CN106153927A - A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously - Google Patents
A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously Download PDFInfo
- Publication number
- CN106153927A CN106153927A CN201610223337.8A CN201610223337A CN106153927A CN 106153927 A CN106153927 A CN 106153927A CN 201610223337 A CN201610223337 A CN 201610223337A CN 106153927 A CN106153927 A CN 106153927A
- Authority
- CN
- China
- Prior art keywords
- myo
- ctni
- ckmb
- time
- microsphere
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
-
- 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
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention belongs to clinical diagnose field, detect time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo particularly to a kind of fast quantification simultaneously.This reagent is made up of test strips and fluorescent liquid two parts.Wherein, test strips includes base plate, Fusion5, nitrocellulose filter and adsorptive pads；Described Fusion5, nitrocellulose filter and adsorptive pads horizontal direction are linked in sequence and are fixed on base plate.The present invention utilizes time-resolved fluorescence microsphere to improve fluorescence intensity, reduces background signal, and tri-indexs of cTnI, CKMB, Myo in simultaneous quantitative detection whole blood, serum or blood plasma, sample only needs 10～20 μ l.This test strips have convenient and swift, simple to operate, the detection time is short, high specificity, highly sensitive, testing result is more accurate, it is adaptable to the quick diagnosis of clinical POCT.
Description
Technical field
The invention belongs to clinical diagnose field, detect cTnI, CKMB, Myo particularly to a kind of fast quantification simultaneously
Time-resolved fluoroimmunoassay chromatography reagent and preparation method thereof.
Background technology
Troponin is to have TnT (cTnT), TnC (cTnC), three subunits of Troponin I (cTnI)
The complex of composition.The molecular weight of cTnI is 22.5kD, N end 26 aminoacid many compared with skeletal muscle TnI of polypeptide, amino acid sequence
Difference reaches 40%, thus antigenicity is significantly different with skeletal muscle.CTnI is applied to acute as a kind of new diagnosis marker
Those are not particularly had the patient of electrocardiographic diagnosis more important by the clinical diagnosis of myocardial infarction (AMI).As AMI, cTnI
Can be discharged into blood from the myocardial cell damaged in 4～8 hours, and beyond normal concentration range.Generally, AMI morbidity 12
～after 18 hours, reach maximum concentration, in 5～10 days, maintain high level.In ACC and American Heart Association
Updated Guidelines shows, owing to reasons such as myocardium high specific and diagnosis efficiencies, cTnI, to detection myocardial dysfunction, has
Higher using value.
Myoglobin (MYO) is the distinctive a kind of low molecule chromoprotein containing heme of striated muscle tissue, main
It is distributed in cardiac muscle and skeletal muscle tissue.When acute myocardial injury, MYO is released in blood at first, occurs in symptom
After 2-3 hour, MYO value exceeds upper limits of normal, within 9-12 hour, reaches peak value, within 24-36 hour, recovers normal.Myoglobin in serum
The index that the early stage that can diagnose as acute myocardial infarction (AIM:acute Myocardial infarction) is the sensitiveest, but
Its specificity is poor, and the disease such as Skeletal muscle injury, wound, renal failure is likely to cause it to raise.Though so MYO is positive
AMI can not be made a definite diagnosis but may be used for getting rid of in early days the important indicator of AMI diagnosis, as MYO is negative, the most substantially get rid of myocardial infarction
May.MYO can be also used for the diagnosis of re-infarction, in conjunction with clinical, as MYO raises again, is considered as prolonging for re-infarction or infarction
Exhibition.
CK-MB is one of three isomers (another two is CK-BB and CK-MM) of creatine kinase (CK).CK is a kind of flesh
The apoenzyme of meat metabolism.CK-MB is made up of (MW=40000 is each) two subunits: subunit M represents that muscle, subunit B represent
Brain.CK-MB is primarily present in cardiac muscle, is present on a small quantity in skeletal muscle.Quantitative determination CK-MB is the one of heart routine examination
Part, and the diagnosis of beneficially AMI.When there is AMI, CK-MB i.e. comes across in blood circulation and illustrates that myocardium receives
Damage.CK-MB concentration raises in 4-8 hour after panic attacks, reaches peak value, the most after 48 hrs in 12-24 hour
Drop to normal condition.CK-MB is the labelling that peri-operation period myocardial infarction selects in 48 hours of first after panic attacks
Thing.CK-MB concentration is additionally operable to evaluate the degree of AMI and blocking again later.Come from diagnosis susceptiveness, specificity and efficiency
Seeing, CK-MB will be better than the CK isozyme according to electrophoresis.The mensuration of CK-MB also contributes to repeatedly filling cardiac muscle under thrombolytic therapy
The effect of fluid injection and the non-invasive evaluation that carries out.The rising of CK-MB level is also relevant with Skeletal muscle injury simultaneously, but is not as
In AMI, CK-MB level has the version raising and declining.
