CN108398557A - A kind of myeloperoxidase fluorescence immune chromatography test paper bar and test card - Google Patents
A kind of myeloperoxidase fluorescence immune chromatography test paper bar and test card Download PDFInfo
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Abstract
The invention discloses a kind of myeloperoxidase fluorescence immune chromatography quantitative testing test paper items, including：Bottom plate, sample pad, bonding pad, nitrocellulose filter and water absorption pad, the sample pad, bonding pad, nitrocellulose filter and water absorption pad overlap be assembled on bottom plate successively, wherein, water absorption pad and bonding pad overlap the both ends for being pressed in nitrocellulose filter respectively, and form detection zone on the surface of nitrocellulose filter；Sample pad is overlapping to be pressed on bonding pad, and myeloperoxidase antibody fluorescence microsphere compound is fixed on the bonding pad；It is coated on nitrocellulose filter in the detection zone and identifies the detection line that the monoclonal antibody of another epitope of myeloperoxidase is constituted and the nature controlling line that sheep anti-mouse igg polyclonal antibody is constituted.It is linearly 3.125 600ng/ml that the present invention, which has highly sensitive, high stability, detection, and no non-specificity, detection time only needs 5min, and can realize fast inspection by bed, greatly improves clinical diagnosis efficiency.
Description
Technical field
The invention belongs to POCT immuno-chromatographic assay technologies fields, and in particular to a kind of myeloperoxidase fluorescence immunoassay layer
Analyse quantitative testing test paper item, the test card comprising the test strips and their preparation method.
Background technology
Coronary atherosclerotic heart disease is that coronary artery occurs atherosclerotic lesion and causes blood vessel
Chamber stenosis or occlusion causes heart disease caused by myocardial ischemia, anoxic or necrosis, abbreviation coronary heart disease（coronary heart
disease,CHD）.CHD has become the highest disease of hair rate disease in global angiocardiopathy at present.China has nearly million people to suffer from
CHD more than about 800,000 people dies of CHD, the lethality of far super tumour every year, and death toll shows a rising trend with the age.Hat
The generation of worry is affected by many factors, such as smoking, gender (male is more than women), gene pleiomorphism, areal variation, age
(mostly at 40 years old or more) etc..CHD occurrence and development mechanism and inflammatory reaction caused by numerous pathological factors and the close phase of oxidative stress
It closes.China CHD has high mortality, high sick hair rate, high burden at present（Society and family）The features such as, it has also become threaten modern state
The primary common disease of people's health and life.
Myeloperoxidase (myelopexoxidase, MPO) is a kind of protease of leukocyte derived prosthetic heme group,
It is the marker of inflammation and oxidative stress.With going deep into for scientific research, it has been found that it is same immunologic defence function of bringing into normal play
When, it is also related with the occurrence and development of other human diseases, such as inflammation, atherosclerosis, vasculitis, lung cancer, colorectal cancer.Closely
Nian Lai, many biomarkers have been used for clinical or are being studied, it is intended to risk of cardiovascular diseases are predicted, because of Atherosclerosis
Change and there are close ties between inflammatory reaction, it is believed that marker of inflammation, which is likely to become, preferably predicts atherosclerosis
Index.There are close ties-MPO to be considered as an atherosclerosis pusher with angiocardiopathy by MPO, leads to peroxidation
It should be catalyzed the product and enzyme of generation, promote patch to be formed and increase with unstability, accelerate progression of atherosclerosis, and then cause
Multiple complications, such as acute coronary syndrome（Acute coronary syndrome, ACS）.Atherosclerosis
（Atherosclerosis, AS）It is the important pathologic basis to form CHD again.Serum MPO contents and coronary artery disease deformation states phase
It closes, it is also related to CHD clinical state seriousness.In scientific research institutions are tested in the world, MPO is by as prediction acute myocardial infarction AMI
Early stage index, it is similar with c reactive protein detection, it is also used as a kind of inflammatory reaction index, level variation can be earlier than cardiac muscle
Ischemic changes.MPO levels measure and its in CHD diagnosis, change of illness state and prognosis evaluation, play the part of extremely critical role.
