CN104237517B - Detect fluorescence immune chromatography test paper of people Lp-PLA2 albumen and preparation method thereof - Google Patents
Detect fluorescence immune chromatography test paper of people Lp-PLA2 albumen and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to fluorescence immune chromatography test paper detecting in people Lp-PLA2 albumen and preparation method thereof. Described test paper detects people Lp-PLA2 albumen by double antibody sandwich method, described double antibody sandwich method adopts a Lp-PLA2 monoclonal antibody that is marked with fluorescent microsphere as capture antibody, does a described Lp-PLA2 monoclonal antibody derive from sequence table SEQ? ID? NO.1 and SEQ? ID? one in sequence described in NO.2; And described double antibody sandwich method adopts the 2nd Lp-PLA2 monoclonal antibody as detecting antibody, does described the 2nd Lp-PLA2 monoclonal antibody derive from sequence table SEQ? ID? NO.1 and SEQ? ID? another one in sequence described in NO.2. Fluorescence immune chromatography test paper of the present invention is easy and simple to handle, quick, detection range is wide, specificity is high, sensitivity is good.
Description
Technical field
The invention belongs to chemiluminescent polypeptide and field of immunology, be specifically related to the relevant phosphorus of human lipoproteinLipase A2(Lp-PLA2) epitope peptide, the Lp-PLA2 for preparing with this epitope peptideSpecific antigen and corresponding monoclonal antibody or polyclonal antibody, described antibody are preparation peoplePurposes on Lp-PLA2 external diagnosis reagent case, people Lp-PLA2 external diagnosis reagent case,And a kind of for quantitatively detecting the fluorescence immune chromatography examination of determinand people Lp-PLA2 albumenPaper and preparation method thereof.
Background technology
Cardiovascular and cerebrovascular disease is acknowledged as one of maximum killer of harm humans health always,Also be murderous main reason in global range. China dies from cardiovascular and cerebrovascular disease every yearNumber up to more than 3,000,000, existing patient exceedes 6,000 ten thousand people. In recent years, along with residenceImproving constantly of people's living standard, the continuous deterioration of natural environment, various crowds' cardiovascular and cerebrovascularThe illness rate of disease, M & M are all the trend rising year by year. Especially person in middle and old agePeople's the incidence of disease is up to 62%, and therefore prophylactic treatment cardiovascular and cerebrovascular disease has become to carve and do not allowedSlow, it is not only related to broad masses of the people's healthy living problem, goes back to a certain extent shadowRinging the overall condition of whole national national quality.
The pathologic basis of cardiovascular and cerebrovascular disease is atherosclerotic. In recent years, research is foundAtherosclerotic is a kind of inflammatory reaction disease, and the product of inflammatory cell and release thereof is recognizedFor being topmost short atherosclerotic factor. In atherosclerosis plaque forming, progress and finalIn the process of breaking, all there is the participation of inflammatory reaction medium.
Therefore, find a kind of medium that can react this dynamic changing process particularly important,Have been found that in the world at present a kind of new inflammatory reaction mark, i.e. the relevant phosphatide of lipoproteinEnzyme A2(Lipoprotein-AssociatedPhospholipaseA2, Lp-PLA2).
Platelet-activating factor acetylhydro-lase, Lp-PLA2 write a Chinese character in simplified form in English, belongs to phospholipase A2 manFamily, is mainly secreted by inflammatory cell (as macrophage and lymphocyte). Lp-PLA2 isThe protein being made up of 441 amino acid, molecular weight is 45KDa. Lp-PLA2 has and fallsThe activity of separating platelet activating factor, is called again platelet-activating factor acetylhydrolase(PAF-AH). 80% Lp-PLA2 and low-density lipoprotein (LDL) combination, can waterSeparate the oxidation lecithin on low-density lipoprotein, generate lysolecithin and oxidized form free-fatAcid, the latter two are proinflammatory materials, therefore Lp-PLA2 has proinflammatory disease and short AtherosclerosisChange the effect forming.
In the time that atherosclerotic inflammation develops into the order of severity, Lp-PLA2 will be by greatlyAmount is released in blood, causes the level in its blood significantly to increase, and therefore Lp-PLA2 existsConcentration in blood can reflect the degree of inflammation of atherosclerotic plaque.
Therefore,, by detecting the Lp-PLA2 in blood, can effectively understand artery congee sampleThe degree of inflammation of plaque and stability thereof. So just can early warning myocardial infarction and cerebral thrombusOccur, to prevention cardiovascular and cerebrovascular, accident has considerable meaning.
Detecting the optimal method of Lp-PLA2 is immunoassays. Therefore, find suitable toolHave immunogenic Lp-PLA2 epitope peptide, prepare specific Lp-PLA2 antigen andAntibody become emphasis.
Determinand in fluorescence immune chromatography method survey blood is easy and simple to handle, quick, only needs 10Minute just can complete sample detection, and detection range is wide, sensitivity is good, can be timely rapidlyThe ground assisted diagnosis state of an illness, monitoring prognosis. Chinese patent application No.200910117820.8 is openA kind of preparation method and quantitative detecting method of fluorescent micro-ball immune chromatography test paper strip; China speciallyProfit application No.200910047352.1 discloses a kind of fluorescence immune chromatography test paper and preparation side thereofMethod and application. But, also there is no at present the fluorescence immune chromatography for people Lp-PLA2 albumenTest paper.
Summary of the invention
For solving existing problem in above-mentioned prior art, the invention provides a kind of forQuantitatively detect fluorescence immune chromatography test paper and the preparation side thereof of people Lp-PLA2 albumen in determinandMethod.
Particularly, the invention provides:
A kind of for quantitatively detecting the fluorescence immune chromatography examination of determinand people Lp-PLA2 albumenPaper, this test paper detects described people Lp-PLA2 albumen by double antibody sandwich method, wherein:
Described double antibody sandwich method adopts a Lp-PLA2 monoclonal that is marked with fluorescent microsphereAntibody is as capture antibody, and a described Lp-PLA2 monoclonal antibody derives from people Lp-PLA2One in epitope peptide (1) and (2); And
Described double antibody sandwich method adopts the 2nd Lp-PLA2 monoclonal antibody anti-as detectingBody, described the 2nd Lp-PLA2 monoclonal antibody derives from people Lp-PLA2 epitope peptide (1)(2) another one in;
Described people Lp-PLA2 epitope peptide (1) and (2) are respectively:
(1)Thr-Lys-Ile-Pro-Arg-Gly-Asn-Gly-Pro-Arg-Ser-Lys-Glu-Ser-Tyr；
(2)Tyr-Lys-Gly-Asp-Ile-Asp-Ser-Asn-Val-Ala-Ile-Asp-Leu-Ser-Asn。
Preferably, a described Lp-PLA2 monoclonal antibody is by a Lp-PLA2 antigenBe prepared from, a Lp-PLA2 antigen is by making described people Lp-PLA2 antigen tableOne in position peptide (1) and (2) and carrier protein couplet are prepared from; And
Described the 2nd Lp-PLA2 monoclonal antibody is to be prepared from by the 2nd Lp-PLA2 antigen, the 2nd Lp-PLA2 antigen is by making described people Lp-PLA2 epitope peptide (1)(2) another one in and carrier protein couplet are prepared from.
Preferably, the particle diameter of described fluorescent microsphere is 320nm to 400nm.
Preferably, the fluorescent material on described fluorescent microsphere is fluorescein isothiocynate, tetremBase rhodamine, TRITC or X-rhodamine, be wherein preferably X-Luo DanBright; The micro-sphere material of described fluorescent microsphere is polystyrene, polymethyl methacrylate or methylThe copolymer of methyl acrylate.
Preferably, the excitation wavelength of described fluorescent microsphere is 350～600nm, is preferably 390nm;Emission wavelength is 500～700nm, is preferably 615nm.
Preferably, described fluorescence immune chromatography test paper has base plate, and on this base plate edgeChromatography direction when use is provided with the way of contact successively: sample pad, pad, reaction film,Absorbent filter, described pad is provided with a described Lp-PLA2 who is marked with fluorescent microsphereMonoclonal antibody, described reaction film comprises detection zone and Quality Control district, described detection zone is coated to some extentState the 2nd Lp-PLA2 monoclonal antibody, described Quality Control district be coated with can with described being marked withThe antiantibody of the one Lp-PLA2 monoclonal antibody specificity combination of fluorescent microsphere.
