CN107907514A

CN107907514A - CTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card, kit and application thereof

Info

Publication number: CN107907514A
Application number: CN201711057387.4A
Authority: CN
Inventors: 丁晓辉
Original assignee: SHANGHAI CHEMTRON BIOTECH CO Ltd
Current assignee: SHANGHAI CHEMTRON BIOTECH CO Ltd
Priority date: 2017-11-01
Filing date: 2017-11-01
Publication date: 2018-04-13

Abstract

The present invention relates to fluorescence immune chromatography field, more particularly to a kind of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card, including cTnI near-infrared fluorescent detection reagent bar arranged in parallel, myoglobins near-infrared fluorescent detection reagent bar and creatine kinase isozyme near-infrared fluorescent detection reagent bar, each reagent strip includes the sample application pad being arranged in order, glass fibre element film, nitrocellulose filter and water suction gasket, two p-wires and a control line are equipped with the nitrocellulose filter, the amount of detection object can be determined using the ratio or the ratio of p-wire and control line of two p-wires, error can be effectively reduced, improve accuracy and the precision of testing result.

Description

CTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection examination Agent card, kit and application thereof

Technical field

The present invention relates to fluorescence immune chromatography field, and in particular to the cTnI/same work of myoglobins/creatine kinase Enzyme near-infrared fluorescent detection reagent card, kit and application thereof.

Background technology

The raising of cTnI, three myoglobins, creatine kinase isozyme indexs is diagnosing myocardial infarction disease Important indicator, clinic can be using three indexs above as the foundation of myocardial infarction quick diagnosis.Therefore, cTnI/flesh is red The detection of albumen/creatine kinase isozyme is of great significance.

Immune diagnostic reagent of the prior art, either qualitative or quantitative reagent, is all based on immune response Principle, i.e., occurred to specifically bind and produce antigen-antibody complex, use tracer-labelling under certain condition with antigen and antibody Remember antigen or antibody, carry out the final detection and analysis realized to reaction product.

In the prior art, immune diagnostic reagent card, sets a p-wire, is determined according to the color of p-wire in sample Whether target antigen is contained.Judged by the color of a p-wire, easily influenced be subject to environmental light brightness and color, And the influence of family subjective judgement is also benefited from, produces large error so that testing result, particularly quantitatively testing result accuracy It is relatively low, bring obstacle for the diagnosis of disease.

The content of the invention

In view of the foregoing deficiencies of prior art, it is an object of the invention to provide a kind of cTnI/flesh red eggs In vain/creatine kinase isozyme near-infrared fluorescent detection reagent card, kit and application thereof, to reduce detection error, improve detection As a result accuracy.

In a first aspect, the present invention provides a kind of cTnI/myoglobins/creatine kinase isozyme near-infrared is glimmering Light detection reagent card, including cTnI near-infrared fluorescent detection reagent bar arranged in parallel, myoglobins near-infrared fluorescent Detection reagent bar and creatine kinase isozyme near-infrared fluorescent detection reagent bar, the cTnI near-infrared fluorescent detection Reagent strip includes sample application pad, glass fibre element film, nitrocellulose filter and the water suction gasket being arranged in order, wherein, the glass Contain the anti-cTnI antibody of fluorescence probe-the first and fluorescence probe-test antigen on glass cellulose membrane, the nitric acid is fine The first p-wire, the second p-wire and control line are disposed with the plain film of dimension, first p-wire has been coated with the second anti-heart Troponin I antibody, second p-wire have been coated with cTnI antigen, and it is anti-that the control line has been coated with the test Former antibody；The myoglobins near-infrared fluorescent detection reagent bar include be arranged in order sample application pad, glass fibre element film, Nitrocellulose filter and water suction gasket, wherein, the anti-myoglobins antibody of fluorescence probe-the first is contained on the glass fibre element film With fluorescence probe-test antigen, the first p-wire, the second p-wire and control are disposed with the nitrocellulose filter Line, first p-wire have been coated with the second anti-myoglobins antibody, and second p-wire has been coated with myoglobins antigen, institute State the antibody that control line has been coated with the test antigen；Creatine kinase isozyme near-infrared fluorescent detection reagent bar includes arranging successively Sample application pad, glass fibre element film, nitrocellulose filter and the water suction gasket of row, wherein, contain on the glass fibre element film The anti-creatine kinase isozyme antibody of fluorescence probe-the first and with fluorescence probe-test antigen, on the nitrocellulose filter successively The first p-wire, the second p-wire and control line are provided with, first p-wire has been coated with the second anti-creatine kinase isozyme Antibody, second p-wire have been coated with creatine kinase isozyme antigen, and the control line has been coated with the anti-of the test antigen Body.

In one embodiment of the present of invention, the test antigen is rabbit igg；The antibody of the test antigen is goat-anti rabbit IgG。

In one embodiment of the present of invention, the first anti-cTnI antibody and/or the second anti-myocardium calcium Protein I antibody is monoclonal antibody, and the first anti-myoglobins antibody and/or the second anti-myoglobins antibody are Dan Ke Grand antibody, the first anti-creatine kinase isozyme antibody and/or the second anti-creatine kinase isozyme antibody are monoclonal Antibody.

In one embodiment of the present of invention, the fluorescence probe is fluorescin.

In one embodiment of the present of invention, the fluorescin is green fluorescent protein.

Second aspect, swashs the present invention provides a kind of cTnI/myoglobins/creatine as described in relation to the first aspect The preparation method of enzyme isoenzyme near-infrared fluorescent detection reagent card, includes the following steps：

(1) cTnI near-infrared fluorescent detection reagent bar is prepared：

1) the specking fluorescence probe-the first on the glass fibre element film of cTnI near-infrared fluorescent detection reagent bar The mixed solution of anti-cTnI antibody and fluorescence probe-test antigen

2) the first pretreatment is carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, And in the first pretreated anti-cTnI antibody-solutions of nitrocellulose filter specking second；

3) the second pretreatment is carried out to nitrocellulose filter that will be corresponding to the T2 lines of specking using the second pretreatment fluid, And in the second pretreated nitrocellulose filter specking cTnI antigenic solution；

4) on the nitrocellulose filter of predeterminated position specking test antigen antibody-solutions；

(2) myoglobins near-infrared fluorescent detection reagent bar is prepared

1) the-the first anti-flesh of specking fluorescence probe on the glass fibre element film of myoglobins near-infrared fluorescent detection reagent bar Lactoferrin antibody and fluorescence probe-test antigen；

2) the first pretreatment is carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, And in the first pretreated anti-myoglobins antibody-solutions of nitrocellulose filter specking second；

3) the second pretreatment is carried out to nitrocellulose filter that will be corresponding to the T2 lines of specking using the second pretreatment fluid, And in the second pretreated nitrocellulose filter specking myoglobins antigenic solution；

(3) creatine kinase isozyme near-infrared fluorescent detection reagent bar is prepared

1) the specking fluorescence probe-the on the glass fibre element film of creatine kinase isozyme near-infrared fluorescent detection reagent article Primary antibody creatine kinase isozyme antibody and with fluorescence probe-test antigen；

2) the first pretreatment is carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, And in the first pretreated anti-creatine kinase isozyme antibody-solutions of nitrocellulose filter specking second；

3) the second pretreatment is carried out to nitrocellulose filter that will be corresponding to the T2 lines of specking using the second pretreatment fluid, And in the second pretreated nitrocellulose filter specking creatine kinase isozyme antigenic solution；

(4) by the cTnI near-infrared fluorescent detection reagent bar prepared, the detection examination of myoglobins near-infrared fluorescent Agent bar and the progress of creatine kinase isozyme near-infrared fluorescent detection reagent bar are arranged in parallel, are installed in reagent card, obtain cardiac muscle Calcium protein I/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card.

