CN106248927A - The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK MB and preparation method - Google Patents
The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK MB and preparation method Download PDFInfo
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Abstract
Present invention time-resolved fluoroimmunoassay chromatography reagent disclosing a kind of Quantitative detection CK MB and preparation method thereof, belongs to clinical diagnose field.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.Detection line and the nature controlling line of rabbit igg antibody composition of CK MB monoclonal antibody 1 it is coated with on described nitrocellulose filter.Fluorescent liquid comprises the fluorescent microsphere of CK MB monoclonal antibody 2 labelling, the fluorescent microsphere of goat anti-rabbit antibody labelling.The present invention utilizes time-resolved fluorescence microsphere to improve fluorescence intensity, reduces background signal, and the CK MB content in simultaneous quantitative detection whole blood, serum or blood plasma, sample only needs 10～20 microlitres.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, the time resolution being specifically related to a kind of Quantitative detection CK-MB is glimmering
Light immunochromatography reagent and preparation method.
Background technology
CK-MB is one of three isomers (another two is CK-BB and CK-MMOL/L) of creatine kinase (CK).CK is one
Plant the apoenzyme of muscle metabolism.CK-MB is made up of (MW=40000 is each) two subunits: subunit M represents 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 heart routine examination
A part, and the diagnosis of beneficially AMI.When there is AMI, CK-MB i.e. comes across in blood circulation and illustrates that myocardium is subject to
Arrive damage.CK-MB concentration raises in 4-8 hour after panic attacks, reaches peak value in 12-24 hour, then little 48
Normal condition is dropped to time after.CK-MB be first after panic attacks in 48 hours peri-operation period myocardial infarction select
Label.CK-MB concentration is additionally operable to evaluate the degree of AMI and blocking again later.From diagnosis susceptiveness, specificity and effect
From the point of view of rate, CK-MB will be better than the CK isozyme according to electrophoresis.The mensuration of CK-MB also contributes under thrombolytic therapy many to cardiac muscle
The effect of secondary infusion liquid and the non-invasive evaluation that carries out.The rising of CK-MB level is also relevant with Skeletal muscle injury simultaneously, but also
Unlike 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 one
Plant the detection based on film using time-resolved fluorescence, for detecting existence and the content of analyte in test sample；Patent (CN
202149903U) invention c reactive protein time-resolved fluoroimmunoassay chromatography detection by quantitative；Patent (CN 202166649U) is invented
Time-resolved fluoroimmunoassay quantitative detecting test strip for neopterin；A kind of time resolution of patent (CN 102879559A) invention
Fluorescence immune chromatography real-time and quantification detectable cassette method；A kind of AFP and Free-β-HCG of patent (CN 103760368A) invention
The double target immue quantitative detection reagent box of time-resolved fluorescence；Patent (CN 204514928U) invention is a kind of based on time-resolved fluorescence
Immunochromatographyassay assay pepsinogen I and the test strips of II and the test strips of its application.
A kind of cardiovascular disease diagnosis of patent (CN 2783324Y) invention and prediction multi-index protein chip detection reagent
Box, for the joint-detection of CRP, Myo, cTnI, cTnT, NT-proBNP, CKMB, CK-MB, GPBB.
At present, the method for CK-MB detection mainly has enzyme linked immunosorbent assay, latex enhancing immune turbidimetry, gold colloidal to exempt from
Epidemic disease chromatography, fluorescence immune chromatography method etc..Enzyme linked immunosorbent assay operation is complicated, detection time length, detection range are narrow；Latex increases
Strong immunoturbidimetry needs to combine large-scale instrument, and cost is high；Colloidal gold immunity chromatography qualitatively or quantitatively detects, and sensitivity is low, line
Property narrow range, it is impossible to meet quantitatively, the requirement of accurately detection；Fluorescence immune chromatography method great majority use fluorescein or other materials
As luminous source, although have the higher range of linearity, 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 sample pad of family uses artificial treatment to cross glass fibre as conjugate release pad, adds imprecision equally.