Time-resolved fluorescence (TRF) analytical technology be using have the lanthanide series of unique fluorescent characteristic and chelating agen thereof as
Tracer, a kind of novel on-radiation trace analysis of foundation.Divide since employing times such as nineteen eighty-three Pettersson
Since distinguishing that fluorescence immunoassay (TRFIA) standard measure measures hCG, TRFIA methodological study and clinical practice quickly grow, and become
Both a new milestone of labelling immunoassay development after RIA, have developed many clocks TRFIA instrument and supporting the most in succession
Commercial kit.TRFIA technology has highly sensitive, standard curve range width, pollution easy and simple to handle, "dead", many
The advantages such as labelling, are widely used in the clinic of tumor, infectious disease, endocrinopathy, autoimmune disease, hereditary
Laboratory diagnosis, it has also become one of analysis means that biomedical research and clinical ultramicron biochemical investigation are commonly used.By TRF, gather
Phenylethylene micro ball and nitrocellulose filter combine, and can be used for rapid in-vitro diagnostic field.
Patent (ZL 03819391.4) relates to a kind of detection based on film using time-resolved fluorescence, is used for detecting survey
The existence of sample analyte in this and content；
Patent (CN 202149903 U) invention c reactive protein time-resolved fluoroimmunoassay chromatography detection by quantitative；Patent (CN
202166649 U) invention time-resolved fluoroimmunoassay quantitative detecting test strip for neopterin；
Patent (CN 102879559 A) invention a kind of time-resolved fluoroimmunoassay chromatography real-time and quantification detection kit side
Method；
The double target detection by quantitative of a kind of AFP and Free-β-HCG time-resolved fluorescence of patent (CN 103760368 A) invention
Test kit；
Patent (CN 204514928 U) invention is a kind of detects pepsinogen I based on time-resolved fluoroimmunoassay chromatography
With the test strips of II and the test strips applying it.
At present, the method for three joint-detection of cardiac muscle mainly has colloidal gold immunity chromatography, micro-fluidic method, fluorescence immunoassay layer
Analysis method etc..Colloidal gold immunity chromatography qualitatively or quantitatively detects, and sensitivity is low, the range of linearity is narrow, it is impossible to meets quantitatively, accurately examine
The requirement surveyed；Micro-fluidic method can detect cardiac muscle three quantitatively, accurately, simultaneously, but generally requires more sample size, Er Qieshi
Agent cost is high；Fluorescence immune chromatography method great majority use fluorescein or other materials as luminous source, although have higher linear
Scope, but disturbed by background signal, it is impossible to enough ensure low side sensitivity.
Although time-resolved fluoroimmunoassay chromatography is widely used for the detection of multiple project, but great majority all use
Time-resolved fluorescence microsphere is fixed in conjugate release pad, due to the release heterogeneity of microsphere, causes imprecision very big,
The detection of the project that sensitivity requirement is high, especially Troponin I cannot be met.Additionally, in order to detect whole blood sample, Duo Shuochang
The conjugate release pad of family uses artificial treatment to cross glass fibre, adds imprecision equally.
In sum, when cardiac muscle three associating detection by quantitative, in the urgent need to one, sample size is few, precision is high, detection
Background signal is low, highly sensitive, range of linearity width, sample pad are not required to process and just can detect the time-resolved fluorescence of whole blood sample and exempt from
Epidemic disease chromatography reagent.
Summary of the invention
It is an object of the invention to provide a kind of fast quantification and detect the time-resolved fluoroimmunoassay of cTnI, CKMB, Myo simultaneously
Chromatography reagent, this reagent can complete the detection of cTnI, CKMB, Myo within the shorter detection time, and the detection time is short, detection spirit
Sensitivity is high.
Present invention also offers fast quantification and detect the time-resolved fluoroimmunoassay chromatography reagent of cTnI, CKMB, Myo simultaneously
Preparation method.
A kind of fast quantification detects the time-resolved fluoroimmunoassay chromatography reagent of cTnI, CKMB, Myo simultaneously, and its feature exists
In: this reagent is made up of test strips and fluorescent liquid two parts.
Described test strips includes base plate (1), Fusion5 (2), nitrocellulose filter (3) and adsorptive pads (4).
Described Fusion5 (2), nitrocellulose filter (3) and adsorptive pads (4) horizontal direction are linked in sequence and are fixed on base plate
On.
Myo monoclonal antibody 1 (5), cTnI monoclonal antibody 1 or polyclone it is coated with on described nitrocellulose filter (3)
The nature controlling line that the detection line of antibody 1 (6), CKMB polyclonal antibody 1 (7) capture molecule composition and rabbit igg antibody (8) are constituted.
Fluorescent liquid (9) comprise the fluorescent microsphere of Myo monoclonal antibody 2 labelling, cTnI monoclonal antibody 2 labelling fluorescence micro-
Ball, the fluorescent microsphere of CKMB monoclonal antibody 2 labelling, the fluorescent microsphere of goat anti-rabbit antibody labelling.
Described time-resolved fluorescence microsphere is selected from the polystyrene microsphere of modified.
Described polystyrene microsphere, surface modification functional group is the one of carboxyl, hydroxyl or epoxy radicals, and particle diameter is 100
～500nm.
The internal chelate filling lanthanide series of described time-resolved fluorescence microsphere.
Described lanthanide series be europium, samarium, one in dysprosium.
