CN106645730A - Kit for determining lipoprotein-associated phospholipase A2 and manufacturing method - Google Patents
Kit for determining lipoprotein-associated phospholipase A2 and manufacturing method Download PDFInfo
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- CN106645730A CN106645730A CN201611162009.8A CN201611162009A CN106645730A CN 106645730 A CN106645730 A CN 106645730A CN 201611162009 A CN201611162009 A CN 201611162009A CN 106645730 A CN106645730 A CN 106645730A
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention relates to the technical field of fluorescence immunochromatography in medical immunology and in particular relates to a kit for determining lipoprotein-associated phospholipase A2 and a manufacturing method. The kit is provided with a test paper card and is characterized in that the test paper card is provided with a PVC (Polyvinyl Chloride) plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad in sequence from bottom to top, wherein a lipoprotein-associated phospholipase A2 monoclonal antibody marked by a rare earth Eu<3+> fluorescent microsphere is adsorbed on the combination pad; the diameter of the fluorescent microsphere is 150nm; the rare earth fluorescent microsphere contains a rare earth lanthanide element Eu<3+> and is stable under a base state; the rare earth fluorescent microsphere sends out fluorescent light with the wavelength of 615nm under the action of a 337nm excitation light source; the monoclonal antibody is a purified and mixed monoclonal antibody and is selected from a monoclonal antibody cell strain aiming at 2 to 6 different antigenic epitopes of the lipoprotein-associated phospholipase A2; the kit has the advantages of simplicity and convenience for operation, rapid reaction, high sensitivity, high specificity and the like.
Description
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, including protein-crosslinking technology, film layer analysis skill
Art, labelling immunoassay technology etc..Specifically one kind can fast and accurately to fat in the samples such as serum, blood plasma and whole blood
Albumen associated phospholipase A2 carries out the platelet-activating factor acetylhydro-lase of quantitative analysis and determines kit and preparation method.
Background technology：
With the further investigation to Pathogenesis of Atherosclerosis, it has been found that occur, develop in atherosclerotic
Evolution process in, have the participation of a large amount of inflammatory mediators of various inflammatory cell cores all the time, inflammation is progression of atherosclerosis
During central factor.Lp-PLA2 is the one kind and atherosclerotic Cardial or cerebral vascular diseases for causing extensive concern in recent years
Closely related phospholipase A2 superfamily, increasing research shows Lp-PLA2 and coronary atherosclerotic heart disease
Being related for (coronary heart disease) stroke event is close.Lp-PLA2 is for the special of the i.e. AS orders of severity of blood vessel endothelium inflammation
Property Inflammation Marker, can effectively early warning, assessment 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, is that life escorts.
Platelet-activating factor acetylhydro-lase (Lp-PLA2) is a kind of new inflammatory reaction mark generally acknowledged in the world at present.
Lp-PLA2 in blood is mainly produced by macrophage, can hydrolyze the phosphatide of oxidation in low-density lipoprotein (LDL), its hydrolysis
Product can promote Atherosclerosis, be the Specific marker for reflecting vascular inflammation.Lp-PLA2 levels raise also with it is dynamic
The ulceration of pulse atherosclerosis patch is related, thus can be used for atherosclerotic plaque unstability evaluation.Lp-PLA2 levels are raised
A kind of independent hazard factor and predictive factor of prediction coronary heart disease, cardiovascular event and palsy risk.
Immuno analytical method is detected such as using the immune response between trace antigen and corresponding antibody with high specificity
Hormone, medicine, protein, polypeptide, enzyme, tumor associated antigen, micro-element, virus, bacterium and metallic element etc. are biological living in vivo
Property material.Immuno analytical method includes labelling immunoassay, non-marked immunoassay and instrument immunoassay.This kit is utilized
The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be belonging to one kind of labelling immunoassay.