In sum, when CK-MB detection by quantitative, in the urgent need to one, sample size is few, precision is high, detection background signal
Low, highly sensitive, range of linearity width, sample pad are not required to process the time-resolved fluoroimmunoassay chromatography examination that just can detect whole blood sample
Agent.
Summary of the invention
It is an object of the invention to provide the time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK-MB, should
Reagent can complete the detection of CK-MB within the shorter detection time, and the detection time is short, and detection sensitivity is high.
Present invention also offers the preparation method of the time-resolved fluoroimmunoassay chromatography reagent of Quantitative detection CK-MB.
The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK-MB is by test strips and fluorescent liquid two parts
Composition.
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.
Detection line and the rabbit igg antibody (6) of CK-MB monoclonal antibody 1 (5) it is coated with on described nitrocellulose filter (3)
The nature controlling line constituted.
Described fluorescent liquid (7) comprises the time-resolved fluorescence microsphere of CK-MB monoclonal antibody 2 labelling, goat anti-rabbit antibody
The time-resolved fluorescence microsphere of 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.
The compound method of described fluorescent liquid is as follows:
(1) preparation of CK-MB monoclonal antibody 2 labelling time-resolved fluorescence microsphere and dilution:
Being dissolved in by time-resolved fluorescence microsphere in 100mmol/L MES buffer, every 1mg time-resolved fluorescence microsphere is corresponding
100-300 μ L MES buffer, adds the final concentration of the final concentration of 0.05%-0.1%, NHS of activator NHS and EDC, EDC
For 0.1%-0.2%；Being subsequently added glycine so that microsphere surface connects arm, every 1mg time-resolved fluorescence microsphere is corresponding
10-100 μ L 50mmol/L glycine；After washing, 10000-15000rpm are centrifuged, again add activator activation, EDC's
The final concentration of 0.1%-0.2% of final concentration of 0.05%-0.1%, NHS；Washing again, 10000-15000rpm are centrifuged, will
Time-resolved fluorescence microsphere borate buffer redissolves, and is subsequently added CK-MB monoclonal antibody 2 and reacts, microsphere and antibody
Mass ratio be 10mg:0.01mg～2.0mg；Add sealer after having reacted to close, the final concentration of 2-of sealer
3%；Closing 2h, washing, 10000-15000rpm are centrifuged, again by the 60-100mmol/L borate buffer that pH is 7-9 and envelope
Close agent redissolve, ultrasonic, final concentration of 2-3%, the CK-MB antibody labeling time-resolved fluorescence microsphere final concentration of sealer is 0.5
～50 μ g/ml, make microsphere be dispersed in buffer, 2-8 DEG C keeps in Dark Place；Fluorescent particle activation process is added amino second
Acid is as arm so that the distance between antibody and microsphere increases, and effectively reduces space steric effect, improves detection system
System sensitivity.
(2) preparation and the dilution same (1) of the fluorescent microsphere of goat anti-rabbit antibody labelling are described；
(3) drip and join:
With pH be 8.0, the Tris buffer of 200mmol/L is by the CK-MB of isodose, goat-anti rabbit bag in step (1) and (2)
The fluorescent microsphere of quilt is diluted respectively, and wherein, the coated fluorescent microsphere of CK-MB dilutes 200～300 times, and goat-anti rabbit is coated
Fluorescent microsphere dilutes 2000～3000 times, is required fluorescence mixed liquor after mixing.
Preferably, using the time-resolved fluorescence microsphere that europium particle chelate is filled, particle diameter is: 100～500nm；
Preferably, microsphere with the ratio of being coated of antibody is: 10mg:0.01mg～2.0mg；
Described washing uses 0.9%NaCl, 0.05%Tween 20, the washing mixed remaining as the PB of 5mmol/L
Agent, its pH is 7.8, and after adding detergent, microsphere final concentration is 2%.