In one embodiment of the present of invention, first pretreatment fluid is to contain 1.5-2g/L arginine, 1.2-1.5g/ L Potassium Hydrogen Phthalates, 0.3-0.5g/L sodium hydroxides, the aqueous solution of 2.0-2.5g/L citric acids.First pretreatment fluid PH be 6.0~6.5.

In one embodiment of the present of invention, second pretreatment fluid is to contain 2-2.5g/L asparagines, 0.5- 0.7g/L disodium hydrogen phosphates, 3-4g/L rhamnoses, the aqueous solution of 3-3.5g/L glycine.The pH of first pretreatment fluid is 7.6~8.0.

The third aspect, the present invention provides cTnI/myoglobins/creatine kinase described in a kind of first aspect Isodynamic enzyme near-infrared fluorescent detection reagent, which is stuck in, prepares cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent Purposes in detection kit.

In one embodiment of the present of invention, the purposes is specially to use the cTnI/myoglobins/flesh Acid kinase isodynamic enzyme near-infrared fluorescent detection kit is to cTnI/myoglobins/creatine kinase isozyme in sample It is detected.

Fourth aspect, the present invention provides a kind of cTnI/myoglobins/creatine kinase isozyme near-infrared is glimmering Light detection kit, including cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent described in first aspect Detection reagent card.

Compared with prior art, the present invention has the advantages that：Using two p-wires ratio or p-wire with The ratio of control line determines the amount of cTnI/myoglobins/creatine kinase isozyme, can effectively reduce error, Testing result is improved, particularly the quantitatively accuracy of testing result；And avoid between control system and test system Interfere with each other；And control system can use the antigen-antibody reaction thing of not disturbed test system, avoid control system and survey Interfering with each other between test system, improves accuracy and the precision of detection.

Brief description of the drawings

Fig. 1 is cTnI near-infrared fluorescent detection reagent bar provided by the invention, myoglobins near-infrared fluorescent is examined The structure diagram of test agent bar and creatine kinase isozyme near-infrared fluorescent detection reagent bar.

Embodiment

Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text In addition explicitly point out, singulative "one", " one " and " this " include plural form.

When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, according to grasp of the those skilled in the art to the prior art and the record of the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

The present invention provides a kind of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent Card, including cTnI near-infrared fluorescent detection reagent bar arranged in parallel, myoglobins near-infrared fluorescent detection reagent bar It is as shown in Figure 1 with creatine kinase isozyme near-infrared fluorescent detection reagent bar, the structure of each reagent strip.Cardiac muscle provided by the invention Calcium protein I/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card employ fluorescence immune chromatography technology and Double antibody sandwich method principle.

On cTnI near-infrared fluorescent detection reagent bar, sample application pad, glass fibre element film, nitrocellulose Film and water suction gasket are arranged in order.Contain the anti-cTnI antibody of fluorescence probe-the first and glimmering on the glass fibre element film Light probe-test antigen.The anti-cTnI antibody of the fluorescence probe-the first and fluorescence probe-test antigen can chromatograph to The nitrocellulose filter.T1 lines, T2 lines and C lines are disposed with nitrocellulose filter.T1 lines and T2 lines are p-wire, C Line line in order to control.T1 lines have been coated with the second anti-cTnI antibody；T2 lines have been coated with cTnI antigen；C lines are coated with The antibody of the test antigen.

The anti-cTnI antibody of fluorescence probe-the first refers to the first anti-cTnI of fluorescence probe mark Antibody.

The fluorescence probe-test antigen refers to the test antigen of fluorescence probe mark.

The test antigen refers to the antigen beyond cTnI antigen, such as IgG.

The test antigen of fluorescence probe mark on sample application pad and the antibody of the coated test antigen of C lines, which are used to verify, to be examined Whether test agent bar fails.

During detection, on cTnI near-infrared fluorescent detection reagent bar, if with the presence of cTnI in sample, The first anti-cTnI antibody binding that can be first with fluorescence probe mark forms fluorescent immunocomplex, when the fluorescence is exempted from When epidemic disease compound is chromatographed to T1 lines, the second anti-cTnI antibody capture for being coated in advance on T1 lines, fluorescence immunoassay is multiple Compound is enriched with T1 lines, and center of a sample's Troponin I concentration is higher, and enrichment of the fluorescent immunocomplex on T1 lines is got over More, T1 line colors are deeper；If center of a sample's Troponin I is less than the anti-cTnI antibody of fluorescence probe-the first, remain First anti-cTnI antibody of remaining fluorescence probe mark, which can be chromatographed to T2 lines, center of a sample's Troponin I concentration, to be got over Low, the anti-cTnI antibody of fluorescence probe-the first of the enrichment on T2 lines is more, and fluorescence signal is stronger.It can use Dry type fluorescence immunity analyzer is detected fluorescence signal.

The testing result of cTnI near-infrared fluorescent detection reagent bar by the ratio of T1/T2 or the ratio of T1/C Lai Characterization.T1 values, T2 values, C values are specific respectively with the fluorescence being enriched with the fluorescence signal value for the fluorescence probe being enriched with T1 lines, T2 lines The fluorescence signal value of the fluorescence probe being enriched with the fluorescence signal value of probe, C lines represents.Dry type fluorescence immunoassay point can be used The fluorescence signal for the fluorescence probe that analyzer is enriched with each line is detected, to draw fluorescence signal value.

The detection of cTnI is carried out using cTnI near-infrared fluorescent detection reagent bar provided by the invention, It is likely to occur following several situations：

(1) when center of a sample's Troponin I from scratch when, cTnI and the first cTnI antigen-antibody The immune complex of formation is more and more, and the first free cTnI antigen-antibody is fewer and fewer, therefore, the capture of T1 lines Fluorescence probe it is more and more, and T2 lines capture fluorescence probe it is fewer and fewer.T1 values from without becoming strong to gradual, T2 from it is extremely strong to Weaken, C is constant, then：T1/T2 is by minimum to becoming larger, and T1/C is by minimum to becoming larger.

(2) when center of a sample's Troponin I is in equivalence zone, cTnI and the first cTnI antigen resist The fluorescent immunocomplex that body is formed is most, and the first free cTnI antigen-antibody is minimum, and T1 values are maximum at this time, T2 Value is minimum, and C values are constant.Then：T1/T2 is become larger greatly by big, and T1/C gradually strengthens.

Therefore, the content of center of a sample's Troponin I is obtained according to the ratio of the ratio of T1/T2 or T1/C.In reality In, the calibration curve of standard items fitting T1/T2 or T1/C ratios can be utilized, is then calculated according to standard curve in sample The content of cTnI.

And cTnI near-infrared fluorescent detection reagent card of the prior art has only been coated with a p-wire, upper The concrete condition stated under situation is as follows：

(1) when center of a sample's Troponin I from scratch when：T1 tends to constant quickly from without becoming strong to gradual；

(2) when center of a sample's Troponin I is in equivalence zone, then T1 is constant.

The amount that cTnI is determined by means of a p-wire is only relied on, error is larger, especially for cTnI Quantitative detection, accuracy are relatively low.

And the ratio or survey of two p-wires of cTnI near-infrared fluorescent detection reagent strip adoption provided by the invention The ratio of examination line and control line determines the amount of cTnI, can effectively reduce error, can be adapted for myocardium calcium egg The quantitative determination of white I.

It is specially the antigen different from cTnI to test antigen.If the reagent card is specifically used for the detection people source heart Troponin I, test antigen select non-human antigen, humanized's sample will not be interfered.Test antigen and test antigen Antibody and the non-cross interference of Ag-Ab system of p-wire system.So that system methodology error is controlled, improve The accuracy of detection and precision.

In one example, the test antigen is rabbit igg；The antibody of the test antigen is goat anti-rabbit igg.

