CN109001449A - Measure the immunochromatographytest test kit and preparation method thereof of platelet-activating factor acetylhydro-lase - Google Patents
Measure the immunochromatographytest test kit and preparation method thereof of platelet-activating factor acetylhydro-lase Download PDFInfo
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- CN109001449A CN109001449A CN201810708123.9A CN201810708123A CN109001449A CN 109001449 A CN109001449 A CN 109001449A CN 201810708123 A CN201810708123 A CN 201810708123A CN 109001449 A CN109001449 A CN 109001449A
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- monoclonal antibody
- pad
- pla2
- mouse
- nitrocellulose filter
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Abstract
The present invention relates to a kind of immunochromatographytest test kits for measuring platelet-activating factor acetylhydro-lase, it is by immuno-chromatographic test paper strip and plastic shell, the immuno-chromatographic test paper strip is by bottom plate, sample pad, reagent pad, the test strips of nitrocellulose filter and absorption pad composition, the sample pad, reagent pad, nitrocellulose filter and absorption pad are successively staggeredly fixed on the bottom plate, the reagent pad is coated with the color fluorescence microballoon of the anti-human Lp-PLA2 labeling of monoclonal antibody of mouse, detection line on nitrocellulose filter is coated with the anti-human Lp-PLA2 monoclonal antibody of mouse, nature controlling line is coated with mountain sheep anti mouse polyclonal antibody, the kit qualitative determination line of demarcation concentration is 200ng/ml, fluorescence immunity analyzer detection sensitivity is 10ng/ml, the range of linearity is 10-1000ng/m L can quickly obtain qualitative results using color fluorescence microballoon and slightly obtain quantitative result late, have the characteristics that it is easy to operate, be swift in response, high sensitivity, qualitative can quantify, help meet the needs of different inspection scenes.
Description
Technical field
The present invention relates to immunochromatography technique fields in Medical Immunology, and specifically one kind can be fast and accurately right
Lp-PLA2 carries out the immunochromatographytest test kit of the measurement platelet-activating factor acetylhydro-lase of qualitative and quantitative analysis simultaneously.
Background technique
With the further investigation to Pathogenesis of Atherosclerosis, it has been found that in atherosclerosis generation, development
Evolution process in, have the participation of the various a large amount of inflammatory mediators of inflammatory cell core always, inflammation is progression of atherosclerosis
Central factor in the process.Lp-PLA2 is the one kind attracted extensive attention in recent years and atherosclerosis Cardial or cerebral vascular diseases
Closely related phospholipase A2 superfamily, more and more researches show that Lp-PLA2 and coronary atherosclerotic heart disease
(coronary heart disease) stroke event is related closely.Lp-PLA2 is for the special of the i.e. AS severity of blood vessel endothelium inflammation
Property Inflammation Marker, can effectively early warning, assess cardiovascular and cerebrovascular embolism class diseases occurrence risk, reduce cardio-cerebral vascular disease patient or
The incidence of the serious adverse events such as people at highest risk's burst heart infarction/cerebral infarction, escorts for life.
Platelet-activating factor acetylhydro-lase (Lp-PLA2) is a kind of new inflammatory reaction marker generally acknowledged in the world at present.
Lp-PLA2 in blood is mainly generated by macrophage, can hydrolyze the phosphatide aoxidized in low-density lipoprotein (LDL), hydrolysis
Product can promote Atherosclerosis, for the Specific marker for reflecting vascular inflammation.Lp-PLA2 level increase also with it is dynamic
The ulceration of pulse atherosclerosis patch is related, thus can be used for the evaluation of atherosclerotic plaque unstability.Lp-PLA2 level increases
Predict coronary heart disease, a kind of independent hazard factor of cardiovascular event and stroke risk and predictive factor.