Described time-resolved fluorescence microsphere markers step is as follows:
(1) preparation of Troponin I monoclonal antibody 2 labelling time-resolved fluorescence microsphere and dilution:
Time-resolved fluorescence microsphere is dissolved in MES buffer, adds activator activation；It is subsequently added glycine, makes
Obtain microsphere surface and connect arm；Washing, centrifugal after, again add activator activation；Again wash, be centrifuged, time resolution is glimmering
Light microsphere with borate buffer redissolve, be subsequently added Troponin I monoclonal antibody 2 and react, borate buffer, microsphere with
In the range of the mass ratio of antibody is 10mg:0.01mg～10mg:2.0mg；Add sealer after having reacted to close；Closed
After, wash, centrifugal, again with borate buffer and sealer redissolve, ultrasonic, make microsphere be dispersed in buffer, 2-8 DEG C
Keep in Dark Place.Selecting suitable buffer to dilute fluorescent microsphere, working concentration is at 0.5～50 μ g/ml, for Troponin I
The assessment of energy.
(2) preparation of the fluorescent microsphere of Myo monoclonal antibody 2 labelling and dilution, the fluorescence of CKMB monoclonal antibody 2 labelling
The preparation of microsphere and dilution, the preparation of fluorescent microsphere of goat anti-rabbit antibody labelling and dilution are with (1) described.
Preferably, using the time-resolved fluorescence microsphere that europium particle chelate is filled, particle diameter is: 100～500nm；
Preferably, borate buffer, microsphere with the mass ratio of antibody are: 10mg:0.01mg～10mg:2.0mg；
Preferably, the working concentration of fluorescent liquid is: 0.5～50 μ g/ml；
A kind of prepare described fast quantification detect simultaneously cTnI, CKMB, Myo time-resolved fluoroimmunoassay chromatography reagent
Method, comprise the steps:
(1) preparation detection line and nature controlling line: nitrocellulose filter (3) is resisted for fixed packet in Immunofluorescence test paper strip
Body, is also the place of immunoreactive generation simultaneously；Detection line (5) is that Myo monoclonal antibody 1 is used citrate or sucrose
Diluted, lines on described nitrocellulose filter (3)；Detection line (6) is by cTnI monoclonal antibody 1 or Anti-TNF-α
Body 1 uses citrate or sucrose diluted, lines on described nitrocellulose filter (3)；Detection line (7) is by CKMB
Polyclonal antibody 1 uses citrate buffer solution or sucrose solution dilution, in line and described nitrocellulose filter (3)；Nature controlling line
(8) it is that rabbit igg antibody is used citrate or sucrose diluted, in line and described nitrocellulose filter (3).To draw
Good nitrocellulose filter is placed in vacuum drying oven, at 37～45 DEG C, dries 24～48 hours.Detection line (5), (6), (7)
Co-located or diverse location, if diverse location, then detects line (5), (6), (7) are respectively positioned on nature controlling line (8) the same side,
The position that i.e. first liquid contact；
(2) assembling of test strips: adhered to Fusion5 (2), celluloid successively by left-to-right at test strips base plate (1)
Film (3), absorbent paper (4).The big plate assembled is cut into little bar, i.e. obtain described fast quantification detect simultaneously cTnI, CKMB,
The time-resolved fluoroimmunoassay chromatograph test strip of Myo.
Preferably, citrate or the dilute middle citrate of sucrose diluent or sucrose account for 5-20%；
Preferably, the film concentration of drawing of detection line and nature controlling line antibody is 0.5～2mg/ml；
Preferably, the film discharge rate of drawing of detection line and nature controlling line antibody is 0.5～2.0 μ l/cm；
Above fast quantification detects the detection method of the time-resolved fluoroimmunoassay chromatography reagent of cTnI, H-FABP simultaneously,
Its step includes:
(1) drafting of standard curve: take cTnI, CKMB and Myo hybrid standard product of 10～20 μ l and the glimmering of 80～500 μ l
Light mixed liquor (cTnI, CKMB, Myo, goat-anti rabbit fluorescent liquid) mixes, and takes mixed liquor 70～100 μ l and is added drop-wise to test strips
On Fusion5, response time 15～20 minutes, read by the time resolution immunochromatography quantitative analysis instrument supporting with test strips
System detection signal 1, system detection signal 2, system detection signal 3, then with each concentration of standard substance as abscissa, each concentration
The meansigma methods of system detection signal is vertical coordinate, and standard substance curve is prepared in matching；
(2) detection of testing sample, takes testing sample and the fluorescence mixed liquor of 80～500 μ l of 10～20 μ l respectively
(cTnI, CKMB, Myo, goat-anti rabbit fluorescent liquid) mixes, and takes mixed liquor 70～100 μ l and is added drop-wise on the Fusion5 of test strips, instead
Between Ying Shi 15～20 minutes, i.e. be can get the concentration of cTnI, CKMB and Myo in testing sample by the value of system detection signal
Value.
In described (1), the concentration of cTnI, CKMB, Myo hybrid standard product is respectively as follows:
CTnI:0,0.1,0.5,1.0,5.0,25.0,50.0ng/ml；
CKMB:0,0.5,2.0,10.0,25.0,50.0,100.0ng/ml；
Myo:0,5,20,100,200,400,800ng/ml.
The invention has the beneficial effects as follows:
(1) present invention uses Fusion5 to be sample pad, has abandoned traditional sample pad and conjugate release pad, it is not necessary to pre-
Process, the detection of whole blood, serum, plasma sample can be carried out, it is possible to improve detection precision and accuracy greatly.