The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK-MB and preparation method, including walking as follows
Rapid:
(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 CK-MB monoclonal antibody 1 is used 50mmol/L Fructus Citri Limoniae
Hydrochlorate or sucrose diluted, line on described nitrocellulose filter (3)；Nature controlling line (6) is to be used by rabbit igg antibody
50mmol/L citrate or sucrose diluted, in line and described nitric acid fibre nature controlling line (6).The cellulose nitrate that will pull
Element film is placed in vacuum drying oven, at 37～45 DEG C, dries 24～48 hours.Detection line (5) is positioned at nature controlling line (6) left side, i.e.
The position that first liquid contact；
(2) assembling of test strips: test strips base plate (1) is adhered to Fusion5 (2), nitrocellulose filter successively by left-to-right
(3), absorbent paper (4).The big plate assembled is cut into little bar, and the time resolution i.e. obtaining described Quantitative detection CK-MB is glimmering
Light immuno-chromatographic test paper strip.
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；
The detection method of the time-resolved fluoroimmunoassay chromatography reagent of above Quantitative detection CK-MB, its step includes:
(1) drafting of standard curve: take the CK-MB standard substance of 10～20 μ l and the fluorescence mixed liquor (CK-of 80～500 μ l
MB, goat-anti rabbit fluorescent liquid) mixing, take mixed liquor 70～100 μ l and be added drop-wise on the Fusion5 of test strips, the response time 15～20
Minute, read system detection signal by the time resolution immunochromatography quantitative analysis instrument supporting with test strips, then with standard
Each concentration of product is abscissa, and the meansigma methods of each concentration systems detection signal is vertical coordinate, and standard substance curve is prepared in matching；
(2) detection of testing sample, takes the testing sample of 10～20 μ l and the fluorescence mixed liquor (CK-of 80～500 μ l respectively
MB, goat-anti rabbit fluorescent liquid) mixing, take mixed liquor 70～100 μ l and be added drop-wise on the Fusion5 of test strips, the response time 15～20
Minute, the concentration value of the CK-MB in testing sample is i.e. can get by the value of system detection signal.
In described (1), the concentration of CK-MB standard substance is respectively as follows: 0,0.5,2.0,10.0,25.0,50.0,100.0ng/
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 structural representation (1 of the time-resolved fluoroimmunoassay chromatography reagent of Quantitative detection CK-MB of the present invention
For base plate, 2 be Fusion5,3 for nitrocellulose filter, 4 for adsorptive pads, 5 for CK-MB detection line, 6 be rabbit igg nature controlling line):
Fig. 2 is the standard substance curve of the present invention (embodiment 1) CK-MB；
Fig. 3 is the standard substance curve of the present invention (embodiment 2) CK-MB；
Fig. 4 is the standard substance curve of the present invention (embodiment 3) CK-MB；
Fig. 5 is that (abscissa is sent out for chemical with the correlation curve of chemoluminescence method CK-MB in Austria of the present invention general (embodiment 1)
Light method detectable concentration (ng/ml), vertical coordinate is detectable concentration of the present invention (ng/ml))；
Fig. 6 is that (abscissa is sent out for chemical with the correlation curve of chemoluminescence method CK-MB in Austria of the present invention general (embodiment 2)
Light method detectable concentration (ng/ml), vertical coordinate is detectable concentration of the present invention (ng/ml))；
Fig. 7 is that (abscissa is sent out for chemical with the correlation curve of chemoluminescence method CK-MB in Austria of the present invention general (embodiment 3)
Light method detectable concentration (ng/ml), vertical coordinate is detectable concentration of the present invention (ng/ml)).
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described:
In the present invention, CK-MB antibody and the supplier of fluorescent microsphere and article No. are as follows
| Antibody Designation | Catalog number (Cat.No.) | Producer |
| CKMB-03 | Nothing | Shenzhen City Fapon Biotech Co., Ltd |
| CKMB-04 | Nothing | Shenzhen City Fapon Biotech Co., Ltd |
| 200nm fluorescent microsphere | FC02F | Bangs laboratories |
Embodiment 1
1, the preparation of CK-MB time-resolved fluoroimmunoassay chromatograph test strip
(1) detect the preparation of line (T) solution: with the citrate solution of 50mmol/L dilution CK-MB monoclonal antibody 1 to
0.5mg/ml；
(2) preparation of nature controlling line (C) solution: with the citrate solution dilution rabbit igg antibody of 50mmol/L to 0.5mg/
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 T (CK-MB captured line (5)), draws C (rabbit igg) line, between T, C in the one end near absorbent paper (4)
Away from for 5mm.