The common method of detection myeloperoxidase has at present：
1, enzyme linked immunosorbent assay（ELISA）：Enzyme-linked immunosorbent assay is based on immunological response, by antigen-antibody
Specific binding acts on a kind of very high detection technique of sensibility for being combined with the efficient catalytic product of zymolyte.This method is sensitive
Degree is high, and up to the level of ng/ml, specificity is high, and activator, inhibitor are influenced hardly in by body fluid, also not by peroxidating
Object enzyme and hemoglobin interference.Since cost is higher, and minute is longer, cumbersome, to the requirement of operating personnel's technology
It is high.
2, flow cytometer measuring method：The content for measuring MPO in neutrophil leucocyte slurry, blood cytolysate is added by whole blood
In, cell is handled using fixer and membrane permeability agent afterwards, is eventually adding fluorescein-labeled antibody MPO monoclonal antibodies,
MPO is dyed using antigen-antibody reaction, specific cell colony is analyzed, analyze the ratio of MPO positive cells and is averaged
Fluorescence intensity.This method high specificity, high sensitivity, but technical program is complicated, detection needs specific apparatus.
3, immunogold staining method (GICA methods)：Colloidal gold immunochromatographimethod technical characterstic is mainly using colloidal gold as marker.It can be weak
Under the conditions of alkali, this negatively charged marker is combined with the positively charged group on antigen-antibody molecule, in conjunction with being to belong to electrostatic
In conjunction with not influencing biomolecule characteristic.The method can only qualitative or sxemiquantitative, data are inaccurate.
4, latex enhancing immune turbidimetry：Detection method wide clinical detection MPO, principle be with polyethylene or
For the present latex particulate of polystyrene polymerization as labeled vector, labelled antigen/antibody connects the antigen/antibody and mesh of present latex particulate
Labeling antibody and antigen-reactive generate latex-antigen-antibody complex.Immunoturbidimetry high sensitivity, the range of linearity is wide, but examines
The survey time is long, is not suitable for by bed.
5, Timed resolved fluoroimmunoassay：It is that three kinds to run neck and neck at present with chemiluminescence, electrochemical luminescence surpass
One of quick immunoassay method.Its principle is marked object using the rare earth ion of longer fluorescence half-life period, due to this label
Object Stokes displacements big (＞ 150nm) and fluorescence lifetime 5~6 orders of magnitude higher than background substance fluorescence lifetime, therefore, when measurement
As long as delaying time of measuring, measuring the signal of marker again after the fluorescence of background substance is fully decayed can effectively eliminate respectively
The interference of kind non-specific fluorescence, obtains very high sensitivity.
The report for carrying out quantitative detection for myeloperoxidase using fluorescent micro-ball immune chromatography technology existing at present, example
Such as, patent application 201120513019.8 discloses a kind of test paper of quick detection myeloperoxidase, specifically discloses use
MPO monoclonal antibodies are marked in the fluorescent microsphere of diameter 210nm, prepare test strips, however, the document does not disclose
The parameters such as the range of linearity, the sensitivity of test strips；For another example, patent application 201611227045.8 discloses a kind of people's marrow peroxidating
The preparation method of object enzyme immuno-chromatographic test paper strip, but test strips qualitative detection aspect is only verified in embodiment part,
Do not record the parameters such as the detection range of linearity, sensitivity equally.
Invention content
The good myeloperoxidase of wide, high sensitivity that the purpose of the present invention is to provide a kind of ranges of linearity, stability is glimmering
Light immunochromatographiassay assay quantitative detection test paper, therefore, inventor carry out fully optimized to the multiple Step By Conditions for preparing test strips,
So as to complete the present invention.