In one example, the described first anti-cTnI antibody and/or the second anti-cTnI antibody For monoclonal antibody.The monoclonal antibody of the gene engineering expression of purifying is selected to compare polyclonal antibody specificity and targeting more Good, quality is more stable.Further, the described first anti-cTnI antibody and the second anti-cTnI antibody are Different monoclonal antibodies, such as the first anti-cTnI antibody can use the anti-cardiac muscle of mouse of Halothane thing engineering Calcium protein I monoclonal antibody (article No. CSB-MA079361A0m), the second anti-cTnI antibody can be won using Zhengzhou Match the anti-cTnI monoclonal antibody (article No. BCW1101062) of mouse of Biotechnology Ltd..

In one example, the fluorescence probe in the anti-cTnI antibody of the fluorescence probe-the first and the fluorescence Fluorescence probe in probe-test antigen is fluorescin.Fluorescin and first can be resisted using carbodiimides method CTnI antibody or test antigen are coupled, to carry out fluorescent marker.The good biocompatibility of fluorescin, mark The bioactivity of the biomolecule is not influenced after biomolecule；Luminous intensity is strong, is easy to detect；With wider excitation wavelength (450~650nm), fluorescent brightness is 20~30 times of fluorescein, and is not easy to be quenched.

In one example, the fluorescin is green fluorescent protein.Had the following advantages using green fluorescent protein：

(1) immunofluorescence present green, can directly observe as a result, detect it is convenient；

(2) after the illumination by the short time, fluorescence duration time length is sent；

(3) it is small by background interference still with the presence of fluorescence after light source is removed.

On myoglobins near-infrared fluorescent detection reagent bar, sample application pad, glass fibre element film, nitrocellulose filter and Water suction gasket is arranged in order.On the glass fibre element film containing the anti-myoglobins antibody of fluorescence probe-the first and fluorescence probe- Test antigen.The anti-myoglobins antibody of the fluorescence probe-the first and fluorescence probe-test antigen can chromatograph fine to the nitric acid The plain film of dimension.T1 lines, T2 lines and C lines are disposed with nitrocellulose filter.T1 lines and T2 lines are p-wire, C lines line in order to control. T1 lines have been coated with the second anti-myoglobins antibody, and T2 lines have been coated with myoglobins antigen, and C lines have been coated with the anti-of the test antigen Body.

The anti-myoglobins antibody of fluorescence probe-the first refers to the first anti-myoglobins antibody of fluorescence probe mark.

The test antigen refers to the antigen beyond cTnI antigen, such as IgG.

During detection, on myoglobins near-infrared fluorescent detection reagent bar, if with the presence of myoglobins in sample, can first The first anti-myoglobins antibody binding with fluorescence probe mark forms fluorescent immunocomplex, when the fluorescent immunocomplex layer When analysis is to T1 lines, the second anti-myoglobins antibody capture for being coated in advance on T1 lines, fluorescent immunocomplex is at T1 lines It is enriched with, myoglobin concentration is higher in sample, and enrichment of the fluorescent immunocomplex on T1 lines is more, and T1 line colors are deeper； If myoglobins is less than the anti-myoglobins antibody of fluorescence probe-the first in sample, the first of remaining fluorescence probe mark Anti- myoglobins antibody can be chromatographed to T2 lines, and myoglobin concentration is lower in sample, the fluorescence probe-the first of the enrichment on T2 lines Anti- myoglobins antibody is more, and fluorescence signal is stronger.Dry type fluorescence immunity analyzer can be used to carry out fluorescence signal Detection.

The testing result of myoglobins near-infrared fluorescent detection reagent bar is by the ratio of T1/T2 or the ratio of T1/C come table Sign.T1 values, T2 values, C values are specific to be visited with the fluorescence being enriched with the fluorescence signal value for the fluorescence probe being enriched with T1 lines, T2 lines respectively The fluorescence signal value of the fluorescence probe being enriched with the fluorescence signal value of pin, C lines represents.Dry type fluoroimmunoassay can be used The fluorescence signal for the fluorescence probe that instrument is enriched with each line is detected, to draw fluorescence signal value.

The detection of myoglobins is carried out using myoglobins near-infrared fluorescent detection reagent bar provided by the invention, may be gone out Now following several situations：

(1) when in sample myoglobins from scratch when, what myoglobins and the first myoglobins antigen-antibody were formed exempts from Epidemic disease compound is more and more, and the first free myoglobins antigen-antibody is fewer and fewer, and therefore, the fluorescence probe of T1 lines capture is got over Come it is more, and T2 lines capture fluorescence probe it is fewer and fewer.T1 values to gradual from without becoming strong, and for T2 from extremely strong to decrease, C is constant, Then：T1/T2 is by minimum to becoming larger, and T1/C is by minimum to becoming larger.

(2) when myoglobins is in equivalence zone in sample, what myoglobins and the first myoglobins antigen-antibody were formed Fluorescent immunocomplex is most, and the first free myoglobins antigen-antibody is minimum, and T1 values are maximum at this time, and T2 values are minimum, and C values are not Become.Then：T1/T2 is become larger greatly by big, and T1/C gradually strengthens.

Therefore, the content of myoglobins in sample is obtained according to the ratio of the ratio of T1/T2 or T1/C.In practical application In, the calibration curve of standard items fitting T1/T2 or T1/C ratios can be utilized, it is red that flesh in sample is then calculated according to standard curve The content of albumen.

And myoglobins near-infrared fluorescent detection reagent card of the prior art has only been coated with a p-wire, in above-mentioned feelings Concrete condition under shape is as follows：

(1) when in sample myoglobins from scratch when：T1 tends to constant quickly from without becoming strong to gradual；

(2) when myoglobins is in equivalence zone in sample, then T1 is constant.

The amount that myoglobins is determined by means of a p-wire is only relied on, error is larger, especially for the quantitative inspection of myoglobins Survey, accuracy is relatively low.

And the ratio or test of two p-wires of myoglobins near-infrared fluorescent detection reagent strip adoption provided by the invention The ratio of line and control line determines the amount of myoglobins, can effectively reduce error, can be adapted for determining for myoglobins It is fixed to measure.

It is specially the antigen different from myoglobins to test antigen.If the reagent card is specifically used for detection people source flesh red eggs In vain, test antigen and select non-human antigen, humanized's sample will not be interfered.Antigen and test antigen-antibody are tested with surveying Try the non-cross interference of Ag-Ab system of linear system system.So that system methodology error is controlled, the standard of detection is improved Exactness and precision.

In one example, the described first anti-myoglobins antibody and/or the second anti-myoglobins antibody are Dan Ke Grand antibody.Select the monoclonal antibody of the gene engineering expression of purifying more preferable compared to polyclonal antibody specificity and targeting, matter Measure more stable.Further, the described first anti-myoglobins antibody and the second anti-myoglobins antibody are different Dan Ke Grand antibody, such as the first anti-myoglobins antibody can use the anti-flesh of mouse of Guangzhou Wondfo Biotech. Co., Ltd. red Protein monoclonal antibody (article No. M103-2), the second anti-myoglobins antibody can use Shanghai Hu Zhen Industrial Co., Ltd.s The anti-myoglobins monoclonal antibody (article No. hz480Hu22) of mouse.

In one example, the fluorescence probe in the anti-myoglobins antibody of the fluorescence probe-the first and the fluorescence are visited Fluorescence probe in pin-test antigen is fluorescin.Carbodiimides method can be used by fluorescin and the first anti-flesh Lactoferrin antibody or test antigen are coupled, to carry out fluorescent marker.The good biocompatibility of fluorescin, mark biology point The bioactivity of the biomolecule is not influenced after son；Luminous intensity is strong, is easy to detect；With wider excitation wavelength (450~ 650nm), fluorescent brightness is 20~30 times of fluorescein, and is not easy to be quenched.