At T line, draw T line, at C line, draw C line.The final concentration of final concentration of 0.5mg/ml, the C line rabbit igg of T line antibody
For 0.5mg/ml, the discharge rate of C, T line is 0.5 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.
2, the detection of CK-MB time-resolved fluoroimmunoassay chromatograph test strip
(1) CK-MB, goat-anti rabbit time-resolved fluorescence microsphere are prepared
100mg time-resolved fluorescence microsphere (particle diameter is about 200nm) is dissolved in the 100mmol/L that 10ml pH is 6.0
In MES buffer, add 5mgNHS and EDC and activate 15 minutes；Washing, 15000rpm are centrifuged, and are subsequently added 5ml 50mmol/L
Glycine so that microsphere surface connects arm, after washing, 15000rpm are centrifuged, adds activator activation, the end of EDC again
Concentration is 0.05%, final concentration of the 0.1% of NHS；Washing again, 15000rpm are centrifuged, and adding 50ml pH is 8.5
Time-resolved fluorescence microsphere is completely dissolved by 60mmol/L borate buffer, is subsequently added 5mg CK-MB monoclonal antibody 2 and carries out
React 2 hours；Add BSA after having reacted to carry out closing 2 hours, final concentration of the 2% of BSA；After closing, washing,
15000rpm is centrifuged, again with the 60mmol/L borate buffer that pH is 8.5 and BSA redissolve, ultrasonic, make microsphere be dispersed in
In buffer, final concentration of the 2% of BSA, CK-MB antibody labeling time-resolved fluorescence microsphere final concentration is in 5 μ g/ml, 4 DEG C of lucifuges
Preserve.
The process that the preparation process of the fluorescent microsphere of goat anti-rabbit antibody labelling prepares microsphere with CK-MB is identical.
(2) drip and join
With pH be 8.0, the Tris buffer of 200mmol/L is by micro-to the CK-MB in step (1), the coated fluorescence of goat-anti rabbit
The each 0.1ml of ball is diluted.Wherein, the coated fluorescent microsphere of CK-MB dilutes 200 times, and the coated fluorescent microsphere of goat-anti rabbit dilutes
2000 times, after mixing, it is required fluorescence mixed liquor.
(3) drafting of standard curve
Diluting CK-MB standard substance with calf serum, concentration is: 0,0.5,2.0,10.0,25.0,50.0,100.0ng/ml.
Take fluorescence mixed liquor mixing in 10 μ l hybrid standard product and 80 μ l step (2) respectively, then take 80 μ l mixed liquors droppings in step 1
CK-MB time-resolved fluoroimmunoassay chromatograph test strip on, after reacting 15 minutes, by with CK-MB time-resolved fluoroimmunoassay
The immunochromatography readout instrument that chromatograph test strip is supporting, reading system detection signal, the Concentration Testing of each hybrid standard product three times,
Concrete numerical value is 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 CK-MB is up to 0.5ng/ml, upper limit of detection is up to 100ng/ml.The present invention
CK-MB detection performance is superior to colloidal gold chromatography, with chemoluminescence method reagent similar nature, embodies time-resolved fluorescence and exempts from
The advantage of epidemic disease chromatography.
(4) ID card is made
With standard substance CK-MB concentration value as abscissa, the system detection signal averaging of each concentration is vertical coordinate, draws mark
Quasi-product curve, concrete as in figure 2 it is shown, burning ID card, and import in necessary instrument.
3, precision detection
With the method for testing in step 2, the standard substance (concentration is 10 and 50ng/ml) of test CK-MB, each standard substance weight
Rechecking and survey 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 the CK-MB test strip of the present invention, the error of CK-MB is less than 10%, accords with completely
Close the requirement less than 15% of the POCT product error.