Preferably, described reaction film is substantially not fluoresce under the wavelength that is greater than 550nmNitrocellulose filter.
Preferably, described base plate does not have photoluminescent property substantially.
Preferably, described antiantibody is sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg listClonal antibody, is wherein preferably sheep anti-mouse igg monoclonal antibody.
The present invention also provide a kind of prepare described for quantitatively detecting determinand peopleThe method of the fluorescence immune chromatography test paper of Lp-PLA2 albumen, it comprises the following steps:
1) provide a Lp-PLA2 monoclonal antibody that is marked with fluorescent microsphere;
2) provide pad, wherein coated described on described pad to be marked with fluorescence micro-The one Lp-PLA2 monoclonal antibody of ball;
3) provide reaction film, chromatography direction interval when wherein edge is used on described reaction filmFix the 2nd Lp-PLA2 monoclonal antibody and antiantibody, to form respectively detection zone and Quality ControlDistrict; With
4) on base plate along use time chromatography direction successively with the way of contact arrange sample pad,Described pad, described reaction film, absorbent filter, thus described fluorescence immune chromatography examination madePaper.
Preferably, described step 1) comprises:
A) activate fluorescent microsphere with carbodiimide, preferably, by the aqueous dispersions of fluorescent microsphereOr MES buffer solution dispersion liquid through ultrasonic wave process and mixes with carbodiimide, thereby activate instituteState fluorescent microsphere;
The fluorescent microsphere of the activation that b) washing step a) obtains, preferably, by step a)The fluorescent microsphere of the activation obtaining is washed with N-hydroxy thiosuccinimide-citrate buffer solutionWash, disperse, and through ultrasonic wave processing;
C) fluorescent microsphere mark the one Lp-PLA2 monoclonal antibody obtaining by step b),Preferably, fluorescent microsphere step b) being obtained and a Lp-PLA2 monoclonal antibodyMix, with the sealing of BSA-monoethanolamine buffer solution, centrifugal, with the dispersion of BSA-Tween solution,Through ultrasonic wave processing, thereby it is anti-to obtain being marked with a Lp-PLA2 monoclonal of fluorescent microsphereBody.
Preferably, in described step 2) in, described first of the fluorescent microsphere that is marked withThe coated concentration of Lp-PLA2 monoclonal antibody is 0.5～2mg/ml.
Preferably, in described step 3), described detection zone and described Quality Control interval are every 3mmTo 8mm, the coated concentration of described the 2nd Lp-PLA2 monoclonal antibody and described antiantibody is dividedBe not 0.5～2mg/ml.
The present invention compared with prior art has the following advantages and good effect:
1. people Lp-PLA2 epitope peptide of the present invention has good antigenicity, makes with itStandby antigen (immunogene) immune animal can produce the monoclonal antibody of high degree of specificity with manyClonal antibody.
2. the Lp-PLA2 monoclonal antibody of preparing with the present invention and polyclonal antibody can heightLp-PLA2 in blood sample is combined specifically.
3. people Lp-PLA2 external diagnosis reagent case of the present invention can be monitored artery congee effectivelyThe degree of inflammation of sample plaque and stability thereof, can early warning myocardial infarction and the generation of cerebral thrombus,The accident of prevention cardiovascular and cerebrovascular.
4. the present invention combines fluorescence analysis with flash chromatography immunological technique, providesFor quantitatively detecting a fluorescence immune chromatography test paper for determinand people Lp-PLA2 albumen,With the people Lp-PLA2 albumen in this detection paper determinand, easy and simple to handle, quick, only needWithin 10 minutes, just can complete sample detection, and detection range is wide, specificity is high, sensitivity is good,Can be rapidly the assisted diagnosis state of an illness in time, monitoring prognosis.
5. the present invention is in the mistake of the fluorescence immune chromatography test paper of the described people Lp-PLA2 albumen of preparationCheng Zhong, gropes by a large amount of tests, has optimized the preparation condition of each side, makes with thisWhen bright fluorescence immune chromatography test paper detects, fluorescence signal-to-background ratio improves greatly, thereby improvesDetection sensitivity and credible result degree; In addition, the present invention is also by detection zone and the matter of test paperThe variation of the fluorescence intensity ratio in control district carrys out the content of Lp-PLA2 in response sample, this and biographyThe chromatographic technique of system is only examined or check the absolute fluorescence intensity of detection zone and is compared, and has reduced to the full extentThe impact of external condition and background etc., has further improved testing result confidence level.
Brief description of the drawings
Fig. 1 schematically shows the fluorescence immune chromatography test paper of one embodiment of the inventionStructural representation.
Detailed description of the invention
Below pass through the description of detailed description of the invention and with reference to accompanying drawing, the present invention done furtherlyBright, but this is not limitation of the present invention, and those skilled in the art are according to of the present invention basicThought, can make various amendments or improvement, but only otherwise depart from basic thought of the present invention,All within the scope of the present invention.
One, people Lp-PLA2 epitope peptide
Described people Lp-PLA2 albumen is known in the art herein, its amino acid sequenceBe known in the art, can in the specialized databases such as NCBI, find.
The invention provides a kind of people Lp-PLA2 epitope peptide (1) and (2), its amino acid orderRow respectively as shown in sequence table SEQ IDNo.1 and SEQIDNo.2, for:
(1)Thr-Lys-Ile-Pro-Arg-Gly-Asn-Gly-Pro-Arg-Ser-Lys-Glu-Ser-Tyr；
(2)Tyr-Lys-Gly-Asp-Ile-Asp-Ser-Asn-Val-Ala-Ile-Asp-Leu-Ser-Asn。
The present inventor gropes through a large amount of theoretical research and experiments, finally screenThere is good antigenic epitope peptide to two kinds.
Lp-PLA2 epitope peptide (1) is with the 54th to 62 of people Lp-PLA2 albumen n endsOne section of peptide section containing 9 amino acid residues is antigenic determinant, and is added with at its C endHydrophilic peptide section Arg-Ser-Lys-Glu-Ser-Tyr, the Lp-PLA2 epitope forming like thisPeptide (1) has that hydrophily is high, antigenicity is strong and be easy to synthetic feature.
Lp-PLA2 epitope peptide (2) is with people Lp-PLA2 PROTEIN C end the 372nd to 385One section of peptide section that contains 14 amino acid residues of position is antigenic determinant, and adds at its N endBe added with Tyr. Adding Tyr at N end is in order to make this epitope peptide pass through BDB(Bis-diazotizedbenzidinedichloride) be linked to carrier protein (the blue egg of for example blood(KLH) in vain) upper, thus carry out Dispersal risk as antigen. Lp-PLA2 epitope peptide (2)Also have that hydrophily is high, antigenicity is strong and is easy to synthetic feature.
At present, the present invention studies discovery, Lp-PLA2 epitope peptide of the present invention have asLower function:
1. there is antigenicity; 2. after being connected with carrier protein, produce as immunogene stimulating animalSpecific antibody; 3. the antibody of preparing with epitope peptide can be specifically and peopleLp-PLA2 combination.
The preparation method of Lp-PLA2 epitope peptide of the present invention can use chemical synthesis: profitWith American AB I431A type polypeptide automatic synthesizer, by solid phase method synthetic antigen epitope peptide.The molecular weight of epitope peptide of the present invention (1) and (2) is respectively 1690.09 and 1623.92, canDetermine with mass spectrum, and measure the epitope peptide order of qualification synthesized by peptide sequenceRow. The purity of peptide section is evaluated with thin-layer chromatography and high performance liquid chromatography, and measures antigen tableThe concentration of position peptide.
Two, Lp-PLA2 antigen
The present invention also provides a kind of Lp-PLA2 antigen, and it is by making people of the present inventionOne and carrier protein couplet in Lp-PLA2 epitope peptide (1) and (2) are prepared from. ToolBody, the invention provides Lp-PLA2 antigen (1) and (2), described Lp-PLA2 antigen (1)By making people Lp-PLA2 epitope peptide of the present invention (1) with carrier protein couplet preparationBecome; Described Lp-PLA2 antigen (2) is by making people Lp-PLA2 epitope peptide of the present invention (2)Be prepared from carrier protein couplet. Lp-PLA2 antigen of the present invention have immunogenicity andSpecificity, is a kind of immunogene, prepares specific Lp-PLA2 thereby can be used to immune animalAntibody. In the present invention, the example of available carrier protein comprises the blue egg of KLH(key hole bloodBovine serum albumin(BSA) (BSA), ovalbumin OVA etc. in vain). Due to KLH(key holeHemocyanin) immunogenicity is strong, and binding site is many, and immune effect is better, and moving with immunityThing affiliation is far away, is difficult for causing cross reaction with it as carrier protein, is therefore preferred.