On creatine kinase isozyme near-infrared fluorescent detection reagent bar, sample application pad, glass fibre element film, cellulose nitrate Plain film and water suction gasket are arranged in order.Resist on the glass fibre element film containing the anti-creatine kinase isozyme of fluorescence probe-the first Body and with fluorescence probe-test antigen.The anti-creatine kinase isozyme antibody of the fluorescence probe-the first and with fluorescence probe-survey Examination antigen can be chromatographed to the nitrocellulose filter.T1 lines, T2 lines and C lines are disposed with nitrocellulose filter.T1 lines and T2 lines are p-wire, C lines line in order to control.T1 lines have been coated with the second anti-creatine kinase isozyme antibody, and T2 lines have been coated with creatine and have swashed Enzyme isoenzyme antigen, C lines have been coated with the antibody of the test antigen.

The anti-creatine kinase isozyme antibody of fluorescence probe-the first refers to the first anti-creatine kinase of fluorescence probe mark Isozyme antibody.

The test antigen refers to the antigen beyond creatine kinase isozyme antigen, such as IgG.

During detection, on creatine kinase isozyme near-infrared fluorescent detection reagent bar, if there is the same work of creatine kinase in sample Enzyme exists, and the first anti-creatine kinase isozyme antibody binding that can be first with fluorescence probe mark forms fluorescent immunocomplex, When the fluorescent immunocomplex is chromatographed to T1 lines, the second anti-creatine kinase isozyme antibody being coated in advance on T1 lines is caught Obtain, fluorescent immunocomplex is enriched with T1 lines, and creatine kinase isozyme concentration is higher in sample, and fluorescent immunocomplex is in T1 Enrichment on line is more, and T1 line colors are deeper；If creatine kinase isozyme is less than the anti-creatine of fluorescence probe-the first in sample Kinase isozyme antibody, then the first anti-creatine kinase isozyme antibody that remaining fluorescence probe marks can be chromatographed to T2 lines, sample Creatine kinase isozyme concentration is lower in this, and the anti-myoglobins antibody of fluorescence probe-the first of the enrichment on T2 lines is more, glimmering Optical signal is stronger.Fluorescence signal can be detected using dry type fluorescence immunity analyzer.

The creatine kinase isozyme near-infrared fluorescent detection reagent bar provided using invention carries out creatine kinase isozyme Detection, in fact it could happen that following several situations：

(1) when in sample creatine kinase isozyme from scratch when, creatine kinase isozyme and the same work of the first creatine kinase The immune complex that enzyme antigen-antibody is formed is more and more, and the first free creatine kinase isozyme antigen-antibody is fewer and fewer, Therefore, the fluorescence probe of T1 lines capture is more and more, and the fluorescence probe of T2 lines capture is fewer and fewer.T1 values become from nothing to gradual By force, T2 is from extremely strong to decrease, and C is constant, then：T1/T2 is by minimum to becoming larger, and T1/C is by minimum to becoming larger.

(2) when creatine kinase isozyme is in equivalence zone in sample, creatine kinase isozyme and the first creatine kinase are same The fluorescent immunocomplex that work enzyme antigen-antibody is formed is most, and the first free creatine kinase isozyme antigen-antibody is minimum, this When T1 values it is maximum, T2 values minimum, C values are constant.Then：T1/T2 is become larger greatly by big, and T1/C gradually strengthens.

Therefore, the content of creatine kinase isozyme in sample is obtained according to the ratio of the ratio of T1/T2 or T1/C.In reality In the application of border, the calibration curve of standard items fitting T1/T2 or T1/C ratios can be utilized, sample is then calculated according to standard curve The content of middle creatine kinase isozyme.

And creatine kinase isozyme near-infrared fluorescent detection reagent card of the prior art has only been coated with a p-wire, Concrete condition under said circumstances is as follows：

(1) when in sample creatine kinase isozyme from scratch when：T1 tends to constant quickly from without becoming strong to gradual；

(2) when creatine kinase isozyme is in equivalence zone in sample, then T1 is constant.

The amount that creatine kinase isozyme is determined by means of a p-wire is only relied on, error is larger, same especially for creatine kinase The quantitative detection of work enzyme, accuracy are relatively low.

And the ratio of two p-wires of creatine kinase isozyme near-infrared fluorescent detection reagent strip adoption provided by the invention Or the ratio of p-wire and control line determines the amount of creatine kinase isozyme, it error can be effectively reduced, can be adapted for The quantitative determination of creatine kinase isozyme.

It is specially the antigen different from creatine kinase isozyme to test antigen.If the reagent card is specifically used for detection people source Creatine kinase isozyme, test antigen select non-human antigen, humanized's sample will not be interfered.Test antigen and test Antigen-antibody and the non-cross interference of Ag-Ab system of p-wire system.So that system methodology error is controlled, carry The accuracy of high detection and precision.

In one example, the described first anti-creatine kinase isozyme antibody and/or the second anti-same work of creatine kinase Enzyme antibody is monoclonal antibody.The monoclonal antibody of the gene engineering expression of purifying is selected compared to polyclonal antibody specificity and target Tropism is more preferable, and quality is more stable.Further, the described first anti-creatine kinase isozyme antibody and the second anti-creatine kinase Isozyme antibody is different monoclonal antibody, such as the first anti-creatine kinase isozyme antibody is with can using Shenzhen person of outstanding talent The anti-creatine kinase isozyme monoclonal antibody (article No. HT1620004) of mouse of Hua Tuo bio tech ltd, described second Anti- creatine kinase isozyme antibody can use the anti-creatine kinase isozyme list of mouse of Shanghai Mai Bai stars bio tech ltd Clonal antibody (article No. M-CK).

In one example, the fluorescence probe in the anti-creatine kinase isozyme antibody of the fluorescence probe-the first and described Fluorescence probe in fluorescence probe-test antigen is fluorescin.Carbodiimides method can be used by fluorescin and the Primary antibody creatine kinase isozyme antibody or test antigen are coupled, to carry out fluorescent marker.The biocompatibility of fluorescin It is good, the bioactivity of the biomolecule is not influenced after mark biomolecule；Luminous intensity is strong, is easy to detect；Swash with wider Wavelength (450~650nm) is sent out, fluorescent brightness is 20~30 times of fluorescein, and is not easy to be quenched.

Present invention also offers a kind of cTnI/myoglobins/creatine kinase isozyme near-infrared described above The preparation method of luciferase assay reagent card, includes the following steps：

(1) cTnI near-infrared fluorescent detection reagent bar is prepared：

1) the specking fluorescence probe-the first on the glass fibre element film of cTnI near-infrared fluorescent detection reagent bar The mixed solution of anti-cTnI antibody and fluorescence probe-test antigen；

(2) myoglobins near-infrared fluorescent detection reagent bar is prepared

In one example, first pretreatment fluid is to contain 1.5-2g/L arginine, 1.2-1.5g/L O-phthalics Potassium hydrogen phthalate, 0.3-0.5g/L sodium hydroxides, the aqueous solution of 2-2.5g/L citric acids.

In one example, second pretreatment fluid is to contain 2-2.5g/L asparagines, 0.5-0.7g/L phosphoric acid hydrogen Disodium, 3-4g/L rhamnoses, the aqueous solution of 3-3.5g/L glycine.

In the preparation, the nitrocellulose filter corresponding to T1 lines is pre-processed using the first pretreatment fluid, and used Second pretreatment fluid pre-processes the nitrocellulose filter corresponding to T2 lines, and it is closely red can to significantly improve cTnI The detection sensitivity of outer luciferase assay reagent card and accuracy.

Present invention also offers a kind of cTnI/myoglobins/creatine kinase isozyme near-infrared described above Luciferase assay reagent, which is stuck in, to be prepared in cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection kit Purposes.

In one example, the purposes is specially to use the cTnI/same work of myoglobins/creatine kinase Enzyme near-infrared fluorescent detection kit is detected cTnI/myoglobins/creatine kinase isozyme in sample.