4, the correlation detection of clinical sample
By the method for testing in step 2, (sample is dense to choose the clinical sample of 40 example external chemoluminescence methods detection CK-MB
The detection range of degree coverage criteria product), use the CK-MB reagent of the present invention to detect.CK-MB with chemoluminescence method detection
Concentration is abscissa, detects CK-MB concentration as vertical coordinate with the present invention, draws sample correlations curve, such as Fig. 5.Wherein, CK-
The relative coefficient R of MB is all higher than 0.95, complies fully with the requirement of clinical trial.
Embodiment 2
1, the preparation of CK-MB time-resolved fluoroimmunoassay chromatograph test strip
(1) detect the preparation of line (T) solution: with the citrate solution of 50mmol/L dilution CK-MB monoclonal antibody 1 to
1mg/ml；
(2) preparation of nature controlling line (C) solution: with the citrate solution dilution rabbit igg antibody of 50mmol/L to 1mg/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 T (CK-MB captured line (5)), draws C (rabbit igg) line, between T, C in the one end near absorbent paper (4)
Away from for 5mm.
At T line, draw T line, at C line, draw C line.Final concentration of 1mg/ml, the C line rabbit igg of T line antibody final concentration of
1mg/ml, C, the discharge rate of T line are 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.
2, the detection of CK-MB time-resolved fluoroimmunoassay chromatograph test strip
(1) CK-MB, goat-anti rabbit time-resolved fluorescence microsphere are prepared
100mg time-resolved fluorescence microsphere (particle diameter is about 200nm) is dissolved in the 100mmol/L that 20ml pH is 6.0
In MES buffer, add 10mgNHS and EDC and activate 15 minutes；Washing, 15000rpm are centrifuged, and are subsequently added 5ml 50mmol/L
Glycine so that microsphere surface connects arm, after washing, 15000rpm are centrifuged, adds activator activation, the end of EDC again
Concentration is 0.1%, final concentration of the 0.1% of NHS；Washing again, 15000rpm are centrifuged, and adding 50ml pH is 8.5
Time-resolved fluorescence microsphere is completely dissolved by 60mmol/L borate buffer, is subsequently added 1mg CK-MB monoclonal antibody 2 and carries out
React 2 hours；Add BSA after having reacted to carry out closing 2 hours, final concentration of the 3% of BSA；After closing, washing,
15000rpm is centrifuged, again with the 60mmol/L borate buffer that pH is 8.5 and BSA redissolve, ultrasonic, make microsphere be dispersed in
In buffer, final concentration of the 3% of BSA, CK-MB antibody labeling time-resolved fluorescence microsphere final concentration is in 5 μ g/ml, 4 DEG C of lucifuges
Preserve.
The process that the preparation process of the fluorescent microsphere of goat anti-rabbit antibody labelling prepares microsphere with CK-MB is identical.
(2) drip and join
With pH be 8.0, the Tris buffer of 200mmol/L is by micro-to the CK-MB in step (1), the coated fluorescence of goat-anti rabbit
The each 0.1ml of ball is diluted.Wherein, the coated fluorescent microsphere of CK-MB dilutes 250 times, and the coated fluorescent microsphere of goat-anti rabbit dilutes
2500 times, after mixing, it is required fluorescence mixed liquor.
(3) drafting of standard curve
Diluting CK-MB standard substance with calf serum, concentration is: 0,0.5,2.0,10.0,25.0,50.0,100.0ng/ml.
Take fluorescence mixed liquor mixing in 10 μ l hybrid standard product and 80 μ l step (2) respectively, then take 80 μ l mixed liquors droppings in step 1
CK-MB time-resolved fluoroimmunoassay chromatograph test strip on, after reacting 15 minutes, by with CK-MB time-resolved fluoroimmunoassay
The immunochromatography readout instrument that chromatograph test strip is supporting, reading system detection signal, the Concentration Testing of each hybrid standard product three times,
Concrete numerical value is as shown in table 3.