To achieve the above object, the technical solution adopted in the present invention is specific as follows：
A kind of myeloperoxidase fluorescence immune chromatography quantitative testing test paper item, including：Bottom plate, sample pad, bonding pad, nitric acid are fine
The plain film of dimension and water absorption pad, the sample pad, bonding pad, nitrocellulose filter and water absorption pad overlap be assembled on bottom plate successively,
In, water absorption pad and bonding pad overlap the both ends for being pressed in nitrocellulose filter respectively, and are formed and examined on the surface of nitrocellulose filter
Survey area；Sample pad is overlapping to be pressed on bonding pad, and MPO antibody-fluorescent microsphere compounds are fixed on the bonding pad；The detection
The detection line for identifying that the monoclonal antibody of another epitope of myeloperoxidase is constituted is coated on nitrocellulose filter in area
The nature controlling line constituted with sheep anti-mouse igg polyclonal antibody, wherein fluorescent microsphere is passed through into carbodiimide（EDC）Activation, with MPO
Antibody coupling forms compound, then by protecting liquid redissolution, confining liquid to close, it is compound to be finally prepared into MPO antibody-fluorescent microballoons
Object.
The fluorescent microsphere is carboxyl, amino or hydroxyl fluorescent microsphere, preferably carboxyl fluorescent microsphere, such as grain size 180nm
Lanthanide series europium（Eu3+）Chelating object.
Preferably, activation is 8-12 times, preferably 10.7 times of fluorescent microsphere volume with the weight of EDC.
Preferably, in coupling process, the weight of MPO antibody is 6-10 times of fluorescent microsphere volume, preferably 8-8.5 times, even
The connection time is 1.2-2h, preferably 1.5h.
Preferably, redissolution protection liquid component include Tris-HCl, PB of 0.01-0.05M, borate buffer solution wherein
One kind, the BSA, PEG, glycine, casein that mass concentration is 0.5% ~ 1% are one or more kinds of, and mass concentration is 1% ~ 5%
Trehalose, sucrose, oligosaccharide are one or more kinds of, and the polysorbas20, Triton X-100, S9 that mass concentration is 0.05% ~ 0.5 are a kind of
Or two or more, 0.05% ~ 0.1% NaN3, the protection liquid is preferably composed of the following components：The Tris- of 0.05M pH 8.0
HCl buffer solutions, 0.5% polysorbas20,0.2% BSA, 0.5% casein, 4% trehalose and 0.05% NaN3。
Preferably, the MPO antibody is by polypeptide 1（SEQ ID No.1：SGCAYQDVGVTCPEQDKY）With polypeptide 2
（SEQ ID No.2：SKNNIFMSNSYPRDFVNC）For haptens, it is prepared into what comlete antigen was obtained through mouse peritoneal immunization
MPO monoclonal antibodies or other identical antibody of performance.
Preferably, the bonding pad is dry glass fibre component after treatment fluid immersion treatment, and treatment fluid includes
Tris-HCl, PB, borate buffer solution one of which, the BSA, PEG, glycine, casein that mass concentration is 0.5% ~ 1% are a kind of
Or it is two or more, the trehalose, sucrose, oligosaccharide that mass concentration is 1% ~ 5% are one or more kinds of, and mass concentration is 0.05% ~
0.5 polysorbas20, Tween 80, Triton X-100, S9 are one or more kinds of, 0.01% ~ 0.05% NaN3, the treatment fluid is excellent
It is the Tris-HCl of 0.01M pH8.0, the BSA that mass concentration is 0.1%, mass concentration to select working concentration composed of the following components
The NaN that polysorbas20 and mass concentration for 0.1% are 5% extra large sucrose and mass concentration is 0.05%3。
The present invention also provides the preparation method of above-mentioned myeloperoxidase fluorescence immune chromatography quantitative testing test paper item, institutes
The method of stating includes：
（1）Prepare MPO antibody-fluorescent microsphere compounds；
（2）Nitrocellulose filter is pasted onto to the centre position of bottom plate, and overlying contact is carried out with the water absorption pad of upper end, will be diluted
T lines and C on nitrocellulose filter of 1-2mg/ml MPO monoclonal antibodies and 1-2mg/ml sheep anti-mouse igg polyclonal antibodies
Line position is crossed, drying；
（3）Glass fibre component is immersed in treatment fluid, is dried after pulling out, by step（1）The MPO antibody-fluorescents of preparation are micro-
Ball compound is sprayed on the glass fibre component of drying, and bonding pad is made in drying；
（4）Group item：By sample pad, water absorption pad and step（3）The bonding pad of preparation overlaps assembling successively, and cutting into strips, is made
Myeloperoxidase fluorescence immune chromatography quantitative testing test paper item.