Three, Lp-PLA2 monoclonal antibody, Lp-PLA2 polyclonal antibody and people Lp-PLA2External diagnosis reagent case
The present invention also provides people Lp-PLA2 monoclonal antibody and people Lp-PLA2 Anti-TNF-αBody, described antibody can utilize respectively Lp-PLA2 antigen of the present invention (1) and (2) (immunogene)Immune animal is prepared and obtains. Preparation method can adopt the ordinary skill in the art, specifically can be referring toEmbodiment 2.
Lp-PLA2 monoclonal antibody of the present invention and polyclonal antibody can be for the preparation of peopleLp-PLA2 external diagnosis reagent case, this kit can be based on immunization method to blood preparationIn Lp-PLA2 detect.
Therefore, the invention provides a kind of people Lp-PLA2 external diagnosis reagent case, it comprisesPeople Lp-PLA2 monoclonal antibody of the present invention or polyclonal antibody.
The known immunization experiment method that can be used for clinical examination mainly comprises following several at present:ELISA method, chemoluminescence method, fluorescent chromatographic method, colloid gold immune determination method etc.
And ELISA method comprises following several types: double antibody sandwich method detectable antigens, dual anti-Former sandwich method detect antibody, indirect method survey antibody, competition law survey antibody, competition law survey antigen,Catch coated method and survey antibody etc.
People Lp-PLA2 external diagnosis reagent case of the present invention preferably adopts ELISA double antibody folderHeart method detects Lp-PLA2 albumen. This kit can comprise coated antibody, binding antibody,The SA of enzyme labeling and/or necessary instrument and reagent etc.
Preferably, described people Lp-PLA2 external diagnosis reagent case adopts people of the present inventionLp-PLA2 monoclonal antibody is as coated antibody. At this, term " coated antibody " refers toBe coated in the antibody on the ELISA Plate of solid phase. In addition described people Lp-PLA2 in-vitro diagnosis examination,Agent box also preferably comprises people Lp-PLA2 polyclonal antibody using as binding antibody, wherein, whenDescribed binding antibody derives from one in people Lp-PLA2 epitope peptide of the present invention (1) and (2)When person, described coated antibody derives from the another one in described epitope peptide (1) and (2). ?This, term " binding antibody " refer in kit can with determined antigen and enzyme-labeled secondary antibodyIn conjunction with specific antibody. Described kit can also comprise the SA of enzyme labeling, and this is years oldTwo antibody can be goat anti-rabbit igg antibody, described enzyme labeling can be horseradish peroxidase,Alkaline phosphatase etc.
Carry out clinical research with people Lp-PLA2 external diagnosis reagent case of the present invention, to 115Example patient and 79 routine control group blood samples detect, and detection sensitivity is 93%, specialDegree is 91%, and accuracy is 92%.
Can learn the people who utilizes Lp-PLA2 antibody of the present invention to prepare based on this resultLp-PLA2 external diagnosis reagent case can be monitored the degree of inflammation of arteriosclerosis, forMiocardial infarction and cerebral thrombus there is considerable forewarning function.
In kit of the present invention, all right required any reagent or the instrument of inclusion test,Such as pre-coated plate, cleaning solution, developer, stop buffer etc.
Four, for quantitatively detecting the fluorescence immune chromatography test paper of people Lp-PLA2 albumen
It is a kind of for quantitatively detecting determinand people Lp-PLA2 albumen that the present invention also providesFluorescence immune chromatography test paper, this test paper detects described people Lp-PLA2 by double antibody sandwich methodAlbumen, wherein:
Described double antibody sandwich method adopts a Lp-PLA2 monoclonal that is marked with fluorescent microsphereAntibody is as capture antibody, and a described Lp-PLA2 monoclonal antibody derives from people Lp-PLA2One in epitope peptide (1) and (2); And
Described double antibody sandwich method also adopts the 2nd Lp-PLA2 monoclonal antibody anti-as detectingBody, described the 2nd Lp-PLA2 monoclonal antibody derives from people Lp-PLA2 epitope peptide (1)(2) another one in;
Described people Lp-PLA2 epitope peptide (1) and (2) are respectively:
(1)Thr-Lys-Ile-Pro-Arg-Gly-Asn-Gly-Pro-Arg-Ser-Lys-Glu-Ser-Tyr；
(2)Tyr-Lys-Gly-Asp-Ile-Asp-Ser-Asn-Val-Ala-Ile-Asp-Leu-Ser-Asn。
In the present invention, in double antibody sandwich method fluorescence immune chromatography test paper, " catch anti-Body " refer to the first antibody of specific recognition determined antigen, it is arranged on pad conventionallyOn; " detection antibody " refers to the antibody that another kind can specific recognition determined antigen, its withCapture antibody is identified respectively the different epitope on determined antigen molecule, and it is fixed on conventionallyOn the detection zone of reaction film.
In the present invention, a described Lp-PLA2 monoclonal antibody can be by firstLp-PLA2 antigen is prepared from, and a Lp-PLA2 antigen can be by making described peopleOne and carrier protein couplet in Lp-PLA2 epitope peptide (1) and (2) are prepared from; AndAnd described the 2nd Lp-PLA2 monoclonal antibody can be by the 2nd Lp-PLA2 antigen preparationBecome, the 2nd Lp-PLA2 antigen can be by making described people Lp-PLA2 epitope peptide (1)(2) another one and carrier protein couplet in are prepared from.
In the present invention, the example of available carrier protein comprises KLH(keyhole limpet hemocyanin),Bovine serum albumin(BSA) (BSA), ovalbumin OVA etc. Due to the blue egg of KLH(key hole bloodIn vain) immunogenicity is strong, and binding site is many, and immune effect is better, and with immune animal relationshipRelation is far away, is difficult for causing cross reaction with it as carrier protein, is therefore preferred.
Preferably, the particle diameter of fluorescent microsphere used in fluorescence immune chromatography test paper of the present inventionFor 320nm to 400nm, be preferably 360nm, the fluorescent material on fluorescent microsphere can be differentThiocyanic acid fluorescein, RB 200, TRITC or X-rhodamineDeng, be wherein preferably X-rhodamine (can purchased from Shanghai Jing Chun company). Fluorescent microsphere micro-Ball material can be by polystyrene, polymethyl methacrylate or methyl methacrylate and itsThe copolymer that its monomer copolymerization forms, the example of other monomer is styrene etc. Fluorescent microsphereExcitation wavelength can be 350～600nm, is preferably 390nm; Emission wavelength can be500～700nm, is preferably 615nm.
In the present invention, maximum excitation wavelength and the emission wavelength difference of fluorescent microsphere are larger,Illustrate that fluorescent microsphere has larger Stokes (Stokes) displacement, like this, fluorescent test paperAmbient interferences is lower, and doing immunochromatography label with this microballoon has stronger advantage.
In a specific embodiment, fluorescence immune chromatography test paper of the present invention has the endPlate, and on this base plate along use time chromatography direction be provided with the way of contact successively: sampleProduct pad, pad, reaction film, absorbent filter, described sample pad is for loading and treat in useTest sample product, described pad is provided with a described Lp-PLA2 who is marked with fluorescent microsphereMonoclonal antibody, described reaction film comprises detection zone and Quality Control district, described detection zone is coated to some extentState the 2nd Lp-PLA2 monoclonal antibody, described Quality Control district be coated with can with described being marked withThe antiantibody of the one Lp-PLA2 monoclonal antibody specificity combination of fluorescent microsphere.
Preferably, the sample pad of fluorescence immune chromatography test paper of the present invention, pad, reactionFilm, absorbent filter can the chromatography direction when using overlap successively and be arranged on base plate (referring toFig. 1). On reaction film, spaced detection zone and Quality Control district can be, but are not limited to, line,The forms such as band, piece, detection zone and Quality Control district preferred interval 3mm to 8mm.
In the present invention, reaction film is preferably under the wavelength that is greater than 550nm and does not substantially send outThe nitrocellulose filter of fluorescence. In addition, base plate does not preferably have photoluminescent property substantially.
