CN105717303A - Method and reagent kit for detecting phosphatidylinositol proteoglycan 3 with fluorescence immunochromatographic method - Google Patents
Method and reagent kit for detecting phosphatidylinositol proteoglycan 3 with fluorescence immunochromatographic method Download PDFInfo
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Abstract
The invention provides a method and a reagent kit for detecting phosphatidylinositol proteoglycan 3 with a fluorescence immunochromatographic method. The method for detecting phosphatidylinositol proteoglycan 3 with the fluorescence immunochromatographic method comprises the steps that the fluorescence characteristic of rare earth fluorescent microspheres is utilized and combined with a mature immunochromatography technology, and fluorescent quantitative determination of phosphatidylinositol proteoglycan 3 is achieved by optimizing all steps. The reagent kit is characterized by being composed of a test paper card and an immunofluorescence chromatography analysis meter. A preparing method of the reagent kit comprises the steps of preparation of a specificity high-appetency monoclonal antibody, antibody labeling with rare earth fluorescent microspheres, preparation of a fluorescent microsphere combined pad and a nitrocellulose membrane sprayed with the antibody, assembling and wrapping of test paper strips, and the like. In the detection process, a sample is operated as a specification, a result is subjected to quantitative determination with the immunofluorescence chromatography analysis meter, and the advantages of being easy, convenient and fast to implement, suitable for field detection and the like are achieved.
Description
Technical field:
The invention belongs to Medical Immunology field, relate to a kind of fluorescence immune chromatography technology, further, the present invention relates to a kind of method of rare-earth fluorescent immunochromatographyassay assay phosphatidylinositol protein polysaccharide 3 and test kit thereof, to realize fast and accurately phosphatidylinositol protein polysaccharide 3 being carried out quantitative analysis.
Background technology:
Primary hepatocarcinoma (primaryhepaticcarcinoma, PHC) is one of malignant tumor that at present mortality rate is the highest in the world, and the five year survival rate after making a definite diagnosis is less than 12%, and its sickness rate worldwide has and increases trend in recent years.Surgical excision is still the Therapeutic Method that current PHC is the most frequently used, but has belonged to middle and advanced stage when Most patients is medical, loses best operative treatment opportunity, thus the early diagnosis level improving PHC is to improve the key of liver cancer treatment and outcome.Alpha-fetoprotein (AFP) is the most widely used hepatic carcinoma label in the current whole world, but PHC patient's AFP sensitivity is only 35%-65%, specificity is less than 80%, especially early hepatocarcinoma positive rate is lower, the method made a definite diagnosis in this, as PHC early stage is not very good, in the urgent need to finding new label.Phosphatidylinositol protein polysaccharide 3 (Glypican-3, GPC3) be it have recently found that the liver cancer serum label with potential value.
GPC3 belongs to film Heparan sulfate class glycoprotein superfamily member, has C-terminal and is connected on cell membrane, and aminoterminal is free in extracellular special construction, it is believed that with location, carry relevant.It is the complicated saccharide complex covalently bound by protein, lipid and sugar three, its aminoterminal (N) is soluble protein, can secreting to peripheral blood when expressing in hepatoma carcinoma cell, this is the molecular biology mechanism being able to detect that GPC3 in peripheral circulation blood.Clinical basic shows, during hepatocyte generation canceration GPC3 can unconventionality expression, the GPC3 of activation can pass through multi-signal pathway promote hepatocyte vicious transformation, strengthen its invasive ability.Single GPC3 or AFP expresses limited at PHC, and combines GPC3 and AFP detection and significantly improve the Sensitivity and Specificity of PHC diagnosis, reaches 84.2% and 95.7% respectively, is significantly higher than AFP or GPC3 and individually detects.PHC diagnostic level can be improved.The diagnostic level of AFP negative PHC can be improved especially with GPC3 detection, PHC early diagnosis and examination are had definite meaning, be the significant index improving PHC early diagnosis and Differential Diagnosis level.
Phosphatidylinositol protein polysaccharide 3 detection method mainly has double-antibody method (ELISA) and Immunohistochemical Method, and ELISA method operating time length, automaticity are low, and detection by quantitative is not accurate enough；Immunohistochemical Method needs tissue slice, and can not carry out sample of tissue in PHC early stage, it is impossible to obtains result in early days and carries out early diagnosis.Therefore exploitation sensitivity is higher, phosphatidylinositol protein polysaccharide 3 product fast and easily, is still clinical diagnosis product research field and needs the major issue of solution badly.