The present invention also provides a kind of myeloperoxidase fluorescence immune chromatography quantitative testing test paper cards, wherein will be above-mentioned
In the rectangle that myeloperoxidase fluorescence immune chromatography quantitative testing test paper item is packed into gets stuck.
Preferably, the rectangle gets stuck including plastics upper casing and plastics lower casing, and nitrocellulose membrane is corresponded on epivalve
Position be equipped with observation window, the position that sample pad is corresponded on epivalve is equipped with well, and well is located among sample pad.
The beneficial effects of the invention are as follows：
The present invention provides a kind of highly sensitive myeloperoxidase based on double-antibody sandwich law technology, fluorescence immune chromatography technology
Enzyme（MPO）Immunochromatographiassay assay quantitative detection test paper.MPO fluorescent chromatographics test strips have the advantage that in the present invention：1）It is quickly accurate
Determine amount：Integrate the characteristics of colloidal gold is quick, enzyme linked immunological is quantitative, chromatographic mass spectrometry accurately detects, realizes that 5min is quickly accurate
Determine amount detection；2）With to inspection：To detecting sample size no requirement (NR), both can single or a small amount of sample with to inspection, also can be a large amount of
Sample detects simultaneously, and can realize Site Detection；3）Configuration requirement is low：It is low to mating instrument and equipment and personnel requirement, operation
Personnel only need short-term cultivation that can skillfully grasp；4）It is cost-effective：Production cost is lower than colloidal gold, more competitive advantage.It is logical
Cross fluorescence immune chromatography readout instrument interpretation is carried out to result, it can be achieved that the subjective element of automation influence, provide it is convenient, quickly, can
The diagnostic result leaned on.The detection of the present invention has highly sensitive, high stability, range of linearity 3.125-600ng/ml, nothing
Non-specificity, detection time only need 5min, and can realize fast inspection by bed, greatly improve clinical diagnosis efficiency.
Description of the drawings
Fig. 1 is the structural schematic diagram of test strips prepared by the embodiment of the present invention 1；
In figure：1. sample pad；2. bonding pad；3. nitrocellulose filter；4. water absorption pad；5. bottom plate.
Fig. 2 is that the concentration-T/C of test strips linear criterion product prepared by the embodiment of the present invention 1 is worth canonical plotting；
Fig. 3 is the photo that test strips show test result in the embodiment of the present invention 1；
Fig. 4 is the fluorescence intensity level for the test strips that in the embodiment of the present invention 2 prepared by different protection liquid；
Fig. 5 is the fluorescence photo for the test strips that in the embodiment of the present invention 2 prepared by different protection liquid；
Fig. 6 is to quantify to obtain when testing result is verified to test strips of the present invention using ELISA method in the embodiment of the present invention 4
Correlation curve.
Specific implementation mode
Below by specific embodiment, the invention will be further described.The MPO monoclonals used in following embodiment are anti-
Body, MPO standard items and MPO quality-control products, fluorescent microsphere, nitrocellulose filter etc. are by Henan Biology Engineering Research Center
It provides.The model of fluorescent chromatographic readout instrument：FIC-2016-A5.