Table 3 standard substance detected value
From the data in table 3, it can be seen that the sensitivity of CK-MB is up to 0.5ng/ml, upper limit of detection is up to 100ng/ml.The present invention
CK-MB detection performance is superior to colloidal gold chromatography, with chemoluminescence method reagent similar nature, embodies time-resolved fluorescence and exempts from
The advantage of epidemic disease chromatography.
(4) ID card is made
With standard substance CK-MB concentration value as abscissa, the system detection signal averaging of each concentration is vertical coordinate, draws mark
Quasi-product curve, concrete as it is shown on figure 3, burning ID card, and import in necessary instrument.
3, precision detection
With the method for testing in step 2, the standard substance (concentration is 10 and 50ng/ml) of test CK-MB, each standard substance weight
Rechecking and survey ten times, concrete detection numerical value is as shown in table 4 below.
Table 4 precision test result table
As table 4 data understand, using the CK-MB test strip of the present invention, the error of CK-MB is less than 10%, accords with completely
Close the requirement less than 15% of the POCT product error.
4, the correlation detection of clinical sample
By the method for testing in step 2, (sample is dense to choose the clinical sample of 40 example external chemoluminescence methods detection CK-MB
The detection range of degree coverage criteria product), use the CK-MB reagent of the present invention to detect.CK-MB with chemoluminescence method detection
Concentration is abscissa, detects CK-MB concentration as vertical coordinate with the present invention, draws sample correlations curve, such as Fig. 6.Wherein, CK-
The relative coefficient R of MB is all higher than 0.95, complies fully with the requirement of clinical trial.
Embodiment 3
1, the preparation of CK-MB time-resolved fluoroimmunoassay chromatograph test strip
(1) detect the preparation of line (T) solution: with the citrate solution of 50mmol/L dilution CK-MB monoclonal antibody 1 to
2mg/ml；
(2) preparation of nature controlling line (C) solution: with the citrate solution dilution rabbit igg antibody of 50mmol/L to 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 T (CK-MB captured line (5)), draws C (rabbit igg) line, between T, C in the one end near absorbent paper (4)
Away from for 5mm.
At T line, draw T line, at C line, draw C line.Final concentration of 2mg/ml, the C line rabbit igg of T line antibody final concentration of
2mg/ml, C, the discharge rate of T line are 2.0 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.
2, the detection of CK-MB time-resolved fluoroimmunoassay chromatograph test strip
(1) CK-MB, goat-anti rabbit time-resolved fluorescence microsphere are prepared
100mg time-resolved fluorescence microsphere (particle diameter is about 200nm) is dissolved in the 100mmol/L that 30ml pH is 6.0
In MES buffer, add 10mgNHS and EDC and activate 15 minutes；Washing, 15000rpm are centrifuged, and are subsequently added 10ml 50mmol/
L glycine so that microsphere surface connects arm, after washing, 15000rpm are centrifuged, adds activator activation, the end of EDC again
Concentration is 0.1%, final concentration of the 0.2% of NHS；Washing again, 15000rpm are centrifuged, and adding 50ml pH is 8.5
Time-resolved fluorescence microsphere is completely dissolved by 60mmol/L borate buffer, is subsequently added 5mg CK-MB monoclonal antibody 2 and carries out
React 2 hours；Add BSA after having reacted to carry out closing 2 hours, final concentration of the 2% of BSA；After closing, washing,
15000rpm is centrifuged, again with the 60mmol/L borate buffer that pH is 8.5 and BSA redissolve, ultrasonic, make microsphere be dispersed in
In buffer, final concentration of the 2% of BSA, CK-MB antibody labeling time-resolved fluorescence microsphere final concentration is in 5 μ g/ml, 4 DEG C of lucifuges
Preserve.
The process that the preparation process of the fluorescent microsphere of goat anti-rabbit antibody labelling prepares microsphere with CK-MB is identical.