(2), outside fluorescent liquid is placed in nitrocellulose filter by the present invention, it is different from traditional method and fluorescent microsphere is fixed on
In conjugate release pad, do not exist fluorescent microsphere discharge inhomogenous phenomenon, equally can greatly improve detection precision and
Accuracy.
(3) present invention uses the time-resolved fluorescence microsphere that europium particle chelate is filled, due to the Stokes displacement that it is big
(excitation wavelength is about 340nm, a length of about the 615nm of transmitted wave, and difference is about 275nm), it is possible to substantially reduce background letter
Number value, improve detection sensitivity.
(4) sample size (10～20 μ l) that the employing of the present invention is few, it is possible to the effective interference getting rid of sample.
Accompanying drawing explanation
Fig. 1 is the time-resolved fluoroimmunoassay chromatography reagent that fast quantification of the present invention detects cTnI, CKMB and Myo simultaneously
Structural representation (1 be base plate, 2 for Fusion5,3 for nitrocellulose filter, 4 for adsorptive pads, 5 for Myo detection line, 6 be cTnI
Detection line, 7 detect line for CKMB, 8 be rabbit igg nature controlling line):
Fig. 2 is the standard substance curve of Myo of the present invention；
Fig. 3 is the standard substance curve of cTnI of the present invention；
Fig. 4 is the standard substance curve of CKMB of the present invention；
Fig. 5 is the present invention general correlation curve with chemoluminescence method Myo difficult to understand；
Fig. 6 is the present invention general correlation curve with chemoluminescence method cTnI difficult to understand；
Fig. 7 is the present invention general correlation curve with chemoluminescence method CKMB difficult to understand；
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described:
In the present invention, H-FABP antibody, cTnI antibody and the supplier of fluorescent microsphere and article No. are as follows
| Antibody Designation | Catalog number (Cat.No.) | Producer |
| Anti-h Myoglobin clone 7001 | 100378 | Medix |
| Anti-h Myoglobin clone 7004 | 100354 | Medix |
| 19C7 monoclonal antibody (cTnI) | 4T21 | Hytest |
| 16A11 monoclonal antibody (cTnI) | 4T21 | Hytest |
| G-129-C polyclonal antibody (cTnI) | G-129-C | Biospacific |
| Anti-h CK-MB clone 7501 | 100085 | Medix |
| Anti-h CK-MB clone 7502 | 100086 | Medix |
| 200nm fluorescent microsphere | FC02F | Bangs laboratories |
Embodiment 1
The preparation of cTnI, CKMB, Myo time-resolved fluoroimmunoassay chromatograph test strip:
(1) preparation of line (T1, T2, T3) solution is detected: dilute cTnI respectively with the citrate solution of 10 times, 50mM mono-
Clonal antibody and polyclonal antibody, CKMB polyclonal antibody, Myo monoclonal antibody to 1～2mg/ml；
(2) preparation of nature controlling line (C) solution: with the citrate solution of 10 times, 50mM dilution rabbit igg antibody to 1～
2mg/ml；
In the upper mode using overlap joint of base plate (1) with gum, first paste nitrocellulose filter (3), nitre the most again
The two ends of acid cellulose film (3) paste Fusion5 film (2) and absorbent paper (4) respectively.At nitrocellulose filter (3) and close
Fusion5 film (2) place, draws T1 (Myo captured line (5)), T2 (cTnI captured line (6)), T3 (CKMB captured line (7)) respectively,
Drawing C (rabbit igg) line near one end of absorbent paper (4), the spacing of T1, T2, T3, C is all 3mm.
At T line, draw T1, T2, T3 respectively, at C line, draw C line.The final concentration of 1mg/ml, C of T1, T2, T3 line antibody
The final concentration of 1mg/ml of line rabbit igg, the discharge rate of C, T line is 1.0 μ l/cm.
Baking 24 hours it is placed in greatly in 37 DEG C of constant temperature ovens by sprayed.After having toasted, in the humidity room less than 40%
Between, it is cut into the test strips of 3.85mm width with cutting cutter, is loaded in plastic housing compacting, be then charged into one bag and be dried
Agent, sealing is placed in drying cupboard preservation, stand-by.The most as shown in Figure 1.
Embodiment 2
The detection of cTnI, CKMB, Myo time-resolved fluoroimmunoassay chromatograph test strip
(1) cTnI, CKMB, Myo, goat-anti rabbit time-resolved fluorescence microsphere are prepared
Time-resolved fluorescence microsphere (particle diameter is about 200nm) is dissolved in 100mM MES buffer (PH is 6.0), adds
Enter activator (NHS, 20mg/ml；EDC, 20mg/ml) activate 15 minutes；Wash, be centrifuged, time-resolved fluorescence microsphere is used
60mM borate buffer (PH is 8.5) redissolves, be subsequently added Troponin I monoclonal antibody 2 carry out reacting 2 hours (microsphere with
The mass ratio of antibody is at 10mg:0.5mg)；Add sealer (BSA, 100mg/ml) after having reacted to carry out closing 2 hours；Close
Later, wash, centrifugal, again with 60mM borate buffer (PH is 8.5) and sealer (BSA, 100mg/ml) redissolution, ultrasonic,
Making microsphere be dispersed in buffer, 2-8 DEG C keeps in Dark Place.