Summary of the invention:
The technical problem to be solved is the deficiency for above-mentioned background technology, utilize the susceptiveness of fluorescence immune chromatography, combined with fluorescent immunochromatographiassays assays instrument, there is provided a kind of highly sensitive, fast and simple, it is possible to the method with fluorescence immune chromatography method detection phosphatidylinositol protein polysaccharide 3 of accurate quantitative analysis and test kit thereof.
For reaching above-mentioned purpose, the technical solution used in the present invention is: the method for a kind of fluorescence immune chromatography method detection phosphatidylinositol protein polysaccharide 3, phosphatidylinositol protein polysaccharide 3 can be carried out quantitative assay by the method, has the advantages such as quick, applicable Site Detection highly sensitive, easy and simple to handle；Another object of the present invention is to provide the test kit of a kind of fluorescence immune chromatography method detection phosphatidylinositol protein polysaccharide 3, it is possible to be applied to situation of all-level hospitals emergency department and basic hospital.
The test kit of fluorescence immune chromatography method of the present invention detection phosphatidylinositol protein polysaccharide 3, it is characterised in that test kit is made up of test card and fluorescence immune chromatography analyser.
Described test card, it is in PVC board, sequentially mutually overlap treated sample pad, be adsorbed with the pad of the antibody of rare-earth fluorescence labeling, the nitrocellulose filter being coated with detection line and nature controlling line and adsorptive pads, reagent paper is formed after assembling, it is then cut into the wide test strips of 4mm, test strips is loaded formation test card in plastic clip.
Preferably, described pad being adsorbed with rare-earth fluorescent microsphere, the diameter of rare-earth fluorescent microsphere is 80-220nm, it is further preferred that the diameter of selected rare-earth fluorescent microsphere is 120-150nm.
Preferably, described rare-earth fluorescent microsphere is doped with rare earth lanthanide, including any one or a few in europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd).
Preferably, described rare-earth fluorescent microsphere contains rare-earth complex, stable under ground state, under the excitation source effect of 320-360nm, it is possible to launch the wave-length coverage fluorescence at 540-580nm.
On described pad, the antibody of rare-earth fluorescent microsphere labelling is phosphatidylinositol protein polysaccharide 3 monoclonal antibody of rare-earth fluorescent microsphere labelling, monoclonal antibody is the monoclonal antibody of mixing after purification, derives from the cell strain of monoclonal antibody of phosphatidylinositol protein polysaccharide 3 epitope different for 2-6.
It is further preferred that the antibody sources of rare-earth fluorescent microsphere labelling is in the monoclonal cell cell strain for 3 different epitopes on pad.
Described fluorescence immune chromatography analyser is a kind of Systems for optical inspection, and for the quantitative judgement to testing result, the detection range to phosphatidylinositol protein polysaccharide 3 is 0-800ng/mL.
The preparation method of phosphatidylinositol protein polysaccharide 3 fluorescence immune chromatography Test paper box of the present invention, comprises the following steps:
A, phosphatidylinositol protein polysaccharide 3 monoclonal antibody preparation process as follows:
Step 1), the acquisition of cell strain of monoclonal antibody: with reference to phosphatidylinositol protein polysaccharide 3 aminoacid sequence, select the peptide sequence about 18 aminoacid of site synthetic that antigenicity is strong, it is linked on KLH, adopts the method for preparing monoclonal antibody of standard to prepare the cell strain of monoclonal antibody of specificity high-affinity.
Step 2), the preparation of monoclonal antibody: adopt the preparation of ascites production technology purification phosphatidylinositol protein polysaccharide 3 monoclonal antibody of standard, be stored in after subpackage-20 DEG C standby.
B, rare-earth fluorescent microsphere aldehyde radical:
Taking 3mg rare-earth fluorescent microsphere, with the carbonate buffer solution of 50mM, pH9.5, adopt centrifuging to wash 3 times, centrifugal speed is 12000rpm, and the time is 10 minutes, is finally resuspended in the above-mentioned carbonate buffer solution of 100 μ l.Add the glucosan of 200 μ l aldehyde radicals, mixing, dark reaction 4 hours under room temperature.In adopting the washing of same centrifuging and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C standby.