Embodiment 1
MPO antibody-fluorescent immunochromatographiassay assay quantitative detection test papers are prepared with the following method：
A. the preparation of EDC methods is used to state MPO antibody-fluorescent microsphere compounds：
15ul fluorescent microspheres are taken to be added in 0.6ml 0.05mol/l pH8.0 borate buffer solutions, vortex shakes mixing；It is added
The EDC of 40ul 4mg/ml, room temperature concussion activation 30min；In microspheres solution after activation, it is anti-that 125 μ g MPO labels are added
Body is coupled 90min；The immune microsphere compound that will be coupled, is redissolved with protection liquid, and the group of the protection liquid becomes
0.05M pH8.0 Tris-HCl buffer solutions, 0.5%T-20,0.2%BSA, 0.5% casein, 4% trehalose and 0.05%NaN3, so
After use confining liquid（Include 10% BSA, 0.05% NaN3）2h is closed, MPO antibody-fluorescent microsphere compounds are obtained.
B. assembling test strips are prepared：
（1）Glass fibre component is immersed in treatment fluid, treatment fluid includes that working concentration is the Tris-that 0.01M, pH are 8.0
HCl buffer solutions, the BSA that mass concentration is 0.1 ~ 0.5%, the polysorbas20 and mass concentration that mass concentration is 0.05% ~ 0.5 are
The NaN that 3% sucrose and mass concentration are 0.05%3, it is dry after immersion, as bonding pad；
（2）MPO monoclonal antibodies are diluted to 2mg/ml with coating dilution, sheep anti-mouse igg antibody is diluted to 1.5mg/ml,
Film liquid amount is 1-2 μ l/cm, using them as detection line（T lines）And nature controlling line（C lines）Uniformly draw in nitrocellulose filter
On, detection line（T lines）And nature controlling line（C lines）Between be divided into 5mm, be placed in drying room, in 37 DEG C of temperature, 10% time drying of humidity is not
Less than 8h；
（3）Sample pad 1, bonding pad 2, nitrocellulose filter 3 and water absorption pad 4 are overlapped successively and are assembled in bottom plate 5（PVC board）On,
Wherein, water absorption pad and bonding pad overlap the both ends for being pressed in nitrocellulose filter respectively, and are formed on the surface of nitrocellulose filter
Detection zone；Sample pad is overlapping to be pressed on bonding pad, and the big plate of test paper is obtained after assembling, cuts into wide 3.9mm, test strips are made, is tied
Structure is as shown in Figure 1.
C. test card is prepared：
It is got stuck using the rectangle with plastics upper casing and plastics lower casing, above-mentioned manufactured test strips, which are packed into interior formation of getting stuck, to be tried
Paper card, wherein be equipped with observation window corresponding to the position of nitrocellulose membrane on epivalve, the position of sample pad is corresponded on epivalve
Equipped with well, well is located among sample pad.
To the test strips functional attributes of above-mentioned preparation（Linearly）It is studied：
The test strips of above-mentioned preparation are restored into room temperature, use a concentration of 3.125,6.25,12.5,25,50,100,200 respectively,
300,400,600ng/ml linear MPO standard items are loaded in test strips, and test card is put into storehouse, is exempted from fluorescence after 5min
Epidemic disease chromatographs the fluorescence intensity level that T lines and C lines are read on readout instrument, and using above-mentioned standard concentration as abscissa, T/C values are as vertical
Coordinate draws standard curve, as shown in Fig. 2, the range of linearity is 3.125-600ng/ml, R2Up to 0.9841.The qualitative inspection of test card
The photo of result is surveyed as shown in figure 3, wherein there are three kinds of sentence read results：1）C lines have fluorescent brightness appearance, and T line unstressed configurations are bright
Degree, judgement result is for feminine gender or less than detection limit（Left side）；2）C and T lines show fluorescent brightness, and judgement result is the positive（In
Between）；3）C line unstressed configuration brightness occurs, and for T lines with or without fluorescent brightness, judgement test strips result is invalid（Right side）.