Conventionally, conventional chromatographic test paper assembly (reaction film, base plate etc.) is at 550nm wavelengthUnder there is obvious fluorescence background, this detection to fluorescence signal produces very large interference. ThisBright by adopting not fluorescent nitrocellulose filter substantially under the wavelength that is greater than 550nmWith the base plate of low photoluminescent property, thereby overcome the defect of conventional fluorescent test paper. In addition, thisBright fluorescent material X-rhodamine used can produce stronger fluorescence signal, thereby further largeImprove greatly fluorescence signal-to-background ratio, made it possible to distinguish well signal and background, and then improved inspectionSurvey sensitivity.
In the present invention, it is normally used that the material of sample pad and pad can adopt this areaMaterial, for example, sample pad and pad can be glass fibre.
Of the present invention can resisting with a Lp-PLA2 monoclonal that is marked with fluorescent microsphereThe antiantibody of body specific binding can be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse iggMonoclonal antibody, is wherein preferably sheep anti-mouse igg monoclonal antibody, with polyclonal antibody phaseRatio, monoclonal antibody specificity is higher.
In a specific embodiment, fluorescence immune chromatography test paper of the present invention is usingTime, in sample pad, drip sample liquid (as the blood sample that contains Lp-PLA2), at capillaryUnder effect, sample liquid moves to absorbent filter one end, pad place and described be marked with glimmeringThe one Lp-PLA2 monoclonal antibody of light microballoon forms immune complex, this immune complexBe moved further, on detection line, be combined formation with described the 2nd Lp-PLA2 monoclonal antibodyThe immune complex of double-antibody sandwich, and do not form the fluorescent microsphere that is marked with of immune complexThe one Lp-PLA2 monoclonal antibody antiantibody on nature controlling line is combined. This process need10 minutes to 15 minutes, afterwards, detect with fluorescence detector, if nature controlling line place is notThere is band, illustrate that test paper lost efficacy; If there is band in nature controlling line place, and detection line place is notThere is band, in interpret sample, do not contain people Lp-PLA2 albumen; If in nature controlling line and inspectionOn survey line, all there is band, in interpret sample, contain people Lp-PLA2 albumen.
In yet another aspect, the invention provides one for the preparation of quantitatively detecting in determinandThe method of the fluorescence immune chromatography test paper of people Lp-PLA2 albumen, it comprises the following steps:
1) provide a Lp-PLA2 monoclonal antibody that is marked with fluorescent microsphere;
2) provide pad, wherein coated described on described pad to be marked with fluorescence micro-The one Lp-PLA2 monoclonal antibody of ball;
3) provide reaction film, chromatography direction interval when wherein edge is used on described reaction filmFix the 2nd Lp-PLA2 monoclonal antibody and antiantibody, to form respectively detection zone and Quality ControlDistrict; With
4) on base plate along use time chromatography direction successively with the way of contact arrange sample pad,Described pad, described reaction film, absorbent filter, thus described fluorescence immune chromatography examination madePaper.
It will be appreciated by persons skilled in the art that can be according to actual needs to above-mentioned stepsOrder adjust, for example step 3) can be before step 1), or in step 1)And 2) between.
Method of the present invention can also comprise the fluorescence immune chromatography test paper of making is cut into suitableWhen the step 5) of width.
The present inventor gropes by a large amount of tests, has optimized preparation use of the present inventionIn the bar of each step of method of fluorescence immune chromatography test paper that detects people Lp-PLA2 albumenPart, thus make the fluorescence immune chromatography test paper of the present invention can be for people Lp-PLA2 albumenAcquisition meets the result that Clinical detection requires, that is, detection range is wide, specificity is high, sensitivityGood.
Therefore, preferably, in the method for the invention, described step 1) comprises:
A) activate fluorescent microsphere with carbodiimide, preferably, by the aqueous dispersions of fluorescent microsphereOr MES buffer solution dispersion liquid through ultrasonic wave process and mixes with carbodiimide, thereby activate instituteState fluorescent microsphere;
The fluorescent microsphere of the activation that b) washing step a) obtains, preferably, by step a)The fluorescent microsphere of the activation obtaining is washed with N-hydroxy thiosuccinimide-citrate buffer solutionWash, disperse, and through ultrasonic wave processing;
C) fluorescent microsphere mark the one Lp-PLA2 monoclonal antibody obtaining by step b),Preferably, fluorescent microsphere step b) being obtained and a Lp-PLA2 monoclonal antibodyMix, with the sealing of BSA-monoethanolamine buffer solution, centrifugal, with the dispersion of BSA-Tween solution,Through ultrasonic wave processing, thereby it is anti-to obtain being marked with a Lp-PLA2 monoclonal of fluorescent microsphereBody.
In a specific embodiment of the present invention, described step 1) comprises:
A) activate fluorescent microsphere with carbodiimide, wherein, get the fluorescent microsphere water of 1 (w/v) %Dispersion liquid, centrifugal 5 to 10 minutes of 10000rpm to 15000rpm low temperature (for example 10 DEG C),Remove supernatant, sediment is distributed to distilled water or the first wash buffer (MES of 0.1M of 500 μ lThe aqueous solution) in, ultrasonic wave (240W) is processed 1 to 2 minute, repeats above process three times,Add carbodiimide 10mg to 50mg, stir 10～15 minutes, thereby activate described fluorescenceMicroballoon;
The fluorescent microsphere of the activation that b) washing step a) obtains, wherein, by step a)The fluorescent microsphere of the activation obtaining under 1000rpm to 15000rpm centrifugal 5 to 10 pointsClock, is distributed to 1ml coupling buffer (the N-hydroxyl sulfo-amber of 20～100mM by sedimentAmber acid imide-citrate buffer solution)) in, ultrasonic wave (240W) is processed 1 to 2 minute,Repeat above process three times;
C) fluorescent microsphere mark the one Lp-PLA2 monoclonal antibody obtaining by step b),Wherein, according to the fluorescent microsphere activating described in 1 μ l to 3 μ l antibody (10mg/ml)/100 μ lRatio, the fluorescent microsphere that step b) is obtained and a Lp-PLA2 monoclonal antibodyMix, room temperature (25 DEG C) is lower stirs 1.5～3 hours (preferably 2 hours), adds 1mlSealing buffer solution (1 (w/v) %BSA-0.05M monoethanolamine), continues to stir 1 hour,Under 10000rpm to 15000rpm centrifugal 5 to 10 minutes, repeated centrifugation 3 times, will precipitateThing is distributed to the whole wash buffers of 500 μ l (0.5 (w/v) %BSA-0.11 (v/v) % Tween solution)In, ultrasonic wave (240W) is processed 1 to 2 minute, is settled to 500 μ l with described whole wash buffer.
Preferably, in the method for the invention, described step 2) comprising: will be marked with fluorescenceThe one Lp-PLA2 of microballoon is antibody diluent (1% (w/v) BSA-0.01M for monoclonal antibodyPBS(pH7.2) buffer solution) dilute, to be diluted to 0.5～2mg/ml, be preferably1mg/ml, is then evenly sprayed on pad with micropipettor, dries afterwards or vacuumFreeze drying. In the step 2 of method of the present invention) in, the described fluorescent microsphere that is marked withThe coated concentration of the one Lp-PLA2 monoclonal antibody is 0.5～2mg/ml, is preferably1.5mg/ml。
Preferably, described step 3) comprises: with metal spraying machine by mono-described the 2nd Lp-PLA2Clonal antibody and antiantibody draw nitrocellulose filter (solid phase carrier) upper using as detection zone andQuality Control district, makes the 3mm to 8mm that is spaced apart in detection zone and Quality Control district, described secondThe concentration of Lp-PLA2 monoclonal antibody and described antiantibody is respectively 0.5～2mg/ml, preferablyFor 1mg/ml.
Preferably, result detects and utilizes special fluorescence detector (can pacify group purchased from ShenzhenBioengineering Co., Ltd, model AQ-3000) Quality Control district and detection zone are detected to inspectionThe ratio of surveying district and Quality Control district fluorescence intensity just becomes with the content of the Lp-PLA2 in testing sampleRatio. Adopt the ratio of detection zone and Quality Control district fluorescence intensity instead of directly adopt the exhausted of detection zoneCan reduce as much as possible the impact of reaction condition, matrix etc. on fluorescent value, and avoid background as far as possibleDisturb.
Further explain and describe content of the present invention by the mode of example below, but theseExample should not be understood to the restriction to protection scope of the present invention.
Embodiment
Except as otherwise noted, the following stated solution is the aqueous solution, and the percentage in solution is equalFor percentage by volume.