The preparation of fluorescent microsphere of Myo monoclonal antibody 2 labelling, the system of fluorescent microsphere of CKMB monoclonal antibody 2 labelling
The process that the preparation process of the fluorescent microsphere of standby, goat anti-rabbit antibody labelling prepares microsphere with cTnI is identical.
(2) drip and join
With PH be 8.0, the Tris buffer of 100mM is by coated glimmering to the cTnI in step (1), CKMB, Myo, goat-anti rabbit
Light microsphere is diluted.Wherein, the coated fluorescent microsphere of cTnI, CKMB, Myo dilutes 200～300 times, and goat-anti rabbit is coated glimmering
Light microsphere dilutes 2000～3000 times, is required fluorescence mixed liquor after mixing.
(3) drafting of standard curve
Dilute cTnI, CKMB, Myo hybrid standard product with calf serum, concentration be respectively as follows: cTnI:0,0.1,0.5,1.0,
5.0、25.0、50.0ng/ml；CKMB:0,0.5,2.0,10.0,25.0,50.0,100.0ng/ml；Myo:0,5,20,100,
200、400、800ng/ml.Take 10 μ l hybrid standard product and the mixing of 80 μ l fluorescence mixed liquors respectively, then take 80 μ l mixed liquor droppings
Carry on cTnI, CKMB, Myo time-resolved fluoroimmunoassay chromatograph test strip in embodiment 1, after reacting 15 minutes, by with
The immunochromatography readout instrument that cTnI, CKMB, Myo time-resolved fluoroimmunoassay chromatograph test strip is supporting, reads system detection respectively
Signal 1, system detection signal 2, system detection signal 3 signal value, the Concentration Testing of each hybrid standard product three times, concrete numerical value
As shown in table 1.
Table 1 standard substance detected value
From the data in table 1, it can be seen that the sensitivity of Myo is up to 5ng/ml, upper limit of detection is up to 800ng/ml；The sensitivity of cTnI
Up to 0.1ng/ml, upper limit of detection is up to 50ng/ml；The sensitivity of CKMB is up to 0.5ng/ml, and upper limit of detection is up to 100ng/
ml。
The combined detection performance of present invention cardiac muscle three is superior to colloidal gold chromatography, with micro-fluidic combined detection reagent phase
Closely, the advantage of time-resolved fluoroimmunoassay chromatography is embodied.
(4) ID card is made:
Respectively with standard substance Myo, cTnI, CKMB concentration value as abscissa, the T/C meansigma methods of each concentration is vertical coordinate, paints
Standard substance curve processed, the most as shown in Figure 2, Figure 3, Figure 4, burning ID card, and import in necessary instrument.
Embodiment 3
Precision detects
By the method for testing in embodiment 2, (Myo concentration is 100 to test the hybrid standard product of Myo, cTnI and CKMB respectively
With the concentration of 400ng/ml, cTnI be 1 and the concentration of 25ng/ml, CKMB is 10 and 50ng/ml), each standard substance duplicate detection
Ten times, concrete detection numerical value is as shown in table 2 below.
Table 2 precision test result table
As table 2 data understand, using three test strip of the present invention, the precision of Myo, cTnI, CKMB is respectively less than
10%, comply fully with the requirement less than 15% of the POCT product precision.
Embodiment 4
The correlation detection of clinical sample
By the method for testing in embodiment 2, choose facing of 40 example external chemoluminescence method detection Myo, cTnI, CKMB respectively
The bed sample detection range of standard substance (concentration of specimens be covered each by), examines with three link detection reagents of Shanghai general biology difficult to understand
Survey.Concrete testing result is as shown in table 3.
The general three joint inspection surveys difficult to understand of table 3 Shanghai detect data with chemoluminescence method
Respectively with Myo, cTnI, CKMB concentration of chemoluminescence method detection as abscissa, Shanghai general biological detection Myo difficult to understand,
CTnI, CKMB concentration is vertical coordinate, draws sample correlations curve.As shown in Fig. 5, Fig. 6, Fig. 7.Wherein, the dependency side of Myo
Journey is y=0.871x+8.437, R2=0.992；The relationship equation of cTnI is y=1.129x+2.018, R2=0.969；CKMB
Relationship equation be y=0.967x-0.887, R2=0.970.The relative coefficient R of three projects is all higher than 0.95, completely
Meet the requirement of clinical trial.
Claims (9)
1. fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and a preparation method of cTnI, CKMB, Myo simultaneously,
It is characterized in that: this reagent is made up of test strips and fluorescent liquid two parts；
Described test strips includes base plate (1), Fusion5 (2), nitrocellulose filter (3) and adsorptive pads (4)；
Described Fusion5 (2), nitrocellulose filter (3) and adsorptive pads (4) horizontal direction are linked in sequence and are fixed on base plate；
Described fluorescent liquid (9) comprise the fluorescent microsphere of Myo monoclonal antibody 2 labelling, cTnI monoclonal antibody 2 labelling fluorescence micro-
Ball, the fluorescent microsphere of CKMB monoclonal antibody 2 labelling, the fluorescent microsphere of goat anti-rabbit antibody labelling.