Fluoroimmunoassay (FIA) and radiommunoassay (RIA) since the advent of the world, experienced the development of decades, but
It is that people increasingly feel FIA because of naturally local too high, interference detection results；RIA adopts isotope marks, has pole to human body
Big harm is simultaneously made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme itself is unstable, pushes away
Wide application is restricted.At the beginning of the eighties, people beginning one's study fluorescent material and the isotope-labelled protein of replaced with rare earth element
Or antibody, TIME RESOLVED TECHNIQUE is incorporated into into field of biological detection, establish new ultramicron time-resolved fluoroimmunoassay point
Analysis technology (Time resolved Fluoroimmunoassay, abbreviation TrFIA).The technology adopts multidisciplinary advanced technology, collection
The characteristics of having tied other immunoassays, in fields such as immunology, molecular biology, cytology and medical science, obtains significant progress
With extensively application.
TrFIA make use of the trivalent rare earth ion with unique fluorescent characteristic and chelate be tracer replace fluorescent material,
Enzyme, isotope, chemiluminescent substance, labelled antibody, antigen, hormone, polypeptide, protein, nucleic acid probe and biological cell treat anti-
System is answered (such as antigen-antibody reaction, nucleic acid probe hybridization, biotin-labeled pentylamine reaction and the killing of target cell pairing effect cell
Effect etc.) occur after, with TrFIA detectors determine product in fluorescence intensity.According to product fluorescence intensity and relatively glimmering
The ratio of luminous intensity, judges the concentration of analyte in reaction system, so as to reach quantitative analysis.In common fluoremetry,
Due to containing various fluorescent components in test sample, (colloidal solid and solvent molecule in sample causes background fluorescence
The non-specific fluorescence that scattered light and Proteins in Serum and other compounds send) intensity is big, disturb strong, becomes fluorescence point
The bottleneck that analysis method is promoted on a large scale.Why TrFIA can become after the new sensitive detection method of the latter of EIA, RIA,
Depend primarily on the wavelength resolution and time-delay technique that adopt in the unique fluorescence feature of lanthanide series, detection and dissociation-
Enhancing technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, has in 17, be usually used in TrFIA mainly have europium (Eu),
Samarium (Sm), terbium (Tb), dysprosium (Dy).Lanthanide series has unique fluorescence radiation feature, compared with common fluorescent, lanthanide ion chela
Compound fluorescence decay time is long, is the 10 of conventional fluorescent3-106Times.If the fluorescence decay time of lanthanide ion chelate is in 60-
900 μ s, conventional Eu3+Fluorescence decay time is 714 μ s, and the fluorescence decay time of fluorogen only has in common fluorescent immunoassay
1-100 μ s, the fluorescence decay time of some protein is only 1-10 μ s in sample, hence with TIME RESOLVED TECHNIQUE, postpones one
Measure after fixing time, just can obtain Eu3+Specific fluorescence signal.Simultaneously because decay time is long, Eu3+Label is in measurement
Between in can be excited repeatedly, ground state is transitted to quickly by excitation state after exciting every time, just have fluorescence to send, then can be weighed again
Newly excite, so it is per second have 1000 times excite so that the relative specific activity of TrFIA fluorescent markers is very high.Lanthanide series is glimmering
The maximum of light spectrum is characterized in that the Stokes displacements between exciting light and launching light are larger, Eu3+Excitation wavelength is 337nm, transmitting
Wavelength is 615nm, and Stokes displacements are up to 278nm；While Eu3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is very
Sharply, instrument adjustment can be made to determine in extremely narrow wave-length coverage, thus almost completely eliminates the interference of background fluorescence, after
And pass through time delay and wavelength resolution, and strong specificity fluorescent and background fluorescence are distinguished into open (therefore referred to as time resolution), make interference
Reach almost nil.
In view of the application of above labeling method and detection technique, this kit has good detection specific, higher
Sensitivity, the simplicity of operation and stable fluorescent marker ensure that the accuracy of detection.