The preparation of embodiment 1:Lp-PLA2 epitope peptide (1) and (2).
Preparation method's chemical synthesis: utilize American AB I431A type polypeptide automatic synthesizer,By solid phase method synthetic Lp-PLA2 epitope peptide (1) and (2) respectively. Epitope peptide pureDegree is evaluated with high performance liquid chromatography, and measures the concentration of peptide section. Epitope of the present inventionThe molecular weight of peptide (1) and (2) is respectively 1690.09,1623.92, utilizes mass spectrum to determine,Measure the peptide sequence of qualification synthesized by peptide sequence.
One, Lp-PLA2 epitope peptide (1) and (2) is synthetic
Above-mentioned peptide section adopts solid phase method synthetic. The synthetic main thought of solid-phase peptide is: first by instituteThe amino acid whose carboxyl of carboxyl terminal that synthesizes peptide chain is same insoluble with covalent bond formMacromolecular compound (resin) is connected, and the amino acid being then combined on solid phase carrier with this is doneFor amino component, through deaminate protecting group and with excessive activated carboxyl component reaction, connectLong peptide chain. Such step can repeatedly go on repeatedly, finally reaches required syntheticThe length of peptide chain. This building-up process is as follows.
The concrete preparation process separately of Lp-PLA2 epitope peptide of the present invention (1) and (2)As follows:
1. raw materials used:
HMPresin (P-hydroxymethyl phenoxy methyl poly vinyl, can purchased from sigma company)
Fmoc-AA (amino acid of 9-fluorenyl methoxy carbonyl acyl group protection, can purchased from Merck company)
NMP(nitrogen methyl pyrrolidone, can be purchased from sigma company)
DCM(carrene, can be purchased from Central Plains chemical company)
MeoH(methyl alcohol, can be purchased from Central Plains chemical company)
Piperidine(piperidines, can be purchased from sigma company)
DMAP(dimethyl aminopyridine, can be purchased from sigma company)
HOBT(hydroxybenzotriazole, can be purchased from sigma company)
DCC(dicyclohexylcarbodiimide, can be purchased from sigma company)
TFA(trifluoroacetic acid, can be purchased from sigma company)
EDT(1,2-dithioglycol, can be purchased from sigma company)
Thio phenyl methyl ether, can be purchased from Guangzhou Wei Bai Chemical Co., Ltd.
Crystallization phenol, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Acetonitrile, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group
2. use instrument:
Polypeptide automatic synthesizer, model 431A, can be purchased from ABI company
Rotary Evaporators, model R-201, can be purchased from Shanghai Shen Shun company
High performance liquid chromatograph, Waters600, can be purchased from Waters company of the U.S.
Freeze drier, model VFD-2000, can be purchased from Beijing rich doctor Kanggong department
3. synthetic method and process:
Take HMP resin 100mg, replacing equivalent is 1.0meq, puts by 0.1mmolIn the reaction chamber of American AB I431A type polypeptide automatic synthesizer, by synthesizer automatically by spyFixed amino acid is by different being linked in sequence, and coupling rate reaches 99%. React as follows:
(1) amino acid whose activation (HOBt/DCC method)
The amino acid of Fmoc protection
(2) connect amino acid to resin
(3) the Fmoc protecting group of deaminate acid
(4) another amino acid whose activation (HOBt/DCC method)
(5) coupling
Peptide-the resin of new coupling
(6) repeating step (3) to (5) is until end of synthesis.
Obtain respectively peptide resin 131mg and the Lp-PLA2 peptide section (2) of Lp-PLA2 peptide section (1)Peptide resin 110mg.
(7) peptide resin:
Use TFA(trifluoroacetic acid) cutting peptide chain, with EDT(2.5 volume %), thio phenylMethyl ether (2.5 volume %) is made scavenger, at room temperature reacts 3.0 hours, removes cutting examinationAgent, then use extracted with diethyl ether, obtain respectively the crude product of Lp-PLA2 peptide section (1) and (2).
Two, the purifying of Lp-PLA2 epitope peptide (1) and (2) crude product:
Adopt high performance liquid chromatography separation and purification:
Condition: chromatographic column: C810 × 100mm, can be purchased from Waters company of the U.S.
Chromatograph: Waters600, Waters company of the U.S.
Mobile phase: A:0.1%TFA(trifluoroacetic acid) aqueous solution
B:0.1%TFA(trifluoroacetic acid) in 60% acetonitrile
Detect wavelength: 214nm
Flow velocity: 4ml/ minute
Gradient: 20-60%B, 30 minutes
HPLC(high performance liquid chromatography) analyze
Chromatographic column: C184.6 × 150mm, can be purchased from Waters company of the U.S.
Mobile phase: A:0.1%TFA(trifluoroacetic acid) aqueous solution
B:0.1%TFA(trifluoroacetic acid) in acetonitrile
Detect wavelength: 214nm
Flow velocity: 1ml/ minute
Gradient: 0-60%B, 30 minutes
Peptide piecewise analysis result shows the pure of Lp-PLA2 epitope peptide of the present invention (1) and (2)Degree is more than 95%.
Three, the qualification of Lp-PLA2 epitope peptide (1) and (2)
1. utilize mass spectrum to measure respectively Lp-PLA2 epitope peptide (1) and (2) of purifying gainedMolecular weight.
(1) reagent raw material
TFA(trifluoroacetic acid, can be purchased from sigma company)
HCCA(alpha-cyano-4-hydroxycinnamic acid, can be purchased from sigma company)
Acetonitrile (can purchased from Chemical Reagent Co., Ltd., Sinopharm Group)
(2) instrument
Ground substance assistant laser desorption ionization time of-flight mass spectrometer MALDI-TOF-MS(model:REFLEXIII, German Bruker company);
(3) matrix liquid: α-CCA is dissolved in the 50%ACN solution containing 0.1%TFA,Make saturated solution, centrifugal, get supernatant;
(4) instrument testing conditions: reflection detection mode; Flight pipe range 3m; Nitrogen laser:Wavelength 337nm, accelerating potential 20KV; Reflected voltage 23KV.
(5) operating procedure: get respectively the sample of the above-mentioned purified polypeptide of 1 μ L (1) and (2), eachFrom mixing with the saturated stromal supernatant mixing equal-volume of 1 μ L, get respectively 1 μ L point at sampleOn target, send in ion gun and detect.
As a result, the molecular weight that records gained Lp-PLA2 epitope peptide (1) is 1690.4,The molecular weight of Lp-PLA2 epitope peptide (2) is 1623.7, with theoretical molecular 1690.09,1623.92 is consistent, proves that synthetic polypeptide is object product.
By peptide sequence measure identify respectively gained Lp-PLA2 epitope peptide (1) and(2) sequence.
(1) principle: the general principle of polypeptid acid sequence analysis is Edman degraded,It is a circulating chemical reaction process. Comprise three main chemical steps: (1) is evenConnection: isothiocyanic acid benzene fat reacts with the N-end residue of proteins and peptides, forms phenylamino sulphurFormyl (PTC) derivative, i.e. PTC-peptide. (2) cyclisation cracking: PTC-peptide cyclisation cracking.(3) transform: thiazole purine ketone phenylamino (ATZ) is converted into different sulphur urine amino acid (the PTH-ammonia of benzeneBase acid). Stay minimizing in solution the peptide of an amino acid residue repeat again above-mentioned anti-Answer process, whole order-checking process is all automatically to carry out by sequenator now.
(2) instrument: American AB I company 491 type protein/polypeptide-terminal amino acid ordersRow analyzer
(3) reagent raw material
Phenyl isothiocyanate PITC, can be purchased from sigma company
Normal heptane, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group
The trimethylamine TMA aqueous solution, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group
TFA(trifluoroacetic acid, can be purchased from sigma company)
Ethyl acetate, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Chlorobutane, can be purchased from sigma company
Acetonitrile, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group
(4) measure
Undertaken by instrument description.
Result: through qualification, the sequence of gained Lp-PLA2 epitope peptide (1) and (2) is respectively:
(1)Thr-Lys-Ile-Pro-Arg-Gly-Asn-Gly-Pro-Arg-Ser-Lys-Glu-Ser-Tyr; With
(2)Tyr-Lys-Gly-Asp-Ile-Asp-Ser-Asn-Val-Ala-Ile-Asp-Leu-Ser-Asn。
This result is consistent with target section of synthesized peptide.