Lp-PLA2 detection method mainly has quantitative detecting method and qualitative checking method, and quantitative detecting method has dual anti-folder
Heart method (ELISA), chemoluminescence method, latex particle enhancing immunoturbidimetry, immunofluorescence technique etc., qualitative method mainly has colloid
Golden immunochromatographic method.Quantitative approach generally needs expensive instrument, and it is all partially long to obtain the result time, and Lp-PLA2 is detected
There is an urgent need to obtain testing result as early as possible for the diseases such as relevant myocardial infarction, heart failure；Although and existing qualitative detection can be used
When it is shorter, but qualitative results are easy also obtain the severity of the state of an illness when obtaining positive findings there are deviation.Such as
What, which is able to solve, is quickly obtained result for the strong diagnosis and treatment of timeliness such as myocardial infarction and obtains quantitative result and determine the later period
Immunotherapy targeted autoantibody is the major issue of current clinical diagnosis product research field urgent need to resolve.
Summary of the invention:
The present invention for shortcoming and defect existing for existing testing product, propose one kind combine it is quick with color micro-sphere
The advantages of qualitative and fluorescent microsphere accurate quantitative analysis, being both able to satisfy only need to be qualitative as fast as possible but to find out result, or lacks
Instrument, without the demand of the special surveys scene such as power supply, and can in conjunction with fluorescence immune chromatography analyzer realization need high sensitivity,
The fluorescence immune chromatography detection kit and preparation method thereof of the measurement Lp-PLA2 of the measurement Lp-PLA2 index of accurate quantitative analysis.
The present invention can be achieved by the following measures:
A kind of immunochromatographytest test kit measuring platelet-activating factor acetylhydro-lase, by immuno-chromatographic test paper strip and plastics
Shell composition, the immuno-chromatographic test paper strip are made of bottom plate, sample pad, reagent pad, nitrocellulose filter and absorption pad, institute
It states sample pad, reagent pad, nitrocellulose filter and absorption pad to be successively staggeredly fixed on the bottom plate, the reagent pad is coated with
The color fluorescence microballoon of the anti-human Lp-PLA2 labeling of monoclonal antibody of mouse, it is anti-human that the detection line on nitrocellulose filter is coated with mouse
Lp-PLA2 monoclonal antibody, nature controlling line are coated with mountain sheep anti mouse polyclonal antibody.
The diameter range of the color fluorescence microballoon of reagent pad of the present invention is 180-200nm；The color fluorescence microballoon
It is any one or a few of the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd) doped with rare earth lanthanide
Mixture；The coloured dye of the color fluorescence microballoon doping contains amino or hydroxy functional group, is acid violet 1, acid violet
7, acid blue 25, any one or a few the mixture in disperse red 58.
Reagent pad of the present invention enamel microballoon label antibody sources in the monoclonal for 2 different epitopes
Antibody cell strain, the coated antibody sources of detection line on the nitrocellulose filter are in the list for 2 different epitopes
Clonal antibody, 4 monoclonal antibody cell strains are made using following steps:
Step 1: the acquisition of cell strain of monoclonal antibody: using Lp-PLA2 protein immunization mouse, anti-using the monoclonal of standard
Preparation prepares the cell strain of monoclonal antibody of specific high-affinity, to the antibody of monoclonal antibody cell strain obtained preparation
Pairing carries out pairing screening two-by-two, preferably goes out 4 plants of monoclonal antibody cell strains for kit according to pairing result, wherein 2 plants are used for
Detection, two plants for capturing；
Step 2: the ratio of the monoclonal antibody of adjustment 4 plants of monoclonal antibody cell strains preparation preferably goes out to be used for final kit system
The ratio of standby monoclonal antibody mixture；
Step 3: a large amount of preparations of monoclonal antibody: being prepared using the ascites production technology of standard and it is mono- to purify Lp-PLA2
Clonal antibody, be stored in after packing -20 DEG C it is spare
Reagent pad of the present invention is made using following steps: glass fibre membrane is soaked in X- containing 1.5%Triton
In the 200mM Tris-HCL treatment fluid of 100,2.0%BSA, pH7.5,4 DEG C are impregnated 3 hours, then take out 37 DEG C of baking oven drying 4
Hour, it is spare, by glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platform, connect with Bio-Jet Quanti300 is non-
The anti-human Lp-PLA2 labeling of monoclonal antibody color fluorescence microballoon of mouse is sprayed onto glass fibre membrane, 37 DEG C of bakings by the micro- quantitation nozzle of touch
It is 2 hours dry.