Present invention also offers a kind of cTnI/myoglobins/creatine kinase isozyme detection kit, including CTnI/myoglobins described above/creatine kinase isozyme near-infrared fluorescent detection reagent card.

In specific examples below and comparative example, using standard samples recovery to cTnI provided by the invention The detection result of near-infrared fluorescent detection reagent card is illustrated.

Embodiment 1

11st, the preparation of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card, specifically Method includes the following steps：

(1) preparation of cTnI near-infrared fluorescent detection reagent bar

111st, the system of the mixed solution of the anti-cTnI antibody of fluorescence probe-the first and fluorescence probe-test antigen It is standby：

The anti-cTnI list of mouse of the desired amount of Halothane thing engineering is added in the PBS of the 0.05M of preset vol Clonal antibody (article No. CSB-MA079361A0m) and green fluorescent protein, then adding EDC resists the mouse of Halothane thing engineering CTnI monoclonal antibody (article No. CSB-MA079361A0m) and green fluorescent protein coupling；By centrifuging, diluting Operating procedure removes EDC, the anti-cTnI antibody-solutions of fluorescence probe-the first of acquisition.In the 0.05M of preset vol The desired amount of rabbit igg and green fluorescent protein are added in PBS, then adding EDC is coupled rabbit igg and green fluorescent protein；Through Cross the operating procedures such as centrifugation, dilution and remove EDC, fluorescence probe-test antigenic solution of acquisition.By the fluorescence probe-the of preparation Primary antibody cTnI antibody-solutions and fluorescence probe-test antigenic solution mixing, 2-8 DEG C of preservation.

112nd, the step 111 gained mixed solution PBS of pH6.8-7.2,0.01-0.05M is diluted, is diluted to OD720 =2.0~4.0, by resulting solution specking to glass fibre element film, specking amount is 2-5mg/ml, and 35-45 DEG C of drying, contains to obtain the final product There is the glass fibre element film of the anti-cTnI antibody of fluorescence probe-the first and fluorescence probe-test antigen.

113rd, the preparation of C lines solution：Goat anti-rabbit igg solution is prepared with the PBS of 0.01-0.05M, pH6.8-7.2, solution Final concentration of 1.0-2.0mg/ml.

114th, the preparation of T1 lines solution：The second anti-cTnI is prepared with the PBS of 0.01-0.05M, pH6.8-7.2 to resist Liquid solution, the anti-cTnI list of mouse of the second anti-cTnI antibody Zhengzhou Bosai Biotechnology Co., Ltd Clonal antibody (article No. BCW1101062), the final concentration of 1.0-2.0mg/ml of solution.

115th, the preparation of T2 lines solution：It is molten that cTnI antigen is prepared with the PBS of 0.01-0.05M, pH6.8-7.2 Liquid, the final concentration of 1.0-2.0mg/ml of solution.

116th, nitrocellulose filter that will be corresponding to the T1 lines of specking is pre-processed using the first pretreatment fluid, The formula of one pretreatment fluid is：Arginine 1.5g/L, Potassium Hydrogen Phthalate 1.2g/L, sodium hydroxide 0.5g/L, citric acid 2.0g/L, solvent are water, pH value=6.5, and pretreatment concretely comprises the following steps：

1161st, toward 5 the first pretreatment fluids of μ l are dropped evenly on T1 lines, room temperature is dried；

1162nd, repeat step 1161 is twice.

117th, nitrocellulose filter that will be corresponding to the T2 lines of specking is pre-processed using the second pretreatment fluid, The formula of two pretreatment fluids is：Asparagine 2.5g/L, disodium hydrogen phosphate 0.5g/L, rhamnose 4g/L, glycine 3.5g/L, it is molten Agent is water, pH value=7.6, and pretreatment concretely comprises the following steps：

1171st, 5 the second pre-treatment buffers of μ l are dropped evenly toward T2 lines, room temperature is dried；

1172nd, repeat step 1171 is twice.

118th, respectively using C lines solution, T1 lines solution, T2 line solution specking C lines, T1 lines, T2 on nitrocellulose filter Line：It is with the PBS buffer of 0.01M, pH7.2 that trace of albumin point membranous system is cleaned, spray each parameter of film instrument is debugged, is connected Inlet/outlet pipeline, C pipelines, T1 pipelines, T2 pipelines are respectively put into C lines, T1 lines, T2 line solution, the spray of regulating system speed and Film-passing speed, so as to can respectively spray the C lines solution, T1 lines solution, T2 line solution of 1-3 μ l, cellulose nitrate per the film strips of 1cm length The distributing order of three lines is on plain film, and T1 lines, T2 lines and C lines are followed successively by since being loaded end, and the film sprayed is put into vacuum Drying is vacuumized in pump, is disposed with the first p-wire, the second p-wire and control line and the first p-wire bag to obtain the final product CTnI antigen, control line coating have been coated with by the second anti-cTnI antibody, second p-wire The nitrocellulose filter of the antibody of the test antigen, it is spare.

119th, pad pasting：On offset plate, from top to bottom, the above-mentioned sample application pad prepared, glass fibre are sticked successively Plain film, nitrocellulose filter, water suction gasket.The big plate of reagent is made.

1110th, cutting：The test strips for being 3-5mm into width by the big plate rip cutting of reagent with cutter, each are 1 person-portion.

(2) preparation of myoglobins near-infrared fluorescent detection reagent bar

111st, the preparation of the mixed solution of the anti-myoglobins antibody of fluorescence probe-the first and fluorescence probe-test antigen：

The mouse of the desired amount of Guangzhou Wondfo Biotech. Co., Ltd. is added in the PBS of the 0.05M of preset vol Anti- myoglobins monoclonal antibody (article No. M103-2) and green fluorescent protein, then adding EDC makes Guangzhou ten thousand inspire confidence in biotechnology The anti-myoglobins monoclonal antibody (article No. M103-2) of mouse of limited company and green fluorescent protein coupling；By centrifuging, The operating procedures such as dilution remove EDC, the anti-myoglobins antibody-solutions of fluorescence probe-the first of acquisition.In the 0.05M of preset vol PBS in add the desired amount of rabbit igg and green fluorescent protein, then adding EDC is coupled rabbit igg and green fluorescent protein； EDC, fluorescence probe-test antigenic solution of acquisition are removed by the operating procedure such as centrifuging, diluting.By the fluorescence probe of preparation- First anti-myoglobins antibody-solutions and fluorescence probe-test antigenic solution mixing, 2-8 DEG C of preservation.

112nd, the step 111 gained mixed solution PBS of pH6.8-7.2,0.01-0.05M is diluted, is diluted to OD720 =2.0~4.0, by resulting solution specking to glass fibre element film, specking amount is 2-5mg/ml, and 35-45 DEG C of drying, contains to obtain the final product There is the glass fibre element film of the anti-myoglobins antibody of fluorescence probe-the first and fluorescence probe-test antigen.

114th, the preparation of T1 lines solution：The second anti-myoglobins antibody is prepared with the PBS of 0.01-0.05M, pH6.8-7.2 Solution, the second anti-myoglobins antibody use the anti-myoglobins monoclonal antibody (article No. of mouse of Shanghai Hu Zhen Industrial Co., Ltd.s Hz480Hu22), the final concentration of 1.0-2.0mg/ml of solution.

115th, the preparation of T2 lines solution：Myoglobins antigenic solution is prepared with the PBS of 0.01-0.05M, pH6.8-7.2, it is molten The final concentration of 1.0-2.0mg/ml of liquid.

1162nd, repeat step 1161 is twice.

1172nd, repeat step 1171 is twice.