Embodiment 2: respectively by Lp-PLA2 epitope peptide (1) and (2) of embodiment 1 gainedBe connected to prepare Lp-PLA2 antigen (1) and (2) with carrier protein, utilize gained antigen (1) and (2)Immune animal respectively, thus utilize antigen (1) to prepare specific monoclonal antibody and polycloneAntibody, and utilize antigen (2) to prepare specific monoclonal antibody and polyclonal antibody.
1. the preparation of antigen: with BDB(Bis-diazotizedbenzidinedichloride) methodBy Lp-PLA2 peptide section (1) and (2) respectively with carrier protein KLH(keyhole limpet hemocyanin) be connectedBe prepared into Lp-PLA2 antigen (1) and (2).
Get Lp-PLA2 peptide section (1) or (2) 10.0mg, with 1ml0.1MPBS buffer solution (pH7.4) dissolve; KLH10mg, with 0.2M borate buffer solution (pH9.0) 20ml dissolving;Then both are mixed, be cooled to 0 DEG C, get BDBCl2110 μ L, react 1.5h under room temperature,Packing after dialysed overnight ,-20 DEG C of preservations.
In the present embodiment, the formula of PBS buffer solution is: the Na of 0.2mol/L2HPO481ml adds the NaH of 0.2mol/L2PO419ml mixes.
The formula of borate buffer solution is: 0.05mol/L borax 80ml, adds 0.2mol/L boronAcid 20ml mixes.
2. immune animal is prepared monoclonal antibody:
2.1. get the Lp-PLA2 antigen (1) of above-mentioned preparation and (2) (immunogene) respectively with etc. bodyAfter long-pending Freund's complete adjuvant (purchased from Shanghai Yuan Ju biotech firm) fully mixes, immune Balb/cMouse, 50 μ g antigens/only, subcutaneous multi-point injection. After 4 weeks, survey serum titer, select immunityReactive good mouse booster immunization again: get antigen and isopyknic incomplete Freund's adjuvant is abundantAfter mixing, antigen dose 25 μ g/, subcutaneous multi-point injection, the number of times of booster immunization is 6 times,Continuous booster immunization twice before merging, extracting spleen cell and Sp2/0 myeloma cell are routinely afterwardsMethod 50%PEG(MW4000) (purchased from Central Plains chemical company) mediation merge,And select to cultivate with HAT conditioned medium (purchased from sigma company). After fusion, put into CO2In incubator, cultivate after 9～11 days for 37 DEG C, in hole, occur larger cell clone. 11 days startScreen with indirect ELISA. Utilize limiting dilution assay to carry out 4 times to the hole of the primary dcreening operation positive(even screening after a large amount of schizogamies of cell) cultivated in cloning, afterwards amplifying cells, freezeDeposit, prepare ascites.
2.2. by Balb/c for mouse norphytane (purchased from sigma company) 0.5ml/ only process,One week pneumoretroperitoneum inoculation hybridoma 2 × 106Individual/only, after 10 days, to collect ascites.
2.3. measure antibody titer: measure and utilize Lp-PLA2 antigen with indirect ELISA method(1) tiring of the monoclonal antibody (1) of preparing, result shows that tiring of monoclonal antibody reaches 1:32000Above.
Utilize tiring of monoclonal antibody (2) prepared by Lp-PLA2 antigen (2) also to utilize identicalMethod measure, more than it is tired and also reaches 1:32000.
3. immune animal is prepared polyclonal antibody:
3.1. select NZw that three monthly ages, body weight are about about 2kg as immunityAnimal. In fundamental immunity, Lp-PLA2 antigen (1) and (2) of the above-mentioned preparation of 1-2mg (are exempted fromEpidemic focus) mix with isopyknic Freund's complete adjuvant respectively-enter at rabbit back after fully emulsifiedThe hypodermic injection of row multiple spot. Every 4 weeks booster immunizations once, antigen and incomplete Freund's adjuvant fillAfter point emulsification, with 100 μ g/ only in back multiple spot hypodermic injection. After last booster immunization the 10thIt arteria carotis bloodletting, separation of serum.
3.2. measure antibody titer: measure and utilize Lp-PLA2 antigen (1) with indirect elisa methodTiring of the polyclonal antibody (1) of preparation, more than result shows that antibody titer reaches 1:16000.
Utilize tiring of polyclonal antibody (2) prepared by Lp-PLA2 antigen (2) also to utilize identicalMethod measure, more than it is tired and also reaches 1:16000.
3.3. get blood and separation of serum: arteria carotis intubate is got blood, separation of serum.
4. separation and purification antibody: after ammonium sulfate precipitation, then through ProteinG(purchased from sigmaCompany) affinity purification.
5. freeze-drying after antibody packing, low temperature is preserved.
Embodiment 3: the specificity identification of people Lp-PLA2 monoclonal antibody (1) and (2)
Detect with ELISA. Respectively with people Lp-PLA2 albumen, fibrinogen, C-Reactive protein (all purchased from Shanghai Lian Shuo company), for the coated elisa plate of detectable antigens, passes throughELISA detects respectively prepared Lp-PLA2 monoclonal antibody (1) and (2) and this peopleThe specific reaction of Lp-PLA2 albumen, makes negative control with normal BALB/c mouse serum,PBS liquid is made blank.
Result: only react with Lp-PLA2 respectively for sun Lp-PLA2 monoclonal antibody (1) and (2)Property (P/N > 2.1), and react negative with fibrinogen, C reactive protein, this is describedBright Lp-PLA2 monoclonal antibody (1) and (2) have respectively specificity.
Embodiment 4: the specificity identification of people Lp-PLA2 polyclonal antibody (1) and (2)
Utilize the method identical with above-mentioned qualification monoclonal antibody specificity to identify.
Result shows: Lp-PLA2 polyclonal antibody (1) and (2) are reacted with Lp-PLA2 respectively and arePositive (P/N > 2.1), and react negative with fibrinogen, C reactive protein, this is describedLp-PLA2 polyclonal antibody (1) and (2) of invention have respectively specificity.
Embodiment 5: utilize Lp-PLA2 monoclonal antibody and Lp-PLA2 Anti-TNF-α systemStandby Lp-PLA2 external diagnosis reagent case.
In the present embodiment, Lp-PLA2 epitope peptide (1) preparation will be utilized in embodiment 2Monoclonal antibody (1) as the coated antibody in this kit; To in embodiment 2, utilizePolyclonal antibody (2) prepared by Lp-PLA2 epitope peptide (2) is as binding antibody.
The preparation of Lp-PLA2 external diagnosis reagent case and operation are as follows:
1. the preparation of various buffer solutions and reagent:
The CB(carbonate buffer solution of A, coated buffer solution: 0.050M, pH9.6)
Na2CO3: 16.0 grams
NaHCO3: 29.0 grams
Distill water-soluble to 1000ml
10 × PBS-Tween20 of B, sample/lavation buffer solution: pH7.2
Na2HPO4·12H2O:58 gram
KH2PO4: 4 grams
NaCl:100 gram
KCl:4 gram
Distill water-soluble to 1000ml
Add Tween20:20ml
C, enzyme labeling thing dilution:
10×PBS-Tween20：10ml
FCS(calf serum): 20ml
Distill water-soluble to 1000ml
Enzyme stabilizers (can purchased from Shanghai Xi Bao company): 1 gram
Biological preservative (can purchased from Shanghai Xi Bao company): 1ml
D, developer A:
Citric acid: 35.5 grams
Urea peroxide: 10 grams
Distill water-soluble to 1000ml
Tween20：10ml
E, developer B:
Citric acid: 120 grams
EDTA-2Na:1 gram
TMB2HCl:2 gram
Distill water-soluble to 1000ml
F, stop buffer: 2MH2SO4
The concentrated sulfuric acid (95-98%): 22.2ml
Distilled water: 177.3ml
Timing slowly splashes into the concentrated sulfuric acid in distilled water, and limit edged shakes up.
2. the preparation of pre-coated plate:
Lp-PLA2 monoclonal antibody (1) is dissolved in to the carbonate buffering of the 0.05M of pH=9.6In liquid, make pre-coated liquid, press in the upper every hole of ELISA Plate (can purchased from Shenzhen Jin Canhua company)0.1 μ g/ hole adds 100 μ l, puts 4 DEG C and places 18-24 hour, takes out, and gets rid of coating buffer, washesWash, after BSA sealing 16 hours, dried overnight, pack into and in aluminide-coating bag, vacuumize sealing, andBe placed in 4 DEG C of preservations.