The color fluorescence microballoon of the anti-human Lp-PLA2 labeling of monoclonal antibody of mouse in the present invention on reagent pad is using following step
It is rapid to be made:
Step 1: the aldehyde radical of color fluorescence microballoon: taking 2mg color fluorescence microballoon, and the carbonate with 100mM, pH9.5 is slow
Fliud flushing is washed 3 times, centrifugal speed 12000rpm using centrifugal process, and the time is 15 minutes, is finally resuspended in the above-mentioned of 100 μ l
In carbonate buffer solution, the glucan of 200 μ l aldehyde radicals is added, mixes, at room temperature dark reaction 4 hours, using same centrifugation
Method wash and be resuspended in the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is spare；
Step 2: 1mg is used for the mouse detected by the color fluorescence microballoon of the preparation anti-human Lp-PLA2 labeling of monoclonal antibody of mouse
Anti-human Lp-PLA2 monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, then with above-mentioned aldehyde radical colour it is glimmering
The mixing of light microballoon, 4 DEG C of reactions are overnight；Then, sodium borohydride is added to final concentration 5mM, 4 DEG C are reacted 4 hours；It adds isometric
Confining liquid (150mM Tris-HCL, pH7.5, contain 2%BSA, 8% sucrose), 4 DEG C closing overnight；Then 150mM Tris- is used
The buffer of HCL, pH7.5 are washed 3 times using centrifugal process, are resuspended in the 200mM Tris-HCL buffer of 100 μ l and (are contained
0.9%NaCL, 2%BSA, 0.5%Tween 20), 4 DEG C be kept in dark place it is spare.
The nitrocellulose filter of the present invention for being coated with detection line and nature controlling line is made by following steps:
Step 1: using coating dilution by the anti-human Lp-PLA2 monoclonal antibody of mouse and mountain sheep anti mouse polyclonal antibody tune respectively
For whole concentration to 2-8mg/ml, film liquid amount is 1-2 μ l/cm, is sprayed on nitric acid using them as detection line is parallel with nature controlling line
It is coated on cellulose membrane, 3-7mm is divided between detection line and nature controlling line, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
Sample pad of the present invention is made by following steps: glass fibre membrane is soaked in containing 1.5%Triton X-
100,2.5%BSA, 0.2M Tris buffer in the treatment fluid of pH7.5, in 4 DEG C of 4 hours of immersion, are subsequently placed in baking oven,
37 DEG C dry 2 hours.
The present invention during use, will test kit first and sample balanced to room temperature, take out test card, put down
It puts；Then 25 μ l serum samples are accurately drawn, sample is drawn 50 μ l samples when being whole blood, is added in sample aperture, then exist immediately
100 μ l Sample dilutions are added in the buffering fluid apertures of lower part, Sample dilution uses physiological saline or PBS；After 5-8 minutes, meat
Eye observation detection line and nature controlling line color change, can be obtained qualitative results；Set the correlation of fluorescence immune chromatography analyzer
Parameter in 15-30 minutes, carries out detection and obtains quantitative result.
The present invention provides a kind of use color fluorescence microballoon, and the Lp-PLA2 prepared using immunochromatography technique is qualitative and fixed
Immunochromatographytest test kit is measured, it, can be to obtain qualitative results and then acquisition standard in very short time for the same kit
Determine amount as a result, meet simultaneously it is earlier obtain PRELIMINARY RESULTS and the below demand of acquisition accurate quantitative result, and can be with
It is poor applied to condition, without the inspection scene of instrument or power supply, with easy to operate, result is quick, measurement is accurate, suitable
The advantages that closing different inspection scenes.