(2) drip and join
With pH be 8.0, the Tris buffer of 200mmol/L is by micro-to the CK-MB in step (1), the coated fluorescence of goat-anti rabbit
The each 0.1ml of ball is diluted.Wherein, the coated fluorescent microsphere of CK-MB dilutes 300 times, and the coated fluorescent microsphere of goat-anti rabbit dilutes
3000 times, after mixing, it is required fluorescence mixed liquor.
(3) drafting of standard curve
Diluting CK-MB standard substance with calf serum, concentration is: 0,0.5,2.0,10.0,25.0,50.0,100.0ng/ml.
Take fluorescence mixed liquor mixing in 10 μ l hybrid standard product and 80 μ l step (2) respectively, then take 80 μ l mixed liquors droppings in step 1
CK-MB time-resolved fluoroimmunoassay chromatograph test strip on, after reacting 15 minutes, by with CK-MB time-resolved fluoroimmunoassay
The immunochromatography readout instrument that chromatograph test strip is supporting, reading system detection signal, the Concentration Testing of each hybrid standard product three times,
Concrete numerical value is as shown in table 5.
Table 5 standard substance detected value
From the data in table 5, it can be seen that the sensitivity of CK-MB is up to 0.5ng/ml, upper limit of detection is up to 100ng/ml.The present invention
CK-MB detection performance is superior to colloidal gold chromatography, with chemoluminescence method reagent similar nature, embodies time-resolved fluorescence and exempts from
The advantage of epidemic disease chromatography.
(4) ID card is made
With standard substance CK-MB concentration value as abscissa, the system detection signal averaging of each concentration is vertical coordinate, draws mark
Quasi-product curve, the most as shown in Figure 4, burning ID card, and import in necessary instrument.
3, precision detection
With the method for testing in step 2, the standard substance (concentration is 10 and 50ng/ml) of test CK-MB, each standard substance weight
Rechecking and survey ten times, concrete detection numerical value is as shown in table 6 below.
Table 6 precision test result table
As table 6 data understand, using the CK-MB test strip of the present invention, the error of CK-MB is less than 10%, accords with completely
Close the requirement less than 15% of the POCT product error.
4, the correlation detection of clinical sample
By the method for testing in step 2, (sample is dense to choose the clinical sample of 40 example external chemoluminescence methods detection CK-MB
The detection range of degree coverage criteria product), use the CK-MB reagent of the present invention to detect.CK-MB with chemoluminescence method detection
Concentration is abscissa, with CK-MB concentration of the present invention as vertical coordinate, draws sample correlations curve, such as Fig. 7.Wherein, CK-MB
Relative coefficient R is all higher than 0.95, complies fully with the requirement of clinical trial.
Claims (8)
1. the time-resolved fluoroimmunoassay chromatography reagent of a Quantitative detection CK-MB, it is characterised in that: this reagent is by reagent paper
Bar and fluorescent liquid two parts composition；Described test strips includes base plate (1), Fusion5 (2), nitrocellulose filter (3) and water suction
Pad (4)；
Described Fusion5 (2), nitrocellulose filter (3) and adsorptive pads (4) horizontal direction are linked in sequence and are fixed on base plate；Institute
State and be coated with the detection line of CK-MB monoclonal antibody 1 (5) on nitrocellulose filter (3) and Quality Control that rabbit igg antibody (6) is constituted
Line；
Described fluorescent liquid (7) comprises the time-resolved fluorescence microsphere of CK-MB monoclonal antibody 2 labelling, goat anti-rabbit antibody labelling
Time-resolved fluorescence microsphere.
The time-resolved fluoroimmunoassay chromatography reagent of Quantitative detection CK-MB the most according to claim 1, its feature exists
In: described time-resolved fluorescence microsphere is selected from the polystyrene microsphere of modified.