Embodiment 2：Reaction condition optimization
The optimization of EDC amounts：
15ul fluorescent microspheres are taken to be added in 0.6ml 0.05mol/l pH8.0 borate buffer solutions, vortex shakes mixing；Successively plus
Enter 4mg/ml EDC it is each 10,20,40,60,80ul, room temperature concussion activation 30min prepares 5 groups of test paper as described in Example 1
Item is loaded detection respectively with linear criterion product, and fitting obtains normal equation, detects the range of linearity and R2Value, as a result such as 1 institute of table
Show：
Table 1
As shown in table 1, linear best under the conditions of 160 μ g of EDC are added, optimum linear range 3.125 ~ 600 ng/ml, R2=
0.9803。
The optimization of coupling protein amount and coupling time：
In microspheres solution after activation, sequentially add 75,100,125,150 μ g MPO labelled antibodies, by 30min, 60min,
90min, 120min time are coupled respectively.The test strips of different coupling protein amounts and coupling time are prepared as described in Example 1,
Detection 3 is repeated to different condition test strips with the quality-control product of 100ng/ml, 3 average T line fluorescent values are as a result taken, as a result such as table
Shown in 2.
Table 2
As shown in table 2, with the increase of protein labeling amount, T line fluorescent values are gradually increasing；As coupling time increases, T line fluorescence
Value is fallen after rising.125 μ g of coupling protein amount, when being coupled 1.5h, T line fluorescent value highests, 125 μ g of best coupling protein amount, coupling
1.5h。
Protect liquid compositional optimization：
The immune microsphere compound that will be coupled is redissolved with 4 kinds of protection liquid respectively：
1）0.05M pH8.0 borate buffers, 1%T-20,0.2%BSA, 0.5% casein, 4% trehalose, 0.05%NaN3；
2）0.05M pH8.0 Tris-HCl buffer solutions, 1%T-20,0.2%BSA, 0.5% casein, 4% trehalose, 0.05%
NaN3；
3）0.05M pH8.0 borate buffers, 0.5%T-20,0.2%BSA, 0.5% casein, 4% trehalose, 0.05%NaN3；
4）0.05M pH8.0 Tris-HCl buffer solutions, 0.5%T-20,0.2%BSA, 0.5% casein, 4% trehalose, 0.05%
NaN3,
4 groups of test strips are prepared as described in Example 1, repeat to detect 3 with the quality-control product of 100ng/ml, take 3 average T lines glimmering
Light value simultaneously observes 4 condition fluorescence phenomenons under uv analyzer, as a result as shown in Figure 4 and Figure 5.
Two kinds of buffer solutions are compared, and Tris-HCl buffer solutions redissolution effect is best, and Tris-HCl buffer solution T lines fluorescent values compare boron
Acid buffer fluorescent value is high（Fig. 4）；By the content of change T-20, clustering phenomena significantly reduces in test strips（Fig. 5）.It is final true
Fixed No. 4 are best protection liquid.
Embodiment 3
Detection of Stability is carried out to test strips prepared by the embodiment of the present invention 1：
Test strips prepared by embodiment 1 are stored in respectively under 37 DEG C and 4 DEG C of environment, by with linear criterion product examine survey 37 DEG C, 4
DEG C preserve test strips, each linear R of condition2Result of variations is shown in Table 3.
Table 3
As can be seen from Table 3, test strips of the invention preserve 180 days linear R under 4 DEG C of preservation conditions2Can reach 0.97 with
On, it has good stability.Under 37 DEG C of preservation conditions of test strips, when preserving 10 days, R2Value is reduced to 0.96, and detection error increases.
1 day is stored at 37 DEG C close to the effect for storing about 45 days at 4 DEG C, thus it is speculated that test strips can at least store 8 under the conditions of 4 DEG C
Month.
