad, bonding pad, coated film and the blotting paper on bottom liner are stated, the cTnI that fluorescent microsphere label is coated on the bonding pad is mono-
Clone's detection antibody, the coated film includes being arranged in parallel and spaced detection zone and check plot, the detection zone are coated with
There is the cTnI monoclonals capture antibody for identifying single epitope, the check plot is coated with sheep anti-mouse igg antibody；The coating
Film is the nitrocellulose membrane of conjugated polymer, and the polymer is to have less than 10% light transmittance less than 450nm wavelength,
Material more than 500nm wavelength with more than 95% light transmittance.
2. the time resolution immuno-chromatographic test paper strip of detection cTnI according to claim 1, which is characterized in that the polymerization
One or two of the object for polystyrene acrylonitrile and makrolon.
3. the time resolution immuno-chromatographic test paper strip of detection cTnI according to claim 1 or 2, which is characterized in that described
Identify single epitope cTnI monoclonals capture antibody and sheep anti-mouse igg antibody concentration be respectively 1-1.5mg/ml and
0.3-0.5mg/ml, the package amount on the coated film are 1-1.5ul/cm.
4. the time resolution immuno-chromatographic test paper strip of detection cTnI according to claim 1 or 2, which is characterized in that described
Bonding pad is nitrocellulose membrane, and the fluorescent microsphere is Eu3+Lanthanide series fluorescent microsphere.
5. the time resolution immuno-chromatographic test paper strip of detection cTnI according to claim 4, which is characterized in that the fluorescence
A diameter of 290nm-350nm of microballoon.
6. the time resolution immuno-chromatographic test paper strip of detection cTnI according to claim 1 or 2, which is characterized in that described
Detection zone is divided into close to the bonding pad, the check plot between the blotting paper, the detection zone and the check plot
0.3-0.5cm。
7. the preparation method of the time resolution immuno-chromatographic test paper strip of claim 1-6 any one of them detection cTnI, special
Sign is, includes the following steps：
(1), the fixed cTnI monoclonals capture antibody for identifying single epitope and sheep anti-mouse igg resist respectively on coated film
Body forms detection zone and check plot；
(2), the cTnI monoclonals detection antibody of fluorescent microsphere label is prepared, and is sprayed on bonding pad；
(3), on bottom liner overlap joint paste sample pad, bonding pad, coated film and blotting paper to get.
8. the preparation method of the time resolution immuno-chromatographic test paper strip of detection cTnI according to claim 7, feature exist
In the preparation method of the cTnI monoclonals detection antibody of fluorescent marker described in step (2) includes the following steps：
(1), at 4 DEG C, cTnI monoclonals is detected using the phosphate buffer of the pH7.2-7.6 of 0.02-0.05mol/L and are resisted
Body dialysed overnight, then the cTnI monoclonals detection antibody after dialysis is adjusted to a concentration of 2-4mg/ml；
(2), microballoon is washed using the MES activation buffers of the pH6.0 of 0.01-0.05mol/L, adds in carbodiimide and N- hydroxyls
Succinimide makes the final concentration of 0.2mol/L of microballoon, reacts at room temperature 15-30 minutes, fully washs microballoon, use 0.02-
The borate buffer of 0.05mol/LpH7.4-7.6 redissolves；
(3), it is 1 by the mass ratio of cTnI monoclonals detection antibody and microballoon:The ratio of 5-6 is added in the microballoon after redissolution
CTnI monoclonals detect antibody, react at room temperature 2 hours, the boric acid for adding in the pH7.4-7.6 of the 0.02mol/L containing 5%BSA delays
Fliud flushing reacts at room temperature 30 minutes, washing, then with containing 0.5%BSA, the pH7.4- of the 0.02mol/L of 0.05%Tween-20
7.6 borate buffer is redissolved to original volume, is sprayed on glass fibre membrane, kept away with the amount of 4ul/cm using quantitative spray film instrument
Light, drying.
9. a kind of time-resolved fluoroimmunoassay chromatography kit for detecting cTnI, which is characterized in that the kit includes plastics
It gets stuck, claim 1~6 any one of them test strips and be set to plastics and get stuck interior buffer solution bag.
10. the time-resolved fluoroimmunoassay chromatography kit of detection cTnI according to claim 9, which is characterized in that institute
State buffer solution be containing 0.5% BSA, 0.05% polysorbas20,0.1-1% reducing agents PBS buffer solution；The reducing agent is
Reduced glutathione or ascorbic acid.
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| PCT/CN2017/098114 WO2018120856A1 (en) | 2016-12-28 | 2017-08-18 | Time-resolved fluorescent immunochromatographic test strip and kit for detecting ctni, and preparation method therefor |
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| WO2018120856A1 (en) | 2018-07-05 |
| CN108254563B (en) | 2019-10-29 |
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