The content of the invention：
The present invention is for shortcoming and defect present in prior art, it is proposed that a kind of utilization fluorescence immune chromatography it is sensitive
Property, the sensitivity that combined with fluorescent immunochromatographiassays assays instrument is realized is high, fast and simple, can be with the lipoprotein of accurate quantitative analysis correlation phosphatide
Enzyme A2 determines kit and preparation method.
The present invention can be reached by following measures：
A kind of platelet-activating factor acetylhydro-lase determines kit, is provided with test card, it is characterised in that the test card is by under
It is supreme to be sequentially provided with：Rare earth is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad
The anti-platelet-activating factor acetylhydro-lase monoclonal antibody-microballoon coupled complex of fluorescent microsphere mark, the rare-earth fluorescent microballoon
A diameter of 100-250nm, rare-earth fluorescent microballoon is stable under ground state containing one or more in rare earth lanthanide,
Launch the fluorescence that wave-length coverage is 550-650nm under the excitation source effect of 300-400nm；The monoclonal antibody is purifying
The monoclonal antibody for mixing afterwards, resists from the monoclonal for 2-6 different platelet-activating factor acetylhydro-lase epitope
Body cell strain.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 120-200nm；The rare-earth fluorescent microballoon
Preferably comprise one or more rare earth lanthanides；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 on pad
The monoclonal cell cell line of individual different epitopes.
Pad of the present invention is obtained using following steps：Glass fibre membrane is soaked in into 150mMTris-HCL process
In liquid (X-100 containing 1.0%Triton, 2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 little
When, it is standby, glass fibre membrane is placed on Bio-DotXYZ3050 three-dimensional specking platforms, connect with Bio-Jet Quanti300 are non-
The anti-platelet-activating factor acetylhydro-lase monoclonal antibody coupled complex spray that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon
To glass fibre membrane, 37 DEG C drying 1 hour after be obtained.
The anti-platelet-activating factor acetylhydro-lase monoclonal of the rare-earth fluorescent microballoon mark in the present invention on pad resists
Body is obtained using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：With platelet-activating factor acetylhydro-lase sterling immune mouse, using mark
Accurate method for preparing monoclonal antibody prepares the cell strain of monoclonal antibody of specific high-affinity, to the monoclonal antibody cell for being obtained
Strain carries out pairing screening, and the monoclonal antibody cell line for kit is preferably gone out according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Anti-grease albumen correlation phosphorus is prepared and purified using the ascites production technology of standard
Lipase A2 monoclonal antibodies, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate of pH 9.5 delays
Liquid is rushed, is washed 3 times using centrifugal process, centrifugal speed is 12000rpm, the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, dark reaction 4 hours under room temperature, using same centrifugal process
Washing and be resuspended in the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The preparation of the anti-platelet-activating factor acetylhydro-lase monoclonal antibody of rare-earth fluorescent microballoon mark：Choose from
The monoclonal antibody of the monoclonal cell cell line of 2 different epitopes, according to mass ratio 1:1 by 2mg lipoprotein correlation phosphorus
The above-mentioned carbonate buffer solution of lipase A2 monoclonal antibodies is then micro- with the rare-earth fluorescent of above-mentioned aldehyde radical in 4 DEG C of dialysed overnights
Ball mixes, and 4 DEG C of reactions are overnight；Then, add sodium borohydride to final concentration 5mM, 4 DEG C are reacted 4 hours；Add isopyknic envelope
Liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is closed, 4 DEG C of closings are overnight；Then 50mM Tris-HCL are used,
The buffer solution of pH7.4 is washed 3 times using centrifugal process, is resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l (containing 1.2%
NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.
The nitrocellulose filter for being coated with detection line and nature controlling line of the present invention is obtained by following steps：
Step 1：Using different thin of anti-platelet-activating factor acetylhydro-lase cell strain of monoclonal antibody used from pad
Born of the same parents' strain, prepares and purifies anti-platelet-activating factor acetylhydro-lase monoclonal antibody using the ascites production technology of standard, is stored in -20
It is DEG C standby；
Step 2：Coating dilution is used respectively by the anti-platelet-activating factor acetylhydro-lase monoclonal antibody in above-mentioned mouse source and goat-anti
Mouse IgG antibody adjusts concentration to 1-3mg/ml, and film liquid amount is 1-3 μ l/cm, is put down them as detection line and nature controlling line
Row is sprayed on nitrocellulose filter and is coated with, detection line and nature controlling line at intervals of 3-7mm, in being subsequently placed in baking oven, 37 DEG C
Drying 2 hours.