Fast quantification the most according to claim 1 detects the time-resolved fluoroimmunoassay chromatography of cTnI, CKMB, Myo simultaneously
Reagent, it is characterised in that: it is coated with Myo monoclonal antibody 1 (5), cTnI monoclonal antibody 1 on described nitrocellulose filter (3)
Or the matter that the detection line of polyclonal antibody 1 (6), CKMB polyclonal antibody 1 (7) capture molecule composition and rabbit igg antibody (8) are constituted
Control line.
Fast quantification the most according to claim 1 detects the time-resolved fluoroimmunoassay chromatography of cTnI, CKMB, Myo simultaneously
Reagent, it is characterised in that: described time-resolved fluorescence microsphere is selected from the polystyrene microsphere of modified.
Fast quantification the most according to claim 2 detects the time-resolved fluoroimmunoassay chromatography of cTnI, CKMB, Myo simultaneously
Reagent, it is characterised in that: described polystyrene microsphere, the one that functional group is carboxyl, hydroxyl or epoxy radicals, grain are modified in surface
Footpath is 100～500nm.
Fast quantification the most according to claim 1 detects the time-resolved fluoroimmunoassay chromatography of cTnI, CKMB, Myo simultaneously
Reagent, it is characterised in that: the internal chelate filling lanthanide series of described time-resolved fluorescence microsphere.
Fast quantification the most according to claim 4 detects the time-resolved fluoroimmunoassay chromatography of cTnI, CKMB, Myo simultaneously
Reagent, it is characterised in that: described lanthanide series be europium, samarium, one in dysprosium.
A kind of fast quantification the most according to claim 1 detects the time-resolved fluoroimmunoassay of cTnI, CKMB, Myo simultaneously
Chromatography reagent, it is characterised in that: described time-resolved fluorescence microsphere markers step is as follows:
(1) preparation of Troponin I monoclonal antibody 2 labelling time-resolved fluorescence microsphere and dilution:
Time-resolved fluorescence microsphere is dissolved in MES buffer, adds activator activation；It is subsequently added glycine so that micro-
Ball surface connects arm；Washing, centrifugal after, again add activator activation；Again wash, be centrifuged, time-resolved fluorescence is micro-
Ball borate buffer redissolves, and is subsequently added Troponin I monoclonal antibody 2 and reacts, borate buffer, microsphere and antibody
Mass ratio be 10mg:0.01mg～10mg:2.0mg in the range of；Add sealer after having reacted to close；After closing,
Washing, centrifugal, again with borate buffer and sealer redissolve, ultrasonic, make microsphere be dispersed in buffer, keep away for 2～8 DEG C
Light preserves；Selecting suitable buffer to dilute fluorescent microsphere, working concentration is at 0.5～50 μ g/ml, for Troponin I performance
Assessment；
(2) preparation of the fluorescent microsphere of Myo monoclonal antibody 2 labelling and dilution, the fluorescent microsphere of CKMB monoclonal antibody 2 labelling
Preparation and dilution, the preparation of fluorescent microsphere of goat anti-rabbit antibody labelling and dilution with (1) described.
8. the fast quantification prepared described in claim 1 detects the time-resolved fluoroimmunoassay of cTnI, CKMB, Myo simultaneously
The method of chromatography reagent, comprises the steps:
(1) preparation detection line and nature controlling line: nitrocellulose filter (3) is used for fixing coated antibody in Immunofluorescence test paper strip,
Also it is the place of immunoreactive generation simultaneously；Detection line (5) is to use citrate or sucrose dilute Myo monoclonal antibody 1
Release liquid dilution, line on described nitrocellulose filter (3)；Detection line (6) is by cTnI monoclonal antibody 1 or polyclonal antibody
1 uses citrate or sucrose diluted, lines on described nitrocellulose filter (3)；Detection line (7) is that CKMB is many
Clonal antibody 1 uses citrate buffer solution or sucrose solution dilution, in line and described nitrocellulose filter (3)；Nature controlling line (8)
It is that rabbit igg antibody is used citrate or sucrose diluted, in line and described nitrocellulose filter (3)；To pull
Nitrocellulose filter be placed in vacuum drying oven, at 37～45 DEG C, dry 24～48 hours；Detection line (5), (6), (7) position
In same position or diverse location, if diverse location, then detect line (5), (6), (7) are respectively positioned on nature controlling line (8) the same side, i.e.
The position that first liquid contact；
(2) assembling of test strips: adhered to Fusion5 (2), nitrocellulose filter successively by left-to-right at test strips base plate (1)
(3), absorbent paper (4)；The big plate assembled is cut into little bar, i.e. obtains described fast quantification and detect cTnI, CKMB, Myo simultaneously
Time-resolved fluoroimmunoassay chromatograph test strip.