4 clinical trial of embodiment
Sample situation：Clinical sample is obtained by relevant hospital, totally 45 parts of plasma samples.
Detecting step：Accurate 7 μ l serum samples of drawing are placed in 1.5ml centrifuge tubes, and 693 μ l Sample dilutions are added（Physiology
Brine）Into centrifuge tube.After setting fluorescent chromatographic readout instrument relevant parameter, after test strips are restored room temperature, in well position
It sets and 70 μ L of measuring samples is added, test card is put into storehouse, test strips read fluorescence intensity in fluorescent chromatographic readout instrument after 5min
Value calculates MPO contents according to standard curve.
With the above-mentioned 45 parts of plasma samples of U.S.'s Cleveland HeartLab ELISA method Parallel testings, MPO contents are measured
Between 0 ~ 600ng/ml, using fluorescent chromatographic method detectable concentration as abscissa (X-axis), ELISA method detection concentration is sat as vertical
It marks (Y-axis), draws scatter plot, carry out linear regression analysis, the results are shown in Figure 6：Equation of linear regression is：y = 1.0164x+
11.825 R2=0.9795, show that both detection methods have good correlation, it is seen then that prepared by the embodiment of the present invention 1
Test strips realize the qualitative detection of MPO well, and accuracy is high, and detection speed is fast, are suitable for clinical detection.
Sensitivity experiment is carried out to the test strips of the present invention
Blank control is done with physiological saline, replication 20 times obtains T/C values, and calculates average value（M）And standard deviation（SD）,
2 regression fits are carried out according to the concentration results between zero-dose and adjacent modular product and obtain linear function, by the knot of M+2SD
Fruit substitutes into above-mentioned equation, finds out corresponding concentration value, as minimum detectability.As a result：Minimum detectability 0.9ng/ml is sensitive
Degree is high.
Reaction time experiment is carried out to the test strips of the present invention
It uses 300 ng/ml, 100ng/ml, 10ng/ml quality-control product serum as sample, quality controlled serum 70 is added in sample-adding hole site
Test card is inserted into fluorescent quantitative detector, every one minute fluorescence intensity, as a result shows by μ L：Three special quality control product examines
It surveys fluorescent value to tend towards stability after 5min or so, ELISA test strip speed of the invention is fast.
Claims (10)
1. a kind of myeloperoxidase fluorescence immune chromatography quantitative testing test paper item, including：Bottom plate, sample pad, bonding pad, nitric acid
Cellulose membrane and water absorption pad, the sample pad, bonding pad, nitrocellulose filter and water absorption pad overlap be assembled on bottom plate successively,
Wherein, water absorption pad and bonding pad overlap the both ends for being pressed in nitrocellulose filter respectively, and are formed on the surface of nitrocellulose filter
Detection zone；Sample pad is overlapping to be pressed on bonding pad, and MPO antibody-fluorescent microsphere compounds are fixed on the bonding pad；The inspection
The detection for identifying that the monoclonal antibody of another epitope of myeloperoxidase is constituted is coated on the nitrocellulose filter surveyed in area
The nature controlling line that line and sheep anti-mouse igg polyclonal antibody are constituted, wherein activate fluorescent microsphere by carbodiimide EDC, with MPO
Antibody coupling forms compound, then by protecting liquid redissolution, confining liquid to close, it is compound to be finally prepared into MPO antibody-fluorescent microballoons
Object.
2. myeloperoxidase fluorescence immune chromatography quantitative testing test paper item according to claim 1, which is characterized in that described
Fluorescent microsphere is carboxyl, amino or hydroxyl fluorescent microsphere, preferably carboxyl fluorescent microsphere.
3. myeloperoxidase fluorescence immune chromatography quantitative testing test paper item according to claim 2, which is characterized in that described
Fluorescent microsphere is the rare earth metal chelating object of grain size 100-300nm, preferably lanthanide series europium（Eu3+）Chelating object.