Sample pad of the present invention is obtained by following steps：Glass fibre membrane is soaked in containing 1.0%TritonX-
100,2.5%BSA, 0.15M Tris buffer solutions, in the treatment fluid of pH7.5, in 4 DEG C 4 hours are soaked, and are subsequently placed in baking oven
In, 37 DEG C dry 2 hours.
Present invention also offers the platelet-activating factor acetylhydro-lase preparation method that a kind of kit as described above is realized, it is special
Levy is to comprise the following steps：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, is kept flat；
Step 2：Card Reader：IC-card is placed on into dry type fluorescence immunity analyzer labeling position, relevant information is read, it is described glimmering
Light immunochromatographiassays assays instrument is a kind of Systems for optical inspection, is 10.0- to the measurement range of platelet-activating factor acetylhydro-lase
1000ng/mL；
Step 3：Sample-adding：Serum/plasma：Take 100 μ L serum/plasmas samples vertically to drop at test card sample-adding, whole blood：
Take 150 μ L whole blood samples vertically to drop at test card sample-adding, be careful not to suck bubble during sampling；
Step 4：Detection, can be detected, test automatically using test automatically or immediately test both of which：By test card
On the carrier of insertion dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis and detects to test card automatically,
Immediately test：Test card room temperature is placed after 15min, is inserted on the carrier of dry type fluorescence immunity analyzer, clicks on test immediately.
The present invention provides lipoprotein prepared by a kind of fluorescence immune chromatography technology of utilization rare earth carboxyl latex microballoon mark
Associated phospholipase A2 determines kit, while being adapted to serum, blood plasma and whole blood sample, and is adapted to clinically single part detection, phase
For the qualitative colloid gold reagent of platelet-activating factor acetylhydro-lase, the platelet-activating factor acetylhydro-lase in energy quantitative determination sample contains
Amount, with more specific Clinical significance of MG, with easy to operate, reaction is quick, sensitivity height, high specificity, be adapted to it is live
The advantages of detecting and be economical and practical.
Description of the drawings：
Accompanying drawing 1 is the structural representation of test card in the present invention.
Accompanying drawing 2 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 3 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 4 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
Specific embodiment：
With reference to the accompanying drawings and examples the present invention is further illustrated：
As shown in Figure 1, present invention firstly provides a kind of platelet-activating factor acetylhydro-lase determines kit, it is provided with box
Test card, the test card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4 and water suction
The anti-platelet-activating factor acetylhydro-lase monoclonal antibody-microballoon idol of rare-earth fluorescent microballoon mark is adsorbed with pad 5, wherein pad
Connection compound, a diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, under ground state
It is stable, the fluorescence of wavelength 615nm is launched under the excitation source effect of 337nm；The monoclonal antibody is to mix after purification
Monoclonal antibody, from the Monoclonal Antibody Cell for 2-6 different platelet-activating factor acetylhydro-lase epitopes
Strain.
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 150nm；The rare-earth fluorescent microballoon preferably comprises dilute
Native lanthanide series europium (Eu3+)；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 different epitopes on pad
Monoclonal cell cell line.