3. binding antibody (Lp-PLA2 polyclonal antibody (2)) and (the horseradish peroxidating of enzyme connection thingThe goat anti-rabbit igg antibody (purchased from Beijing company of Zhong Shan Golden Bridge) of thing enzyme labeling) thinner ratioExample is determined by square formation titration experiments.
4. the composition of kit:
Pre-coated plate: 48/96 hole
Lp-PLA2 calibration object (raw material is purchased from Shanghai Lian Shuo company): 5: 5 × 1.0ml(is denseDegree is respectively 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 0ng/ml)
Lp-PLA2 binding antibody: 1 × 10ml(dilutes through 1:5000)
Enzyme connection thing: 1 × 10ml(dilutes through 1:5000)
Concentrated cleaning solution (25 × PBS-Tween20): 1 × 20ml
Developer A:1 × 6.0ml
Developer B:1 × 6.0ml
Stop buffer: 1 × 6.0ml
5. the operating procedure of kit:
In each hole of pre-coated plate, add respectively blood sample to be checked and standard items 100 μ l/ holes,Be diplopore, hatch 60 minutes for 37 DEG C, with 1 × lavation buffer solution washing 5 times, pat dry. ?In each hole, add Lp-PLA2 binding antibody 100 μ l/ holes, hatch 30 minutes for 37 DEG C, with 1 ×Lavation buffer solution washing 5 times, pats dry. In each hole, add again enzyme connection thing 100 μ l/ holes, 37DEG C hatch 30 minutes, with 1 × lavation buffer solution washing 5 times, pat dry. Add developer A,B liquid, the each 50 μ l in every hole, mix, and hatch 15 minutes for 37 DEG C. Adding stop buffer 50 μ l/ holes stopsReaction, joins detector (model RT-6000, can purchased from Lei Du company) dual wavelength with enzyme(450nm, 620nm) detects absorbance.
6. result is judged:
Table 1: standard items concentration and corresponding mean light absorbency (OD) value
| Concentration ng/ml | 0 | 125 | 250 | 500 | 1000 |
| Average OD value | 0.057 | 0.304 | 0.458 | 0.823 | 1.345 |
With standard items concentration and corresponding absorbance drawing standard curve, calibration curveR2=0.988。
Calculate the Lp-PLA2 concentration results in the sample detecting according to calibration curve.
115 routine cardiovascular and cerebrovascular patients and 79 routine healthy persons are carried out to serum in a manner describedLp-PLA2 detects, and the Lp-PLA2 content in patients serum is apparently higher than normal healthy controls group,Difference has statistical significance (P < 0.01), in table 2.
Table 2: two groups of sample Lp-PLA2 concentration ratios
Taking 350ng/ml as dividing value distinguish yin and yang attribute, to above-mentioned to 115 routine cardiovascular and cerebrovascular sufferersPerson and 79 routine healthy persons detect the result of serum Lp-PLA2 and add up, result demonstration, thisThe detection sensitivity (107/115) of invention kit is 93%, and specificity (72/79) is 91%,Accuracy ((107+72)/194) is 92%. The results are shown in Table 3.
Table 3: the performance evaluation of kit of the present invention
Embodiment six: for detection of the fluorescence immune chromatography of people Lp-PLA2 albumen in determinandThe preparation of test paper.
One, be marked with preparation and the coated pad of the monoclonal antibody of fluorescent microsphere
1, be marked with the preparation of the monoclonal antibody of fluorescent microsphere
1.1, the activation of fluorescent microsphere:
Get fluorescent microsphere (purchased from the Guangzhou Growth hormone secretagogue company) water of 500 μ l, content 1 (w/v) %Dispersion liquid, at 10 DEG C, with 13000rpm centrifugal 10 minutes, removes supernatant, by sedimentBe distributed in the distilled water or first wash buffer (the MES aqueous solution of 0.1M) of 500 μ l, superSound wave (240W) is processed 2 minutes, repeats above process three times, adds carbodiimide (to purchaseFrom Shanghai Jing Chun company) 50mg, stirs 15 minutes, thereby activates described fluorescent microsphere.
1.2, with the fluorescent microsphere labelled antibody having activated:
By the fluorescent microsphere having activated under 13000rpm centrifugal 10 minutes, remove supernatant, willSediment be distributed to 1ml coupling buffer (the N-hydroxy thiosuccinimide of 50mMCitrate buffer solution) in, ultrasonic wave (240W) is processed 2 minutes, repeats above process threeInferior, obtain the buffer solution 1ml that is dispersed with fluorescent microsphere. According to 2 μ l antibody (10mg/ml)/100 μ lThe ratio of the fluorescent microsphere having activated, adds the Lp-PLA2 preparing by embodiment 2 whereinMonoclonal antibody (1), stirs 2 hours at normal temperatures, adds 1ml sealing buffer solution (1 (w/v) %BSA-0.05M monoethanolamine), continue to stir 1 hour, afterwards, centrifugal under 13000rpm10 minutes, repeated centrifugation 3 times, was distributed to the whole wash buffer of 500 μ l by sedimentIn (0.5 (w/v) %BSA-0.11 (v/v) % Tween solution), ultrasonic wave (240W) is processed2 minutes, be settled to 500 μ l with above-mentioned whole wash buffer.
2, coated pad
The Lp-PLA2 monoclonal antibody (1) that is marked with fluorescent microsphere of above-mentioned preparation is used to antibodyDilution (1% (w/v) BSA-0.01MPBS(pH7.2) buffer solution) be diluted to 1.5mg/ml,Obtain working solution, then use micropipettor (purchased from labsystems company) by 4 μ l/cmAmount be evenly sprayed on pad, use afterwards 37 DEG C of oven for drying, under 45% humidity, protectDeposit for subsequent use.
Two, the preparation of reaction film
The Lp-PLA2 monoclonal antibody (2) and the sheep anti-mouse igg Dan Ke that prepare according to embodiment 2Grand antibody (purchased from Beijing company of Zhong Shan Golden Bridge) divides with the PBS buffer solution of 50mMpH7.2Be not diluted to 1mg/ml, by detection line and the Quality Control of metal spraying machine (purchased from Hangzhou Feng Hang company)Line spacing parameter is set to 6mm, and package amount is set to respectively to 1.0 μ l/cm, uses metal spraying machineLp-PLA2 monoclonal antibody (2) and sheep anti-mouse igg monoclonal on drawing on nitrocellulose filterAntibody, normal temperature dries for subsequent use.
Three, the assembling of test paper and cutting
On base plate, overlap joint is pasted sample pad, pad, reaction film and water suction filter mutually successivelyPaper, obtains test paper plate, is cut to the test strips that width is 5mm.
Four, the preparation of Lp-PLA2 fluorescence immunoassay test card:
The test paper of above-mentioned well cutting is fixed on plastic bottom card, and test paper surface compresses with face card,Face is stuck on the sample pad of test strips and the position of reaction film and has well and observation window. DetectAfter card assembles, pack in aluminium foil bag, add drier sealing to preserve, can under drying at room temperature conditionTo preserve more than 1 year.
Five, the detection of sample
Lp-PLA2 standard items (purchased from Shanghai Lian Shuo company) are used sample diluting liquid(1% (w/v) BSA-0.01MPBS(pH7.2) buffer solution) be mixed with following series concentrationCalibration object: 1200ng/ml, 600ng/ml, 300ng/ml, 150ng/ml, 75ng/ml, 0ng/ml,The above calibration object of 50 μ l is added drop-wise to respectively on well, after 10 minutes, (purchases with fluorescence detectorFrom Anqun Bioengineering Co., Ltd., Shenzhen, model AQ-3000) detect, can be at detection lineWith on nature controlling line position, collect fluorescence. Taking sample concentration as abscissa, detection line and nature controlling lineThe ratio of the fluorescence intensity at place is that ordinate is drawn calibration curve, R2Be 0.987. Nature controlling line is usedJudge and detection line signal is done to corresponding correction in test paper validity, as bar does not appear in nature controlling lineBand, illustrates that test paper lost efficacy.
The blood sample to be checked of getting 50 μ l, is added drop-wise on well, after 10 minutes, uses fluorescence detectorDetect, if band appears in detection line, in interpret sample, containing Lp-PLA2, its concentration can be complied withObtain according to calibration curve.