4. myeloperoxidase fluorescence immune chromatography quantitative testing test paper item according to claim 1, which is characterized in that activation
It is 8-12 times of fluorescent microsphere volume with the weight of EDC, preferably 10.7 times.
5. myeloperoxidase fluorescence immune chromatography quantitative testing test paper item according to claim 1, which is characterized in that coupling
In the process, the weight of MPO antibody is 6-10 times of fluorescent microsphere volume, preferably 8-8.5 times, coupling time 1.2-2h, preferably
1.5h。
6. myeloperoxidase fluorescence immune chromatography quantitative testing test paper item according to claim 1, which is characterized in that multiple
The molten group with protection liquid is divided into Tris-HCl, PB of 0.01-0.05M, borate buffer solution one of which, mass concentration 0.5%
~ 1% BSA, PEG, glycine, casein are one or more kinds of, the trehalose, sucrose, oligosaccharide that mass concentration is 1% ~ 5%
One or more, polysorbas20 that mass concentration is 0.05% ~ 0.5, Tween 80, Triton X-100, S9 are one or more kinds of,
The NaN of 0.05%-0.1%3, the protection liquid is preferably composed of the following components：The Tris-HCl buffer solutions of 0.05M pH 8.0,
0.5% polysorbas20,0.2% BSA, 0.5% casein, 4% trehalose and 0.05% NaN3。
7. myeloperoxidase fluorescence immune chromatography quantitative testing test paper item according to claim 1, which is characterized in that institute
It is to be used as haptens by polypeptide 1 and polypeptide 2 to state MPO antibody, is prepared into comlete antigen, then obtained through mouse peritoneal immunization
The sequence of MPO monoclonal antibodies or other identical antibody of performance, the polypeptide 1 is SEQ ID No.1：
The sequence of SGCAYQDVGVTCPEQDKY, the polypeptide 2 are SEQ ID No.2：SKNNIFMSNSYPRDFVNC.
8. the preparation side of the myeloperoxidase fluorescence immune chromatography quantitative testing test paper item described in any one of claim 1-7
Method, the method includes：
（1）Prepare MPO antibody-fluorescent microsphere compounds；
（2）Nitrocellulose filter is pasted onto to the centre position of bottom plate, and overlying contact is carried out with the water absorption pad of upper end, will be diluted
T lines and C on nitrocellulose filter of 1-2mg/mlMPO monoclonal antibodies and 1-2mg/ml sheep anti-mouse igg polyclonal antibodies
Line position is crossed, drying；
（3）Glass fibre component is handled and dried using treatment fluid, by step（1）The MPO antibody-fluorescent microballoons of preparation are compound
Object is sprayed on the glass fibre component of drying, and bonding pad is made in drying；
（4）Group item：By sample pad, water absorption pad and step（3）The bonding pad of preparation overlaps assembling successively, and cutting into strips, is made
Myeloperoxidase fluorescence immune chromatography quantitative testing test paper item.
9. a kind of myeloperoxidase fluorescence immune chromatography quantitative testing test paper card, which is characterized in that by the claims 1-7
Any one of described in myeloperoxidase fluorescence immune chromatography quantitative testing test paper item be packed into rectangle gets stuck.
10. myeloperoxidase fluorescence immune chromatography quantitative testing test paper card according to claim 9, which is characterized in that institute
Rectangle to be stated to get stuck including plastics upper casing and plastics lower casing, the position that nitrocellulose membrane is corresponded on epivalve is equipped with observation window,
It is equipped with well corresponding to the position of sample pad on epivalve, well is located among sample pad.
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| CN109324181A (en) * | 2018-10-22 | 2019-02-12 | 河北特温特生物科技发展有限公司 | A kind of preparation method of immunochromatography sealant compositions, purposes and immune chromatography reagent kit |
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