Embodiment 1：
Platelet-activating factor acetylhydro-lase is determined each part of test card in kit and can be obtained by following measures：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M Tris buffer solutions,
In the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, in being subsequently placed in baking oven, 37 DEG C dry 2 hours.2nd, fluorescent microsphere is adsorbed
The preparation of the pad 3 of labelled antibody：
Glass fibre membrane is soaked in 150mM Tris-HCL treatment fluids (X-100 containing 1.0%Triton, 2.5%
BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby, and glass fibre membrane is placed on into Bio-
On DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare earth Eu with Bio-Jet Quanti300 noncontacts3+Fluorescence is micro-
The anti-platelet-activating factor acetylhydro-lase monoclonal antibody coupled complex of ball mark is sprayed onto glass fibre membrane, and 37 DEG C dry 1 hour
After be obtained.
The aldehyde radical of rare-earth fluorescent Nano microsphere：5mg rare-earth fluorescent Nano microspheres are taken, with 20mM, the carbonate of pH9.5 delays
Liquid is rushed, is washed 3 times using centrifugal process, centrifugal speed is 12000rpm, the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, dark reaction 4 hours under room temperature, using same centrifugal process
Washing and be resuspended in the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Rare earth Eu3+The preparation of the anti-platelet-activating factor acetylhydro-lase monoclonal antibody of fluorescent microsphere mark：Choose from 2
The monoclonal antibody of the monoclonal cell cell line of different epitopes, according to mass ratio 1:1 by 2mg anti-grease albumen correlation phosphatide
The above-mentioned carbonate buffer solution of enzyme A2 monoclonal antibodies in 4 DEG C of dialysed overnights, then with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical
Mixing, 4 DEG C of reactions are overnight；Then, add sodium borohydride to final concentration 5mM, 4 DEG C are reacted 4 hours；Add isopyknic closing
Liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 50mM Tris-HCL are used,
The buffer solution of pH7.4 is washed 3 times using centrifugal process, is resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l (containing 1.2%
NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.
3rd, it is coated with the preparation of the nitrocellulose filter 4 of detection line and nature controlling line：
Using the different cell line of anti-platelet-activating factor acetylhydro-lase cell strain of monoclonal antibody used from pad, adopt
Anti- platelet-activating factor acetylhydro-lase monoclonal antibody is prepared and purified with the ascites production technology of standard, be stored in -20 DEG C it is standby；
Coating dilution is used respectively by the anti-platelet-activating factor acetylhydro-lase monoclonal antibody in above-mentioned mouse source and goat anti-mouse igg
Antibody adjusts concentration to 1mg/ml, and film liquid amount is 1 μ l/cm, and they are sprayed on into nitre as detection line is parallel with nature controlling line
It is coated with acid cellulose film, at intervals of 4mm, in being subsequently placed in baking oven, 37 DEG C dry 2 hours for detection line and nature controlling line.
The assembling of test card：Paste treated sample pad 2 successively in PVC board 1, be adsorbed with rare-earth fluorescence labeling
The pad 3 of antibody, the nitrocellulose filter 4 and adsorptive pads 5 that are coated with detection line and nature controlling line, obtain test paper big after assembling
Plate, cuts into as requested 4mm width, test paper is loaded in plastic clip and forms test card.
The equipment selected in above steps and raw material preferably following raw material：
Platelet-activating factor acetylhydro-lase specific pairs antibody；Platelet-activating factor acetylhydro-lase quality-control product：Britain's Landau reality
Test diagnosis Co., Ltd；Rare-earth fluorescent microballoon：Shanghai Zhen Zhun bio tech ltd；Celluloid (NC) film：
Millipore Products；Bovine serum albumin(BSA) (BSA), polyethylene glycol PEG20000, caseinhydrolysate：Sigma products, its
Its common agents is AR.
Embodiment 2：Accuracy test
From above-mentioned test card and fluorescence immune chromatography analyzer (model：NEO-007), fluorescence immunity analyzer parameter
Setting：After test card technological parameter is set on fluorescence immunity analyzer, the above-mentioned test card for assembling is taken, used respectively
10th, 100,200,400,600,800, the platelet-activating factor acetylhydro-lase calibration object of 1000ng/mL, is measured with test card,
The fluorescence intensity level of each calibration object is obtained, result is input in the parameter of analyzer, complete the setting of the parameter of analyzer.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 300 parts of latex enhancing immune turbidimetry definite value samples
Sheet, wherein 100 parts of serum sample, 100 parts of plasma sample, 100 parts of whole blood sample, platelet-activating factor acetylhydro-lase content distribution area
Between between 10.0-1000ng/mL.