118th, respectively using C lines solution, T1 lines solution, T2 line solution specking C lines, T1 lines, T2 on nitrocellulose filter Line：It is with the PBS buffer of 0.01M, pH7.2 that trace of albumin point membranous system is cleaned, spray each parameter of film instrument is debugged, is connected Inlet/outlet pipeline, C pipelines, T1 pipelines, T2 pipelines are respectively put into C lines, T1 lines, T2 line solution, the spray of regulating system speed and Film-passing speed, so as to can respectively spray the C lines solution, T1 lines solution, T2 line solution of 1-3 μ l, cellulose nitrate per the film strips of 1cm length The distributing order of three lines is on plain film, and T1 lines, T2 lines and C lines are followed successively by since being loaded end, and the film sprayed is put into vacuum Drying is vacuumized in pump, is disposed with the first p-wire, the second p-wire and control line and the first p-wire bag to obtain the final product Myoglobins antigen has been coated with by the second anti-myoglobins antibody, second p-wire, the control line be coated with it is described The nitrocellulose filter of the antibody of antigen is tested, it is spare.

(3) preparation of creatine kinase isozyme near-infrared fluorescent detection reagent bar

111st, the mixed solution of the anti-creatine kinase isozyme antibody of fluorescence probe-the first and fluorescence probe-test antigen Prepare：

The desired amount of Shenzhen Hao Dihuatuo bio tech ltd is added in the PBS of the 0.05M of preset vol The anti-creatine kinase isozyme monoclonal antibody (article No. HT1620004) of mouse and green fluorescent protein, then adding EDC makes Shenzhen The anti-creatine kinase isozyme monoclonal antibody (article No. HT1620004) of mouse of Hao Dihuatuo bio tech ltd of city and green Color fluorescin is coupled；EDC is removed by the operating procedure such as centrifuging, diluting, the anti-creatine kinase of fluorescence probe-the first of acquisition is same Work enzyme antibody solution.The desired amount of rabbit igg and green fluorescent protein are added in the PBS of the 0.05M of preset vol, is then added EDC is coupled rabbit igg and green fluorescent protein；EDC, fluorescence probe-survey of acquisition are removed by the operating procedure such as centrifuging, diluting Try antigenic solution.The anti-creatine kinase isozyme antibody-solutions of the fluorescence probe-the first of preparation and fluorescence probe-test antigen are molten Liquid mixes, 2-8 DEG C of preservation.

112nd, the step 111 gained mixed solution PBS of pH6.8-7.2,0.01-0.05M is diluted, is diluted to OD720 =2.0~4.0, by resulting solution specking to glass fibre element film, specking amount is 2-5mg/ml, and 35-45 DEG C of drying, contains to obtain the final product There is the glass fibre element film of the anti-creatine kinase isozyme antibody of fluorescence probe-the first and fluorescence probe-test antigen.

114th, the preparation of T1 lines solution：The second anti-same work of creatine kinase is prepared with the PBS of 0.01-0.05M, pH6.8-7.2 Enzyme antibody solution, the second anti-creatine kinase isozyme antibody use the anti-creatine of mouse of Shanghai Mai Bai stars bio tech ltd Kinase isozyme monoclonal antibody (article No. M-CK), the final concentration of 1.0-2.0mg/ml of solution.

115th, the preparation of T2 lines solution：Creatine kinase isozyme antigen is prepared with the PBS of 0.01-0.05M, pH6.8-7.2 Solution, the final concentration of 1.0-2.0mg/ml of solution.

1162nd, repeat step 1161 is twice.

1172nd, repeat step 1171 is twice.

118th, respectively using C lines solution, T1 lines solution, T2 line solution specking C lines, T1 lines, T2 on nitrocellulose filter Line：It is with the PBS buffer of 0.01M, pH7.2 that trace of albumin point membranous system is cleaned, spray each parameter of film instrument is debugged, is connected Inlet/outlet pipeline, C pipelines, T1 pipelines, T2 pipelines are respectively put into C lines, T1 lines, T2 line solution, the spray of regulating system speed and Film-passing speed, so as to can respectively spray the C lines solution, T1 lines solution, T2 line solution of 1-3 μ l, cellulose nitrate per the film strips of 1cm length The distributing order of three lines is on plain film, and T1 lines, T2 lines and C lines are followed successively by since being loaded end, and the film sprayed is put into vacuum Drying is vacuumized in pump, is disposed with the first p-wire, the second p-wire and control line and the first p-wire bag to obtain the final product Creatine kinase isozyme antigen, the control have been coated with by the second anti-creatine kinase isozyme antibody, second p-wire Line has been coated with the nitrocellulose filter of the antibody of the test antigen, spare.

(4) preparation of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card

Assembling：The cTnI near-infrared fluorescent detection reagent bar prepared, myoglobins near-infrared fluorescent are detected Reagent strip and the progress of creatine kinase isozyme near-infrared fluorescent detection reagent bar are arranged in parallel, and correspondence is installed to every 1 plastic clip In, obtain cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card.

12nd, examined using cTnI/myoglobins provided in this embodiment/creatine kinase isozyme near-infrared fluorescent Test agent card is detected cTnI antigen/myoglobins/creatine kinase isozyme.

121st, calibration curve is made.

Respectively by concentration for 0ng/ml, 0.01ng/ml, 0.02ng/ml, 0.05ng/ml, 0.10ng/ml, 0.20ng/ml, The cTnI antigenic solution of 0.50ng/ml, 1.0ng/ml, 2.0ng/ml, 5.0ng/ml, 10.0ng/ml are added dropwise in sample-adding On gasket, each concentration sets 5 repetitions (testing result takes the average value of 5 repetitions), after film layer is analysed 10 minutes, uses dry type Fluorescence immunity analyzer gathers the fluorescence signal of T1 lines and T2 lines.Detection range of the dry type fluorescence immunity analyzer to fluorescence signal It is AD values 0-10000.The value of T1/T2 is calculated according to the testing result of dry type fluorescence immunity analyzer.Built according to the value of T1/T2 lines Vertical calibration curve, wherein Y-axis are the value of T1/T2, and X-axis is standard items actual value.

Respectively by concentration for 0ng/ml, 0.05ng/ml, 1.0ng/ml, 2.0ng/ml, 5.0ng/ml, 10.0ng/ml, The myoglobins antigenic solution of 50.0ng/ml, 100.0ng/ml, 1000.0ng/ml are added dropwise on sample application pad, and each concentration is set 5 repetitions (testing result takes the average value of 5 repetitions), after film layer is analysed 10 minutes, are adopted using dry type fluorescence immunity analyzer Collect the fluorescence signal of T1 lines and T2 lines.Dry type fluorescence immunity analyzer is AD values 0-10000 to the detection range of fluorescence signal.Root The value of T1/T2 is calculated according to the testing result of dry type fluorescence immunity analyzer.Calibration curve, wherein Y are established according to the value of T1/T2 lines Axis is the value of T1/T2, and X-axis is standard items actual value.

Respectively by concentration for 0U/L, 0.01U/L, 0.02U/L, 0.05U/L, 0.1U/L, 0.5U/L, 1.0U/L, 5.0U/L, The creatine kinase isozyme antigenic solution of 10U/L, 20U/L, 50U/L, 100U/L are added dropwise on sample application pad, and each concentration sets 5 A repetition (testing result takes the average value of 5 repetitions), after film layer is analysed 10 minutes, is gathered using dry type fluorescence immunity analyzer The fluorescence signal of T1 lines and T2 lines.Dry type fluorescence immunity analyzer is AD values 0-10000 to the detection range of fluorescence signal.According to The testing result of dry type fluorescence immunity analyzer calculates the value of T1/T2.Calibration curve, wherein Y-axis are established according to the value of T1/T2 lines For the value of T1/T2, X-axis is standard items actual value.

122nd, cTnI/myoglobins manufactured in the present embodiment/creatine kinase isozyme near-infrared fluorescent detection examination Agent card is detected sample to be tested.