The time-resolved fluoroimmunoassay chromatography reagent of Quantitative detection CK-MB the most according to claim 2, its feature exists
In described polystyrene microsphere, the one that functional group is carboxyl, hydroxyl or epoxy radicals is modified on surface, particle diameter be 100～
500nm。
The time-resolved fluoroimmunoassay chromatography reagent of Quantitative detection CK-MB the most according to claim 1, its feature exists
In: the internal chelate filling lanthanide series of described time-resolved fluorescence microsphere.
The time-resolved fluoroimmunoassay chromatography reagent of Quantitative detection CK-MB the most according to claim 4, its feature exists
In: described lanthanide series be europium, samarium, one in dysprosium.
The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK-MB the most according to claim 1, it is special
Levying and be, the preparation process of described fluorescent liquid is as follows:
(1) preparation of CK-MB monoclonal antibody 2 labelling time-resolved fluorescence microsphere and dilution:
Time-resolved fluorescence microsphere is dissolved in 100mmol/L MES buffer, every 1mg time-resolved fluorescence microsphere correspondence 100-
300 μ L MES buffer, the final concentration of 0.05%-0.1%'s, NHS of addition activator NHS and EDC, EDC is final concentration of
0.1%-0.2%；Being subsequently added glycine so that microsphere surface connects arm, every 1mg time-resolved fluorescence microsphere is corresponding
10-100 μ L 50mmol/L glycine；After washing, 10000-15000rpm are centrifuged, again add activator activation, EDC's
The final concentration of 0.1%-0.2% of final concentration of 0.05%-0.1%, NHS；Washing again, 10000-15000rpm are centrifuged, will
Time-resolved fluorescence microsphere borate buffer redissolves, and is subsequently added CK-MB monoclonal antibody 2 and reacts, microsphere and antibody
Mass ratio be 10mg:0.01mg～2.0mg；Add sealer after having reacted to close, the final concentration of 2-of sealer
3%；Closing 2h, washing, 10000-15000rpm are centrifuged, again by the 60-100mmol/L borate buffer that pH is 7-9 and envelope
Close agent redissolve, ultrasonic, final concentration of 2-3%, the CK-MB antibody labeling time-resolved fluorescence microsphere final concentration of sealer is 0.5
～50 μ g/ml, make microsphere be dispersed in buffer, 2-8 DEG C keeps in Dark Place；
(2) preparation and the dilution same (1) of the fluorescent microsphere of goat anti-rabbit antibody labelling are described；
(3) drip and join:
With pH be 8.0, the Tris buffer of 200mmol/L is by coated to the CK-MB of isodose in step (1) and (2), goat-anti rabbit
Fluorescent microsphere is diluted respectively, and wherein, the coated fluorescent microsphere of CK-MB dilutes 200～300 times, the coated fluorescence of goat-anti rabbit
Microsphere dilutes 2000～3000 times, is required fluorescence mixed liquor after mixing.
The time-resolved fluoroimmunoassay chromatography reagent of a kind of Quantitative detection CK-MB the most according to claim 6.It is special
Levy and be: described washing uses 0.9%NaCl, 0.05%Tween 20, the washing mixed remaining as the PB of 5mmol/L
Agent, its pH is 7.8, and after adding detergent, microsphere final concentration is 2%.
8. a preparation method for the time-resolved fluoroimmunoassay chromatography reagent of Quantitative detection CK-MB, 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 that CK-MB monoclonal antibody 1 is used 50mmol/L citric acid
Salt or sucrose diluted, line on described nitrocellulose filter (3)；Nature controlling line (6) is to be used by rabbit igg antibody
50mmol/L citrate or sucrose diluted, line and described nature controlling line (6), be placed in the nitrocellulose filter pulled
Vacuum drying oven, at 37～45 DEG C, dries 24～48 hours, and detection line (5) is positioned at nature controlling line (6) left side, and i.e. liquid is first
The position of contact；
(2) assembling of test strips: test strips base plate (1) by left-to-right adhere to successively Fusion5 (2), nitrocellulose filter (3),
Absorbent paper (4), is cut into little bar by the big plate assembled, and the time-resolved fluorescence i.e. obtaining described Quantitative detection CK-MB is exempted from
Epidemic disease chromatograph test strip.
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