C, rare-earth fluorescent microsphere labelling the preparation of phosphatidylinositol protein polysaccharide 3 monoclonal antibody:
By 1mg phosphatidylinositol protein polysaccharide 3 monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnight, then mixing with the rare-earth fluorescent microsphere of above-mentioned aldehyde radical, 4 DEG C of reactions are overnight.Then, adding sodium borohydride to final concentration 10mM, 4 DEG C are reacted 4 hours；Add isopyknic confining liquid (150mMTris-HCL, pH7.5, containing 2.5%BSA, 3% sucrose), close overnight for 4 DEG C；Then adopting centrifuging to wash 3 times with the buffer of 150mMTris-HCL, pH7.5, be resuspended in the 150mMTris-HCL buffer of 100 μ l (containing 2%NaCL, 1%BSA, 0.3%Tween20), 4 DEG C keep in Dark Place standby.
D, absorption fluorescent microsphere labelling the preparation of pad of antibody:
Being soaked in by glass fibre membrane (containing 1.5%TritonX-100,2%BSA, pH7.5) in 200mMTris-HCL treatment fluid, 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 hours, standby.By glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platform, with the Bio-JetQuanti300 noncontact quantitation nozzle that declines, phosphatidylinositol protein polysaccharide 3 monoclonal antibody of rare-earth fluorescent microsphere labelling is sprayed onto glass fibre membrane, dry 4 hours for 37 DEG C, standby.
E, it is coated with the preparation of nitrocellulose filter of detection line and nature controlling line:
Step 1), adopt and the different sequence site of phosphatidylinositol protein polysaccharide 3 monoclonal antibody on acquisition pad, synthetic peptide sequence, with reference to said monoclonal antibody preparation flow obtain phosphatidylinositol protein polysaccharide 3 monoclonal antibody, be stored in-20 DEG C standby.
Step 2), phosphatidylinositol protein polysaccharide 3 monoclonal antibody and goat anti-mouse igg antibody are adjusted concentration to 8mg/ml with being coated diluent respectively, film liquid measure is 1.5 μ l/cm, using them, as detecting, line is parallel with nature controlling line to be sprayed on nitrocellulose filter and is coated, detection line and nature controlling line are spaced apart 4.5mm, it is subsequently placed in baking oven, dries 4 hours for 37 DEG C.
F, sample pad preparation:
Glass fibre membrane is soaked in 0.2MTris buffer (pH7.5), 1.5%TritonX-100, in the treatment fluid of 2%BSA, soaks 4 hours in 4 DEG C, be subsequently placed in baking oven, dry 4 hours for 37 DEG C.
G, phosphatidylinositol protein polysaccharide 3 fluorescence immune chromatography Test paper card assembling
PVC board is pasted treated sample pad successively, is adsorbed with the pad of the antibody of rare-earth fluorescent microsphere labelling, the nitrocellulose filter being coated with detection line and nature controlling line and adsorptive pads, the big plate of reagent paper is obtained after assembling, cut into 4mm width as requested, reagent paper is loaded formation test card in plastic clip.
The method of a kind of fluorescence immune chromatography method detection phosphatidylinositol protein polysaccharide 3, comprises the following steps: 1) detectable and sample are balanced to room temperature, take out test card, keep flat；2) accurately drawing 100 μ l serum samples, 150 μ l samples drawn by sample when being whole blood, join in sample aperture, quantitatively judge result with fluorescence immune chromatography analyser in 15-30 minute；3) after setting instrument relevant parameter, test card being put into storehouse to detect, instrument would indicate that the quantified results of sample concentration.
The present invention provides a kind of phosphatidylinositol protein polysaccharide 3 fast quantification immunochromatographytest test kit utilizing rare-earth fluorescent immunochromatography technique to prepare, it is suitable for serum and whole blood sample simultaneously, and it is suitable for single part detection clinically, phosphatidylinositol protein polysaccharide 3 content in energy detection by quantitative sample, has clear and definite Clinical significance of MG.Test kit have easy and simple to handle, react quick, highly sensitive, high specificity, be suitable for Site Detection and the advantage such as economical and practical.
Accompanying drawing explanation
Fig. 1 is the accuracy analysis figure of the result of the test of the present invention.