During detection：First detection reagent and sample are balanced to room temperature, test card is taken out, is kept flat；Then Card Reader：IC-card is put
Put in dry type fluorescence immunity analyzer labeling position, read relevant information, step 3：Sample-adding：Serum/plasma：Take 100 μ L serum/
Plasma sample is vertically dropped at test card sample-adding；Whole blood：Take 150 μ L whole blood samples vertically to drop at test card sample-adding, take
It is careful not to suck bubble during sample, detection, can be using test automatically or immediately test both of which is detected；Automatically test：
Test card is inserted on the carrier of dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned to test card automatically
Analysis detection, immediately test：Test card room temperature is placed after 15min, is inserted on the carrier of dry type fluorescence immunity analyzer, is clicked on
Immediately test.Test result analysis：
After the completion of prepared by clinical sample detection reagent, all clinical samples are detected by detection method, and analyze inspection
Survey result.
Result of the test：
As shown in Figure 2, as Y-axis, the test value with contradistinction system draws scatterplot to the detected value with experimental system as X-axis
Figure, and carry out correlation analysis.Clinical sample detection is less than to 200 parts of clinical definite value pattern detections, sample mean deviation
10%, maximum deviation is less than 20%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection kit for preparing
Can be good, it is suitable for clinical detection, meet the differentiation needs of the different detection occasions of different clients.
It is quickly fixed that the present invention provides a kind of platelet-activating factor acetylhydro-lase of utilization rare-earth fluorescent immunochromatography technique preparation
Amount immunochromatographytest test kit, while being adapted to serum/plasma and whole blood sample, and is adapted to clinically single part detection, relatively
Platelet-activating factor acetylhydro-lase content in the qualitative colloid gold reagent of platelet-activating factor acetylhydro-lase, energy quantitative determination sample,
With more specific Clinical significance of MG, with easy to operate, reaction is quick, sensitivity height, high specificity, suitable Site Detection
And it is economical and practical the advantages of.
Claims (7)
1. a kind of platelet-activating factor acetylhydro-lase determines kit, is provided with test card, it is characterised in that the test card by down to
On be sequentially provided with：It is glimmering rare earth to be adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad
The anti-platelet-activating factor acetylhydro-lase monoclonal antibody-microballoon coupled complex of light microballoon mark, the rare-earth fluorescent microballoon
A diameter of 100-250nm, rare-earth fluorescent microballoon is stable under ground state containing one or more in rare earth lanthanide, in 300-
Launch the fluorescence that wave-length coverage is 550-650nm under the excitation source effect of 400nm；The monoclonal antibody is to mix after purification
The monoclonal antibody of conjunction, it is thin from the monoclonal antibody for 2-6 different platelet-activating factor acetylhydro-lase epitope
Born of the same parents' strain.
2. a kind of platelet-activating factor acetylhydro-lase according to claim 1 determines kit, it is characterised in that the combination
The diameter of the rare-earth fluorescent microballoon of pad is 100-200nm；The rare-earth fluorescent microballoon is doped with rare earth lanthanide Eu3+。
3. a kind of platelet-activating factor acetylhydro-lase according to claim 1 determines kit, it is characterised in that the rare earth
Fluorescent microsphere Eu containing rare earth lanthanide3+；The antibody sources of rare-earth fluorescent microballoon mark are in for 2 not synantigens on pad
The monoclonal cell cell line of epi-position.