Prepare first group of cTnI antigen concentration be respectively 0.01ng/ml, 0.05ng/ml, 0.10ng/ml, The sample to be tested of 1.5ng/ml, 5.0ng/ml, using PBS buffer as control.

It is respectively 1.0ng/ml, 5.0ng/ml, 10.0ng/ml, 50.0ng/ to prepare second group of myoglobins antigen concentration The sample to be tested of ml, 100.0ng/ml, using PBS buffer as control.

Prepare the 3rd group of creatine kinase isozyme antigen concentration be respectively 0.01U/L, 0.02U/L, 0.05U/L, 0.1U/L, The sample to be tested of 5.0U/L, 10U/L, 50U/L, using PBS buffer as control.

First group of sample to be tested is added dropwise to the cTnI near-infrared fluorescent detection examination in above-mentioned detection reagent card respectively On the sample application pad of agent bar, the myoglobins near-infrared fluorescent detection in above-mentioned detection reagent card is added dropwise in second group of sample to be tested On the sample application pad of reagent strip, the 3rd group of sample to be tested is added dropwise in the creatine kinase isozyme near-infrared of above-mentioned detection reagent card On the sample application pad of luciferase assay reagent bar, each sample sets 5 repetitions (testing result takes the average value of 5 repetitions), film layer After analysis 10 minutes, by the T1/T2 values obtained when detecting sample compared with standard curve, detected value is obtained, by detected value and reality Actual value is compared, and obtaining accuracy influences deviation.

The content data of the cTnI antigen for the detection that first group of sample to be tested is obtained is respectively 0.0101ng/ Ml, 0.0502ng/ml, 0.1003ng/ml, 1.5002ng/ml, 5.001ng/ml, are not detected by myocardium calcium egg in blank control White I antigen.

The content data of the myoglobins antigen for the detection that second group of sample to be tested is obtained be respectively 1.001ng/ml, 5.003ng/ml, 10.002ng/ml, 50.001ng/ml, 100.002ng/ml, are not detected by myoglobins in blank control and resist It is former.

3rd group of creatine kinase isozyme antigen concentration be respectively 0.0101U/L, 0.0201U/L, 0.0502U/L, The sample to be tested of 0.1001U/L, 5.002U/L, 10.003U/L, 50.004U/L,

The above results show, the accuracy of cTnI antigen detection antigen provided by the invention influence deviation≤ 10%, the reagent card of the present embodiment can accurate detectable concentration as low as 0.01ng/ml cTnI antigen.Myoglobins The accuracy of antigen detection influences deviation≤10%, and the reagent card of the present embodiment can accurate detectable concentration as low as 1.00ng/ The myoglobins antigen of ml.The accuracy of creatine kinase isozyme antigen detection influences deviation≤10%, the examination of the present embodiment Agent card can accurate detectable concentration as low as 0.01U/L creatine kinase isozyme antigen.

Embodiment 2

21st, the preparation of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card, specifically Method includes the following steps：

Specific preparation method is described with reference to step (1)-(4) in embodiment 1, wherein, difference is, in the present embodiment In the first pretreatment fluid formula be：Arginine 1.75g/L, Potassium Hydrogen Phthalate 1.5g/L, sodium hydroxide 0.3g/L, lemon Sour 2.5g/L, solvent are water, pH value=6.0.Second pretreatment fluid formula is：Asparagine 2.0g/L, disodium hydrogen phosphate 0.7g/ L, rhamnose 3.5g/L, glycine 3.0g/L, solvent are water, pH value=8.0.

22nd, examined using cTnI/myoglobins provided in this embodiment/creatine kinase isozyme near-infrared fluorescent Test agent card is detected sample to be tested

The content data of the cTnI antigen for the detection that first group of sample to be tested is obtained is respectively 0.0102ng/ Ml, 0.0501ng/ml, 0.1002ng/ml, 1.5003ng/ml, 5.002ng/ml, are not detected by myocardium calcium egg in blank control White I antigen.

The content data of the myoglobins antigen for the detection that second group of sample to be tested is obtained be respectively 1.002ng/ml, 5.001ng/ml, 10.003ng/ml, 50.002ng/ml, 100.004ng/ml, are not detected by myoglobins in blank control and resist It is former.

3rd group of creatine kinase isozyme antigen concentration be respectively 0.0102U/L, 0.0203U/L, 0.0501U/L, The sample to be tested of 0.1004U/L, 5.001U/L, 10.004U/L, 50.002U/L,

Embodiment 3

31st, the preparation of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card, specifically Method includes the following steps：

Specific preparation method is described with reference to step (1)-(4) in embodiment 1, wherein, difference is, in the present embodiment In the first pretreatment fluid formula be：Arginase 12 .0g/L, Potassium Hydrogen Phthalate 1.35g/L, sodium hydroxide 0.4g/L, lemon Sour 2.25g/L, solvent are water, pH value=6.3.Second pretreatment fluid formula is：Asparagine 2.25g/L, disodium hydrogen phosphate 0.6g/L, rhamnose 3g/L, glycine 3.25g/L, solvent are water, pH value=7.8.

32nd, examined using cTnI/myoglobins provided in this embodiment/creatine kinase isozyme near-infrared fluorescent Test agent card is detected sample to be tested

The content data of the cTnI antigen for the detection that first group of sample to be tested is obtained is respectively 0.0101ng/ Ml, 0.0503ng/ml, 0.1004ng/ml, 1.5002ng/ml, 5.004ng/ml, are not detected by myocardium calcium egg in blank control White I antigen.

The content data of the myoglobins antigen for the detection that second group of sample to be tested is obtained be respectively 1.001ng/ml, 5.002ng/ml, 10.004ng/ml, 50.003ng/ml, 100.002ng/ml, are not detected by myoglobins in blank control and resist It is former.

3rd group of creatine kinase isozyme antigen concentration be respectively 0.0101U/L, 0.0204U/L, 0.0503U/L, The sample to be tested of 0.1002U/L, 5.002U/L, 10.001U/L, 50.004U/L,

Comparative example 1

41st, the preparation of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card is contrasted

Specific preparation method is described with reference to step (1)-(4) in embodiment 1, wherein, difference is, in the present embodiment In kit in nitrocellulose filter on T1 lines do not pre-processed by the first treatment fluid, T2 lines are not by second Liquid pretreatment is managed, other methods are same as Example 1 with condition.

42nd, the detection of cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card is contrasted Effect

421st, it is glimmering using contrast cTnI/myoglobins/creatine kinase isozyme near-infrared provided in this embodiment Light detection reagent card is detected cTnI/myoglobins/creatine kinase isozyme antigen

4211st, calibration curve is made

4212nd, cTnI/myoglobins that comparative example 1 provides/creatine kinase isozyme near-infrared fluorescent detection examination The testing result of agent card

First group of sample to be tested is added dropwise to the cTnI near-infrared fluorescent detection in comparative example detection reagent card respectively On the sample application pad of reagent strip, second group of sample to be tested is added dropwise in the myoglobins near-infrared fluorescent of comparative example detection reagent card On the sample application pad of detection reagent bar, the 3rd group of sample to be tested is added dropwise in the creatine kinase isozyme of comparative example detection reagent card On the sample application pad of near-infrared fluorescent detection reagent bar, each sample sets 5 repetitions, and (testing result takes being averaged for 5 repetitions Value), after film layer is analysed 10 minutes, by the T1/T2 values obtained when detecting sample compared with standard curve, detected value is obtained, will be examined Measured value and actual value are compared, and obtaining accuracy influences deviation.

The content data of the cTnI antigen for the detection that first group of sample to be tested is obtained be respectively be not detected by, It is not detected by, is not detected by, 1.8002ng/ml, 6.001ng/ml, blank control and 0.01ng/ml, 0.05ng/ml, CTnI antigen is not detected by 0.10ng/ml.