Fast quantification the most according to claim 8 detects the time-resolved fluoroimmunoassay chromatography of cTnI, CKMB, Myo simultaneously
The preparation method of reagent, it is characterised in that: in described citrate or sucrose diluent, citrate or sucrose account for 5-20%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610223337.8A CN106153927A (en) | 2016-04-12 | 2016-04-12 | A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610223337.8A CN106153927A (en) | 2016-04-12 | 2016-04-12 | A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106153927A true CN106153927A (en) | 2016-11-23 |
Family
ID=57353626
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610223337.8A Pending CN106153927A (en) | 2016-04-12 | 2016-04-12 | A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106153927A (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106980019A (en) * | 2017-03-30 | 2017-07-25 | 王晓雨 | POCT quantitative detection systems |
| WO2018120855A1 (en) * | 2016-12-28 | 2018-07-05 | 广州瑞博奥生物科技有限公司 | Time-resolved fluorescent immunochromatographic test strip and kit for detecting myo, and preparation method therefor |
| CN108254563A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of cTnI and preparation method thereof |
| CN108254550A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of CK-MB and preparation method thereof |
| CN108318690A (en) * | 2018-03-13 | 2018-07-24 | 深圳市第二人民医院 | A kind of immunofluorescence chromatographic test paper and its preparation method and application |
| CN109632758A (en) * | 2019-01-25 | 2019-04-16 | 黄飚 | A kind of method of Quality Control in time-resolved fluoroimmunoassay |
| CN109725151A (en) * | 2018-12-28 | 2019-05-07 | 江苏众红生物工程创药研究院有限公司 | Human muscle hemoglobin Test paper card and its clinical application |
| CN110221066A (en) * | 2019-07-05 | 2019-09-10 | 吉林大学 | A kind of trichinosis fluorescence immune chromatography test strip and the preparation method and application thereof |
| CN110333212A (en) * | 2019-08-13 | 2019-10-15 | 江苏省苏微微生物研究有限公司 | A time-resolved fluorescence immunoassay analyzer with multi-channel detection function and its application |
| CN110702901A (en) * | 2019-10-10 | 2020-01-17 | 南京欧凯生物科技有限公司 | Fluorescence immunochromatography test paper for detecting cardiac troponin I |
| CN111308103A (en) * | 2020-03-18 | 2020-06-19 | 厦门稀土材料研究所 | Cardiopulmonary quintuple detection kit, rare earth nano-fluorescence detection card and detection method |
| CN111349168A (en) * | 2018-12-20 | 2020-06-30 | 东莞市朋志生物科技有限公司 | Anti-human CKMB antibody and application thereof |
| CN111521825A (en) * | 2020-05-18 | 2020-08-11 | 巴迪泰（广西）生物科技有限公司 | Cardiac troponin I detection kit and detection method |
| CN111537751A (en) * | 2020-05-18 | 2020-08-14 | 巴迪泰（广西）生物科技有限公司 | Myocardial troponin I, creatine kinase isoenzyme and myoglobin joint detection kit and detection method |
| CN111579800A (en) * | 2020-05-18 | 2020-08-25 | 巴迪泰（广西）生物科技有限公司 | Hypersensitive procalcitonin detection reagent and preparation method and use method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005041893A2 (en) * | 2003-10-31 | 2005-05-12 | Queststar Medical, Inc. | Detection of acute myocardial infarction biomarkers |
| CN103267855A (en) * | 2013-05-06 | 2013-08-28 | 南京凯基生物科技发展有限公司 | CTnI, Myo and CK-MB three-in-one colloidal gold test strip, kit thereof, and making methods of test strip and kit |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
| CN104198723A (en) * | 2014-08-11 | 2014-12-10 | 南京普朗医疗设备有限公司 | Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm |
-
2016
- 2016-04-12 CN CN201610223337.8A patent/CN106153927A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005041893A2 (en) * | 2003-10-31 | 2005-05-12 | Queststar Medical, Inc. | Detection of acute myocardial infarction biomarkers |
| CN103267855A (en) * | 2013-05-06 | 2013-08-28 | 南京凯基生物科技发展有限公司 | CTnI, Myo and CK-MB three-in-one colloidal gold test strip, kit thereof, and making methods of test strip and kit |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
| CN104198723A (en) * | 2014-08-11 | 2014-12-10 | 南京普朗医疗设备有限公司 | Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108254563B (en) * | 2016-12-28 | 2019-10-29 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, the kit and preparation method thereof of cTnI |
| WO2018120855A1 (en) * | 2016-12-28 | 2018-07-05 | 广州瑞博奥生物科技有限公司 | Time-resolved fluorescent immunochromatographic test strip and kit for detecting myo, and preparation method therefor |
| CN108254563A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of cTnI and preparation method thereof |
| CN108254562A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of MYO and preparation method thereof |
| CN108254550A (en) * | 2016-12-28 | 2018-07-06 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, kit of CK-MB and preparation method thereof |
| CN108254562B (en) * | 2016-12-28 | 2019-11-08 | 广州瑞博奥生物科技有限公司 | Detect time-resolved fluoroimmunoassay chromatograph test strip, the kit and preparation method thereof of MYO |
| CN106980019A (en) * | 2017-03-30 | 2017-07-25 | 王晓雨 | POCT quantitative detection systems |
| CN108318690A (en) * | 2018-03-13 | 2018-07-24 | 深圳市第二人民医院 | A kind of immunofluorescence chromatographic test paper and its preparation method and application |
| CN111349168A (en) * | 2018-12-20 | 2020-06-30 | 东莞市朋志生物科技有限公司 | Anti-human CKMB antibody and application thereof |