Six, Lp-PLA2 fluorescence immunoassay test paper performance evaluation
1. evaluate the index of test paper performance
1) range of linearity: each concentration calibration object duplicate detection 3 times, draw calibration curve, warpData fitting and statistical analysis, test paper linear detection range of the present invention is 15.6ng/ml-1200ng/ml。
2) minimum detectability: Lp-PLA2 null value blood sample (without Lp-PLA2 composition) (is purchasedFrom Shanghai Lian Shuo company) be divided into 20 parts and detect, calculating concentration mean value and 2 times of standardsPoor sum, obtains test paper lowest detection of the present invention and is limited to 0.1ng/ml.
3) precision: detect respectively with Lp-PLA2 fluorescence immunoassay test paper of the present inventionLp-PLA2 concentration is respectively the blood sample of 800ng/ml, 300ng/ml, 100ng/ml, repeatsDetect 10 times, carry out withinrun precision mensuration. Carry out every day to the sample of above-mentioned 3 concentrationMeasure, 1 day 1 time, survey continuously 20 days, carry out betweenrun precision mensuration, result is as following table 4Shown in:
Table 4
Batch in the CV(coefficient of variation) and batch between CV be all less than 8%, this reagent accurate is describedWell.
In addition, as seen from the above table, the range of linearity of this detection paper Lp-PLA2 is wide, sensitivityGood.
Claims (17)
1. the fluorescence immune chromatography for quantitative detection determinand people Lp-PLA2 albumenTest paper, this test paper detects described people Lp-PLA2 albumen by double antibody sandwich method, wherein:
Described double antibody sandwich method adopts a Lp-PLA2 monoclonal that is marked with fluorescent microsphereAntibody is as capture antibody, and a described Lp-PLA2 monoclonal antibody derives from people Lp-PLA2One in epitope peptide (1) and (2); And
Described double antibody sandwich method adopts the 2nd Lp-PLA2 monoclonal antibody anti-as detectingBody, described the 2nd Lp-PLA2 monoclonal antibody derives from people Lp-PLA2 epitope peptide (1)(2) another one in;
Described people Lp-PLA2 epitope peptide (1) and (2) are respectively:
(1)Thr-Lys-Ile-Pro-Arg-Gly-Asn-Gly-Pro-Arg-Ser-Lys-Glu-Ser-Tyr；
(2)Tyr-Lys-Gly-Asp-Ile-Asp-Ser-Asn-Val-Ala-Ile-Asp-Leu-Ser-Asn。
2. fluorescence immune chromatography test paper according to claim 1, wherein said firstLp-PLA2 monoclonal antibody is prepared from by a Lp-PLA2 antigen, and this is first years oldLp-PLA2 antigen is by making the one in described people Lp-PLA2 epitope peptide (1) and (2)Be prepared from carrier protein couplet; And
Described the 2nd Lp-PLA2 monoclonal antibody is to be prepared from by the 2nd Lp-PLA2 antigen, the 2nd Lp-PLA2 antigen is by making described people Lp-PLA2 epitope peptide (1)(2) another one in and carrier protein couplet are prepared from.
3. fluorescence immune chromatography test paper according to claim 1, wherein said fluorescence is micro-The particle diameter of ball is 320nm to 400nm.
4. fluorescence immune chromatography test paper according to claim 1, wherein said fluorescence is micro-Fluorescent material on ball is fluorescein isothiocynate, RB 200, tetramethyl isothiocyanic acidRhodamine or X-rhodamine; The micro-sphere material of described fluorescent microsphere is polystyrene, poly-methylThe copolymer of methyl acrylate or methyl methacrylate.
5. fluorescence immune chromatography test paper according to claim 4, wherein said fluorescence is micro-Fluorescent material on ball is X-rhodamine.
6. fluorescence immune chromatography test paper according to claim 1, wherein said fluorescence is micro-The excitation wavelength of ball is 350～600nm; Emission wavelength is 500～700nm.
7. fluorescence immune chromatography test paper according to claim 6, wherein said fluorescence is micro-The excitation wavelength of ball is 390nm; Emission wavelength is 615nm.
8. fluorescence immune chromatography test paper according to claim 1, wherein said test paper toolHave base plate, and on this base plate along use time chromatography direction successively with way of contact settingHave: sample pad, pad, reaction film, absorbent filter, described pad is provided with describedA Lp-PLA2 monoclonal antibody that is marked with fluorescent microsphere, described reaction film comprises detectionDistrict and Quality Control district, described detection zone is coated with described the 2nd Lp-PLA2 monoclonal antibody, instituteState Quality Control district be coated with can with a described Lp-PLA2 monoclonal that is marked with fluorescent microsphereThe antiantibody of antibody specific binding.
9. fluorescence immune chromatography test paper according to claim 8, wherein said reaction filmFor not fluorescent nitrocellulose filter under the wavelength that is greater than 550nm.
10. fluorescence immune chromatography test paper according to claim 8, wherein said base plate is notThere is photoluminescent property.
11. fluorescence immune chromatography test papers according to claim 8, wherein said antiantibodyFor sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody.
12. fluorescence immune chromatography test papers according to claim 11, wherein said anti-Body is sheep anti-mouse igg monoclonal antibody.
Prepare according to the fluorescence immunoassay described in any one in claim 1 to 12 for 13. 1 kindsThe method of chromatographic test paper, it comprises the following steps:
1) provide a Lp-PLA2 monoclonal antibody that is marked with fluorescent microsphere;
2) provide pad, wherein coated described on described pad to be marked with fluorescence micro-The one Lp-PLA2 monoclonal antibody of ball;
3) provide reaction film, chromatography direction interval when wherein edge is used on described reaction filmFix the 2nd Lp-PLA2 monoclonal antibody and antiantibody, to form respectively detection zone and Quality ControlDistrict; With
4) on base plate along use time chromatography direction successively with the way of contact arrange sample pad,Described pad, described reaction film, absorbent filter, thus described fluorescence immune chromatography examination madePaper.
14. methods according to claim 13, wherein said step 1) comprising:
A) activate fluorescent microsphere with carbodiimide;
The fluorescent microsphere of the activation that b) washing step a) obtains;
C) fluorescent microsphere mark the one Lp-PLA2 monoclonal antibody of using step b) to obtain.
15. methods according to claim 14, wherein said step 1) comprising:
A) by the aqueous dispersions of fluorescent microsphere or MES buffer solution dispersion liquid through ultrasonic wave processingAnd mix with carbodiimide, thereby activate described fluorescent microsphere;
B) by steps A) the N-hydroxyl sulfo-succinyl for fluorescent microsphere of the activation that obtainsThe washing of imines-citrate buffer solution, dispersion, and through ultrasonic wave processing;
C) by step B) fluorescent microsphere and the Lp-PLA2 monoclonal antibody that obtainMix, with the sealing of BSA-monoethanolamine buffer solution, centrifugal, with the dispersion of BSA-Tween solution,Through ultrasonic wave processing, thereby it is anti-to obtain being marked with a Lp-PLA2 monoclonal of fluorescent microsphereBody.
16. methods according to claim 13, wherein in described step 2) in, instituteThe coated concentration of a Lp-PLA2 monoclonal antibody that is marked with fluorescent microsphere of stating is 0.5～2mg/ml。
17. methods according to claim 13, wherein in described step 3) in, instituteState detection zone and described Quality Control interval every 3mm to 8mm, described the 2nd Lp-PLA2 Dan KeThe coated concentration of grand antibody and described antiantibody is respectively 0.5～2mg/ml.
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| CN104614534A (en) * | 2015-02-09 | 2015-05-13 | 杨子学 | Rapid chromatography detection card and kit for simultaneously determining lipoprotein-associated phospholipase A2 and C reactive protein in blood plasma |
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| CN105652008B (en) * | 2016-03-31 | 2017-10-10 | 广州市微米生物科技有限公司 | People's Lp PLA2 biotinstreptatins fluorescence immune chromatography detection card and preparation method thereof |
| CN107490692A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of fluorescence immune chromatography method for quantitatively detecting hs-CRP and lipoprotein phospholipase A2 |
| 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 |
| CN106841612B (en) * | 2016-12-27 | 2021-04-02 | 福建普立辰生物技术有限公司 | Preparation method of human lipoprotein-associated phospholipase A2 immunochromatography test strip |
| WO2021128065A1 (en) * | 2019-12-25 | 2021-07-01 | 广州菲康生物技术有限公司 | Human fgf-23 fluorescence immunochromatographic test paper and human fgf-23 fluorescence immunochromatographic test kit |
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