Detailed description of the invention:
Attached drawing 1 is the structural schematic diagram of test strips in the present invention.
Attached drawing 2 is the monoclonal antibody pairing the selection result schematic diagram of embodiment 1 in the present invention.
Attached drawing 3 is the qualitative detection analysis result schematic diagram of embodiment 2 in the present invention.
Attached drawing 4 is the quantitative detection accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Appended drawing reference: PVC board 1, sample pad 2, bonding pad 3, nitrocellulose filter 4, water absorption pad 5.
Specific embodiment:
The present invention is further illustrated with reference to the accompanying drawings and examples:
As shown in Fig. 1, present invention firstly provides a kind of qualitative and quantitative determination Lp-PLA2 immunochromatographies to detect examination
Agent box, the kit include immuno-chromatographic test paper strip and plastic shell, and the immuno-chromatographic test paper strip is by bottom plate 1, sample pad
2, the test strips that reagent pad 3, nitrocellulose filter 4 and absorption pad 5 form, the sample pad 2, reagent pad 3, nitrocellulose filter
4 and absorption pad 5 successively staggeredly be fixed on the bottom plate 1, the reagent pad 3 is coated with the anti-human Lp-PLA2 monoclonal antibody of mouse
The color fluorescence microballoon of label, the detection line on nitrocellulose filter 4 are coated with the anti-human Lp-PLA2 monoclonal antibody of mouse, Quality Control
Line is coated with mountain sheep anti mouse polyclonal antibody.
The diameter of the color fluorescence microballoon of reagent pad 3 of the present invention is preferably 180-200nm；The color fluorescence microballoon
Be preferably doped with rare earth lanthanide, be the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd) any one or it is several
The mixture of kind；The coloured dye that the color fluorescence microballoon preferably adulterates contains amino or hydroxy functional group, is acid violet
1, any one or a few the mixture such as acid violet 7,25 disperse red 58 of acid blue.
Kit preparation method of the present invention is not restricted to above-mentioned concrete scheme, institute referring to foregoing summary
It is identical as the content of present invention for having the predictable fine tuning carried out on this basis all to treat as.
Embodiment 1:
The anti-human Lp-PLA2 monoclonal antibody of mouse that color fluorescence microballoon in the present invention on reagent pad combines is arranged by following
It applies obtained:
1, the acquisition of cell strain of monoclonal antibody: Lp-PLA2 protein immunization mouse is used, using the monoclonal antibody system of standard
Preparation Method prepares the cell strain of monoclonal antibody of at least 100 plants or more of specific high-affinity, to monoclonal antibody cell obtained
Strain prepares corresponding anti-monoclonal, is matched two-by-two with the method for 96 orifice plates, ELISA, carries out just to more than 100 plants of monoclonal antibodies
Sieve chooses 10 pairs of best antibody pair of reaction, amounts to 13 plants of cell strains, wherein being used for 5 plants of reagent pad, is used for nitrocellulose filter
8 plants.Further with above-mentioned 13 plants of monoclonal antibodies, aforementioned agents box preparation method is referred to according to primary dcreening operation result, prepares 40 groups of inspections
Test agent box further screens it according to developing time, colored intensity, background noise degree etc., preferably obtains and be used for reagent
The monoclonal antibody cell strain of pad and nitrocellulose filter is each 2 plants (attached drawings 2).
2, the determination of monoclonal antibody mixture: the ratio of the monoclonal antibody of adjustment 4 plants of monoclonal antibody cell strains preparation, it is final preferably to go out
The ratio of monoclonal antibody mixture for the preparation of final kit.
3, it a large amount of preparations of monoclonal antibody: is prepared using the ascites production technology of standard and purifies Lp-PLA2 monoclonal
Antibody, be stored in after packing -20 DEG C it is spare.