The content data of the myoglobins antigen for the detection that second group of sample to be tested is obtained is respectively to be not detected by, do not examine Measure, be not detected by, being not detected by, 105.002ng/ml, in blank control and 1.00ng/ml, 5.00ng/ml, 10.00ng/ Myoglobins antigen is not detected by ml, 50.00ng/ml.

3rd group of creatine kinase isozyme antigen concentration is respectively to be not detected by, be not detected by, being not detected by, not detecting Arrive, the sample to be tested of 6.002U/L, 11.003U/L, 51.004U/L, in blank control and 0.010U/L, 0.020U/L, 0.050U/L is not detected by creatine kinase isozyme antigen.

The above results show that the reagent card of this comparative example can be resisted with the detectable concentration as low as cTnI of 1.5ng/ml It is former.The reagent card of this comparative example can be with the myoglobins antigen of detectable concentration as low as 100.00ng/ml.The reagent card of this comparative example Can be with the creatine kinase isozyme antigen of detectable concentration as low as 0.1U/L.

In conclusion detection kit provided by the present invention has good sensitivity and accuracy, effectively overcome Various shortcoming of the prior art and have high industrial utilization.

The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as Into all equivalent modifications or change, should by the present invention claim be covered.

Claims

A kind of 1. cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card, it is characterised in that Including cTnI near-infrared fluorescent detection reagent bar arranged in parallel, myoglobins near-infrared fluorescent detection reagent bar and Creatine kinase isozyme near-infrared fluorescent detection reagent bar, the cTnI near-infrared fluorescent detection reagent bar include according to Sample application pad, glass fibre element film, nitrocellulose filter and the water suction gasket of secondary arrangement, wherein, on the glass fibre element film Containing the anti-cTnI antibody of fluorescence probe-the first and fluorescence probe-test antigen, on the nitrocellulose filter successively The first p-wire, the second p-wire and control line are provided with, first p-wire has been coated with the second anti-cTnI and has resisted Body, second p-wire have been coated with cTnI antigen, and the control line has been coated with the antibody of the test antigen；Institute State sample application pad, glass fibre element film, nitrocellulose that myoglobins near-infrared fluorescent detection reagent bar includes being arranged in order Film and water suction gasket, wherein, visited on the glass fibre element film containing the anti-myoglobins antibody of fluorescence probe-the first and fluorescence Pin-test antigen, is disposed with the first p-wire, the second p-wire and control line on the nitrocellulose filter, and described One p-wire has been coated with the second anti-myoglobins antibody, and second p-wire has been coated with myoglobins antigen, the control line It has been coated with the antibody of the test antigen；Creatine kinase isozyme near-infrared fluorescent detection reagent bar includes the sample-adding being arranged in order Gasket, glass fibre element film, nitrocellulose filter and water suction gasket, wherein, on the glass fibre element film containing fluorescence probe- First anti-creatine kinase isozyme antibody and with fluorescence probe-test antigen, be disposed with the nitrocellulose filter One p-wire, the second p-wire and control line, first p-wire has been coated with the second anti-creatine kinase isozyme antibody, described Second p-wire has been coated with creatine kinase isozyme antigen, and the control line has been coated with the antibody of the test antigen.
2. cTnI/myoglobins according to claim 1/creatine kinase isozyme near-infrared fluorescent detection examination Agent card, it is characterised in that the test antigen is rabbit igg；The antibody of the test antigen is goat anti-rabbit igg.
3. cTnI/myoglobins according to claim 1/creatine kinase isozyme near-infrared fluorescent detection examination Agent card, it is characterised in that the first anti-cTnI antibody and/or the second anti-cTnI antibody are Dan Ke Grand antibody, the first anti-myoglobins antibody and/or the second anti-myoglobins antibody are monoclonal antibody, described first Anti- creatine kinase isozyme antibody and/or the second anti-creatine kinase isozyme antibody are monoclonal antibody.
4. cTnI/myoglobins according to claim 1/creatine kinase isozyme near-infrared fluorescent detection examination Agent card, it is characterised in that the fluorescence probe is fluorescin.
5. cTnI/myoglobins according to claim 4/creatine kinase isozyme near-infrared fluorescent detection examination Agent card, it is characterised in that the fluorescin is green fluorescent protein.
6. cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent inspection as described in claim any one of 1-5 The preparation method of test agent card, includes the following steps：

(1) cTnI near-infrared fluorescent detection reagent bar is prepared：

1)-the first anti-heart of specking fluorescence probe on the glass fibre element film of cTnI near-infrared fluorescent detection reagent bar The mixed solution of Troponin I antibody and fluorescence probe-test antigen

2) the first pretreatment carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, and The first pretreated anti-cTnI antibody-solutions of nitrocellulose filter specking second；

3) the second pretreatment carried out to nitrocellulose filter that will be corresponding to the T2 lines of specking using the second pretreatment fluid, and Second pretreated nitrocellulose filter specking cTnI antigenic solution；

4) on the nitrocellulose filter of predeterminated position specking test antigen antibody-solutions；

(2) myoglobins near-infrared fluorescent detection reagent bar is prepared

1) the anti-flesh red eggs of specking fluorescence probe-the first on the glass fibre element film of myoglobins near-infrared fluorescent detection reagent bar Bai Kangti and fluorescence probe-test antigen；

2) the first pretreatment carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, and The first pretreated anti-myoglobins antibody-solutions of nitrocellulose filter specking second；

3) the second pretreatment carried out to nitrocellulose filter that will be corresponding to the T2 lines of specking using the second pretreatment fluid, and Second pretreated nitrocellulose filter specking myoglobins antigenic solution；

4) on the nitrocellulose filter of predeterminated position specking test antigen antibody-solutions；

(3) creatine kinase isozyme near-infrared fluorescent detection reagent bar is prepared

1) specking fluorescence probe-the first is anti-on the glass fibre element film of creatine kinase isozyme near-infrared fluorescent detection reagent bar Creatine kinase isozyme antibody and with fluorescence probe-test antigen；

2) the first pretreatment carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, and The first pretreated anti-creatine kinase isozyme antibody-solutions of nitrocellulose filter specking second；

3) the second pretreatment carried out to nitrocellulose filter that will be corresponding to the T2 lines of specking using the second pretreatment fluid, and Second pretreated nitrocellulose filter specking creatine kinase isozyme antigenic solution；

4) on the nitrocellulose filter of predeterminated position specking test antigen antibody-solutions；

(4) by the cTnI near-infrared fluorescent detection reagent bar prepared, myoglobins near-infrared fluorescent detection reagent bar It is arranged in parallel with the progress of creatine kinase isozyme near-infrared fluorescent detection reagent bar, it is installed in reagent card, obtains myocardium calcium egg White I/ myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card.
7. preparation method according to claim 6, it is characterised in that further include any one of following characteristics or multinomial： First pretreatment fluid is to contain 1.5-2g/L arginine, 1.2-1.5g/L Potassium Hydrogen Phthalates, 0.3-0.5g/L hydrogen-oxygens Change sodium, the aqueous solution of 2-2.5g/L citric acids；Second pretreatment fluid is to contain 2-2.5g/L asparagines, 0.5-0.7g/L Disodium hydrogen phosphate, 3-4g/L rhamnoses, the aqueous solution of 3-3.5g/L glycine.
8. such as claim 1-6 any one of them cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent Detection reagent is stuck in the purposes prepared in cTnI/myoglobins/creatine kinase isozyme detection kit.
9. purposes according to claim 8, it is characterised in that the purposes is specially to use cTnI/flesh red eggs In vain/creatine kinase isozyme near-infrared fluorescent detection kit is to cTnI/myoglobins/creatine kinase in sample Isodynamic enzyme is detected.
10. a kind of cTnI/myoglobins/creatine kinase isozyme detection kit, it is characterised in that including right It is required that 1-6 any one of them cTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card.