| CN109725151A (en) * | 2018-12-28 | 2019-05-07 | 江苏众红生物工程创药研究院有限公司 | Human muscle hemoglobin Test paper card and its clinical application |
| CN109725151B (en) * | 2018-12-28 | 2021-08-27 | 江苏众红生物工程创药研究院有限公司 | Human myoglobin detection test paper card and clinical application thereof |
| CN109632758A (en) * | 2019-01-25 | 2019-04-16 | 黄飚 | A kind of method of Quality Control in time-resolved fluoroimmunoassay |
| CN110221066A (en) * | 2019-07-05 | 2019-09-10 | 吉林大学 | A kind of trichinosis fluorescence immune chromatography test strip and the preparation method and application thereof |
| CN110333212A (en) * | 2019-08-13 | 2019-10-15 | 江苏省苏微微生物研究有限公司 | A time-resolved fluorescence immunoassay analyzer with multi-channel detection function and its application |
| CN110702901A (en) * | 2019-10-10 | 2020-01-17 | 南京欧凯生物科技有限公司 | Fluorescence immunochromatography test paper for detecting cardiac troponin I |
| CN111308103A (en) * | 2020-03-18 | 2020-06-19 | 厦门稀土材料研究所 | Cardiopulmonary quintuple detection kit, rare earth nano-fluorescence detection card and detection method |
| CN111308103B (en) * | 2020-03-18 | 2022-07-22 | 厦门稀土材料研究所 | Cardiopulmonary quintuple detection kit, rare earth nano-fluorescence detection card and detection method |
| CN111521825A (en) * | 2020-05-18 | 2020-08-11 | 巴迪泰（广西）生物科技有限公司 | Cardiac troponin I detection kit and detection method |
| CN111537751A (en) * | 2020-05-18 | 2020-08-14 | 巴迪泰（广西）生物科技有限公司 | Myocardial troponin I, creatine kinase isoenzyme and myoglobin joint detection kit and detection method |
| CN111579800A (en) * | 2020-05-18 | 2020-08-25 | 巴迪泰（广西）生物科技有限公司 | Hypersensitive procalcitonin detection reagent and preparation method and use method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106153927A (en) | A kind of fast quantification detects time-resolved fluoroimmunoassay chromatography reagent and the preparation method of cTnI, CKMB, Myo simultaneously | |
| CN106248927A (en) | The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK MB and preparation method | |
| CN104714015B (en) | Detection kit and detection method for heart-type fatty acid binding protein | |
| CN102323422B (en) | Immunochromatographic test strip for semi-quantitatively and simultaneously detecting cTnI and Myo and preparation method thereof | |
| CN108254550B (en) | Detect time-resolved fluoroimmunoassay chromatograph test strip, the kit and preparation method thereof of CK-MB | |
| CN106248958A (en) | The fluorescence immune chromatography reagent of a kind of detection by quantitative cTnI and preparation method | |
| CN105891508A (en) | TRF (time-resolved fluorescence) immunochromatography reagent for rapidly and quantitatively detecting H-FABP (heart fatty acid-binding protein) and preparation method | |
| CN102662055B (en) | Immune fluorescent test strip component for quickly quantitatively detecting troponin I, detection card component comprising immune fluorescent test strip component and preparation methods for immune fluorescent test strip component and detection card component | |
| CN102087214A (en) | Fluorescent quantitative detection instrument | |
| CN108333357A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits | |
| CN101377506A (en) | Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| CN106959372A (en) | Serum amyloid A protein and the two-in-one measure kit of C reactive proteins and preparation method | |
| CN207248894U (en) | Bladder chalone C time resolution detection card and kit | |
| CN106706926A (en) | Serum amyloid A testing kit and manufacturing method | |
| CN109142758A (en) | It is a kind of to detect the immuno-chromatographic test paper strip of glycosylated hemoglobin, kit and preparation method thereof | |
| CN102116770A (en) | Immunochromatography rapid kit and production method thereof | |
| CN109187981A (en) | A kind of quantum dot immune chromatograph test strip of quick detection beta-amyloid protein and application | |
| CN102135498B (en) | Semi-quantitative colloidal metal detection technology taking multi-capture property as characteristic and preparation method and use thereof | |
| CN106841631A (en) | Cardiac muscle troponin I/N ends Natriuretic Peptide/D dimer is three-in-one to determine kit and preparation method | |
| CN202216908U (en) | Fluorescent quantitative detector | |
| CN106771239A (en) | Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method | |
| CN110133258A (en) | A kind of time-resolved fluoroimmunoassay chromatography reagent strip of quick detection cyclosporin | |
| CN111983243A (en) | Amino-terminal brain natriuretic peptide precursor determination kit, preparation method and detection method | |
| CN107664700A (en) | Cardiac muscle troponin I and creatine kinase isozyme and myoglobins three-in-one detection reagent box and preparation method thereof | |
| CN208060389U (en) | A kind of multi objective time-resolved fluoroimmunoassay for myocardial infarction Quantitative detection chromatographs kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 201201 Shanghai City, Pudong New Area Ruiqinglu No. 526 Applicant after: Shanghai Aopu biomedical Co., Ltd Address before: 201201 Shanghai City, Pudong New Area Ruiqinglu No. 526 Applicant before: Shanghai Upper Bio-tech Pharma Co., Ltd. |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161123 |