Embodiment 2: color micro-sphere qualitative test and fluorescence immunoassay accuracy test
Mentioned reagent box and fluorescence immune chromatography analyzer (model: NEO-007) are selected,
The setting of fluorescence immunity analyzer parameter: it after setting kit technological parameter on fluorescence immunity analyzer, takes
Above-mentioned assembled kit, respectively with the Lp-PLA2 calibration object of 10,25,100,200,400,600,800,1000ng/ml,
It is measured with kit, obtains the fluorescence intensity level of each calibration object, result is input in the parameter of analyzer, complete analysis
The final setting of the parameter of instrument.
Predominantly detect material: 200 parts of clinical samples are obtained by relevant hospital, related using the lipoprotein of desai diagnostic system
Phospholipase A2 assay kit carries out definite value, wherein 100 parts of serum sample, and 100 parts of whole blood sample, Lp-PLA2 content distribution area
Between between 10-1000ng/mL.
Detection method:
Step 1: will test kit and sample is balanced to room temperature, take out test card, lay flat；
Step 2: accurate to draw 25 μ l serum samples, sample is drawn 50 μ l samples when being whole blood, is added in sample aperture, then
100 μ l Sample dilutions are added in the buffering fluid apertures of lower part immediately, Sample dilution uses physiological saline or PBS；
After step 3:8 minutes, detection line and nature controlling line color change are visually observed, obtains qualitative results；
Step 4: setting the relevant parameter of fluorescence immune chromatography analyzer, in 15-30 minutes, carry out detection and quantified
As a result.
Test result analysis:
After the completion of the preparation of clinical sample detection reagent, all clinical samples are detected by detection method, and analyze inspection
Survey result.
Test result:
1, as shown in Fig. 3, qualitative results are analyzed, referring to the term of reference of Lp-PLA2, drafting 200ng/ml is
Line of demarcation, the following are feminine genders, and the above are the positives.Data analysis shows that, pilot system determine negative and positive number of samples and
The testing result consistency of commercial kit is more than 91.50%, shows that prepared detection kit is functional, Neng Gouman
The qualitative detection demand that foot quickly detects.
2, as shown in Fig. 4, quantitative result is analyzed, using the detected value of experimental system as Y-axis, with contradistinction system
Test value is X-axis, draws scatter plot, and carry out correlation analysis.Clinical sample is detected to 200 parts of clinical definite value pattern detections,
Sample mean deviation is less than 18%, and maximum deviation is less than 30%, R2 > 0.97, consistency coefficient > 0.90.Testing result shows
The detection kit of preparation is functional, is suitable for clinical detection, can satisfy the demand that client detects accurate quantitative analysis.
The present invention provides a kind of use color fluorescence microballoon, and the Lp-PLA2 prepared using immunochromatography technique is qualitative and fixed
Immunochromatographytest test kit is measured, which is 200ng/ml, fluorescence immunity analyzer detection
Sensitivity is 10ng/ml, range of linearity 10-1000ng/ml, can quickly obtain qualitative results simultaneously using color fluorescence microballoon
Slightly obtain quantitative result late, have the characteristics that it is easy to operate, be swift in response, high sensitivity, help to meet different inspection scenes
Demand.
Claims (4)
1. it is a kind of measure platelet-activating factor acetylhydro-lase immunochromatographytest test kit, by immuno-chromatographic test paper strip and plastics outside
Shell composition, the immuno-chromatographic test paper strip is made of bottom plate, sample pad, reagent pad, nitrocellulose filter and absorption pad, described
Sample pad, reagent pad, nitrocellulose filter and absorption pad are successively staggeredly fixed on the bottom plate, it is characterised in that the reagent
Pad is coated with the color fluorescence microballoon of the anti-human Lp-PLA2 labeling of monoclonal antibody of mouse, the detection line coating on nitrocellulose filter
There is the anti-human Lp-PLA2 monoclonal antibody of mouse, nature controlling line is coated with mountain sheep anti mouse polyclonal antibody.