4. a kind of platelet-activating factor acetylhydro-lase according to claim 1 determines kit, it is characterised in that the combination
Pad is obtained using following steps：Glass fibre membrane is soaked in into (X- containing 1.0%Triton in 150mM Tris-HCL treatment fluids
100,2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby, by glass fibre membrane
It is placed on Bio-DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare earth with Bio-Jet Quanti300 noncontacts
Eu3+The anti-platelet-activating factor acetylhydro-lase monoclonal antibody coupled complex of fluorescent microsphere mark is sprayed onto glass fibre membrane, 37 DEG C
Drying is obtained after 1 hour.
5. a kind of platelet-activating factor acetylhydro-lase according to claim 1 determines kit, it is characterised in that on pad
The rare-earth fluorescent microballoon mark platelet-activating factor acetylhydro-lase monoclonal antibody using following steps be obtained：
Step 1：The acquisition of cell strain of monoclonal antibody：With platelet-activating factor acetylhydro-lase sterling immune mouse, using standard
Method for preparing monoclonal antibody prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line to being obtained enters
Row pairing screening, the monoclonal antibody cell line for kit is preferably gone out according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Prepared and purified lipoprotein associated phospholipase A2 using the ascites production technology of standard
Monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate buffer solution of pH9.5,
Washed 3 times using centrifugal process, centrifugal speed is 12000rpm, the time is 5 minutes, is finally resuspended in the above-mentioned carbonate of 100 μ l
In buffer solution, the glucan of 500 μ l aldehyde radicals is added, mixed, dark reaction 4 hours under room temperature, washed using same centrifugal process
Be resuspended in the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The preparation of the platelet-activating factor acetylhydro-lase monoclonal antibody of rare-earth fluorescent microballoon mark：Choose from 2 not
The monoclonal antibody of the monoclonal cell cell line of synantigen epi-position, according to mass ratio 1:1 by 2mg platelet-activating factor acetylhydro-lases
Then the above-mentioned carbonate buffer solution of monoclonal antibody mixes in 4 DEG C of dialysed overnights with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical,
4 DEG C of reactions are overnight；Then, add sodium borohydride to final concentration 5mM, 4 DEG C are reacted 4 hours；Add isopyknic confining liquid
(50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 50mM Tris-HCL, pH7.4 are used
Buffer solution washed 3 times using centrifugal process, be resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l (containing 1.2%NaCL,
0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.
6. a kind of platelet-activating factor acetylhydro-lase according to claim 1 determines kit, it is characterised in that the coating
The nitrocellulose filter for having detection line and nature controlling line is obtained by following steps：
Step 1：Using the cell line different from platelet-activating factor acetylhydro-lase cell strain of monoclonal antibody used on pad, adopt
Prepared with the ascites production technology of standard and purified lipoprotein associated phospholipase A2 monoclonal antibodies, be stored in -20 DEG C it is standby；
Step 2：Coating dilution is used respectively by the anti-platelet-activating factor acetylhydro-lase monoclonal antibody in above-mentioned mouse source and sheep anti-Mouse
IgG antibody adjusts concentration to 1mg/ml, and film liquid amount is 1 μ l/cm, and they are sprayed on as detection line is parallel with nature controlling line
It is coated with nitrocellulose filter, at intervals of 4mm, in being subsequently placed in baking oven, 37 DEG C dry 2 hours for detection line and nature controlling line.
7. a kind of platelet-activating factor acetylhydro-lase according to claim 1 determines kit, it is characterised in that the sample
Pad is obtained by following steps：Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M
Tris buffer solutions, in the treatment fluid of pH7.5, in 4 DEG C 4 hours are soaked, and in being subsequently placed in baking oven, 37 DEG C dry 2 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201611162009.8A CN106645730A (en) | 2016-12-15 | 2016-12-15 | Kit for determining lipoprotein-associated phospholipase A2 and manufacturing method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611162009.8A CN106645730A (en) | 2016-12-15 | 2016-12-15 | Kit for determining lipoprotein-associated phospholipase A2 and manufacturing method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109001449A (en) * | 2018-07-02 | 2018-12-14 | 威海纽普生物技术有限公司 | Measure the immunochromatographytest test kit and preparation method thereof of platelet-activating factor acetylhydro-lase |
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