2. a kind of immunochromatographytest test kit for measuring platelet-activating factor acetylhydro-lase according to claim 1,
It is characterized in that the diameter range of the color fluorescence microballoon of the reagent pad is 180-200nm；The color fluorescence microballoon doping
There is rare earth lanthanide, is europium (Eu), any one or a few the mixture in samarium (Sm), erbium (Er), neodymium (Nd)；The coloured silk
The coloured dye of color fluorescent microsphere doping contains amino or hydroxy functional group, is acid violet 1, acid violet 7, acid blue 25, divides
Dissipate any one or a few the mixture in red 58.
3. a kind of immunochromatographytest test kit for measuring platelet-activating factor acetylhydro-lase according to claim 1, special
Sign be the reagent pad enamel microballoon label antibody sources in the Monoclonal Antibody Cell for 2 different epitopes
Plant, the coated antibody sources of detection line on the nitrocellulose filter are in the monoclonal antibody for 2 different epitopes
Cell strain.
4. a kind of immunochromatography of the measurement platelet-activating factor acetylhydro-lase as described in any one of claim 1-3 detects examination
The preparation method of agent box, it is characterised in that 4 cell strains for different epitopes are made using following steps:
Step 1: the acquisition of cell strain of monoclonal antibody: Lp-PLA2 protein immunization mouse is used, using the monoclonal antibody system of standard
Preparation Method prepares the cell strain of monoclonal antibody of specific high-affinity, two-by-two to the antibody of monoclonal antibody cell strain obtained preparation
Pairing carries out pairing screening, preferably goes out 4 plants of monoclonal antibody cell strains for kit according to pairing result, wherein 2 plants are used to detect,
Two plants for capturing；
Step 2: the ratio of the monoclonal antibody of adjustment 4 plants of monoclonal antibody cell strains preparation preferably goes out for the preparation of final kit
The ratio of monoclonal antibody mixture；
Step 3: a large amount of preparations of monoclonal antibody: being prepared using the ascites production technology of standard and purify Lp-PLA2 monoclonal
Antibody, be stored in after packing -20 DEG C it is spare.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810708123.9A CN109001449A (en) | 2018-07-02 | 2018-07-02 | Measure the immunochromatographytest test kit and preparation method thereof of platelet-activating factor acetylhydro-lase |
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| CN201810708123.9A CN109001449A (en) | 2018-07-02 | 2018-07-02 | Measure the immunochromatographytest test kit and preparation method thereof of platelet-activating factor acetylhydro-lase |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424923A (en) * | 2015-09-10 | 2016-03-23 | 南京微测生物科技有限公司 | Color-fluorescence dual-function immunochromatography test strip and preparation method thereof |
| CN106645730A (en) * | 2016-12-15 | 2017-05-10 | 威海纽普生物技术有限公司 | Kit for determining lipoprotein-associated phospholipase A2 and manufacturing method |
| WO2018102982A1 (en) * | 2016-12-06 | 2018-06-14 | 亳州市新健康科技有限公司 | Poct fluorescence quantitative detection kit for hpv16 e7 protein and application thereof |
-
2018
- 2018-07-02 CN CN201810708123.9A patent/CN109001449A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105424923A (en) * | 2015-09-10 | 2016-03-23 | 南京微测生物科技有限公司 | Color-fluorescence dual-function immunochromatography test strip and preparation method thereof |
| WO2018102982A1 (en) * | 2016-12-06 | 2018-06-14 | 亳州市新健康科技有限公司 | Poct fluorescence quantitative detection kit for hpv16 e7 protein and application thereof |
| CN106645730A (en) * | 2016-12-15 | 2017-05-10 | 威海纽普生物技术有限公司 | Kit for determining lipoprotein-associated phospholipase A2 and manufacturing method |
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