Detailed description of the invention
Embodiment 1: the preparation of phosphatidylinositol protein polysaccharide 3 fluorescence immune chromatography detection kit
1 main material:
1.1, the anti-Shanghai trade Co., Ltd of phosphatidylinositol protein polysaccharide 3 specific pairs antibody: Ai Bo；Phosphatidylinositol protein polysaccharide 3 quality-control product: Sino Biological Inc.；Rare-earth fluorescent microsphere: Zhen Zhun bio tech ltd, Shanghai；Celluloid (NC) film: Millipore Products；Bovine serum albumin (BSA), Polyethylene Glycol PEG20000, caseinhydrolysate: Sigma product.Other common agents is analytical reagent.
1.2, fluorescence immune chromatography analyser: model: HF201, Shenzhen Highcreation Technology Co., Ltd..
2 methods
1), the aldehyde radical of rare-earth fluorescent Nano microsphere:
Taking 3mg rare-earth fluorescent microsphere, with the carbonate buffer solution of 50mM, pH9.5, adopt centrifuging to wash 3 times, centrifugal speed is 12000rpm, and the time is 10 minutes, is finally resuspended in the above-mentioned carbonate buffer solution of 100 μ l.Add the glucosan of 200 μ l aldehyde radicals, mixing, dark reaction 4 hours under room temperature.In adopting the washing of same centrifuging and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C standby.
2), the preparation of rare-earth fluorescent Nano microsphere labelling phosphinositides Dan Baiduotang proteoglycan PG 3 monoclonal antibody:
By 1mg phosphatidylinositol protein polysaccharide 3 monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnight, then mixing with the rare-earth fluorescent microsphere of above-mentioned aldehyde radical, 4 DEG C of reactions are overnight.Then, adding sodium borohydride to final concentration 10mM, 4 DEG C are reacted 4 hours；Add isopyknic confining liquid (150mMTris-HCL, pH7.5, containing 2.5%BSA, 3% sucrose), close overnight for 4 DEG C；Then adopting centrifuging to wash 3 times with the buffer of 150mMTris-HCL, pH7.5, be resuspended in the 150mMTris-HCL buffer of 100 μ l (containing 2%NaCL, 1%BSA, 0.3%Tween20), 4 DEG C keep in Dark Place standby.
3) preparation of the pad of fluorescent microsphere traget antibody, is adsorbed:
Being soaked in by glass fibre membrane (containing 1.5%TritonX-100,2%BSA, pH7.5) in 200mMTris-HCL treatment fluid, 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 hours, standby.By glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platform, with the Bio-JetQuanti300 noncontact quantitation nozzle that declines, phosphatidylinositol protein polysaccharide 3 monoclonal antibody of rare-earth fluorescent microsphere labelling is sprayed onto glass fibre membrane, dry 4 hours for 37 DEG C, standby.
4) preparation of the nitrocellulose filter of detection line and nature controlling line, it is coated with:
Step 1), adopt and the different sequence site of phosphatidylinositol protein polysaccharide 3 monoclonal antibody on acquisition pad, synthetic peptide sequence, with reference to said monoclonal antibody preparation flow obtain phosphatidylinositol protein polysaccharide 3 monoclonal antibody, be stored in-20 DEG C standby.
Step 2), phosphatidylinositol protein polysaccharide 3 monoclonal antibody and goat anti-mouse igg antibody are adjusted concentration to 8mg/ml with being coated diluent respectively, film liquid measure is 21.5 μ l/cm, using them, as detecting, line is parallel with nature controlling line to be sprayed on nitrocellulose filter and is coated, detection line and nature controlling line are spaced apart 4.5mm, it is subsequently placed in baking oven, dries 4 hours for 37 DEG C.
5), the preparation of sample pad:
Glass fibre membrane is soaked in 0.2MTris buffer (pH7.5), 1.5%TritonX-100, in the treatment fluid of 2%BSA, soaks 4 hours in 4 DEG C, be subsequently placed in baking oven, dry 4 hours for 37 DEG C.
6), the assembling of phosphatidylinositol protein polysaccharide 3 fluorescence immune chromatography Test paper card
PVC board is pasted treated sample pad successively, is adsorbed with the pad of the antibody of rare-earth fluorescence labeling, the nitrocellulose filter being coated with detection line and nature controlling line and adsorptive pads, the big plate of reagent paper is obtained after assembling, cut into 4mm width as requested, reagent paper is loaded formation test card in plastic clip.
7), the setting of fluorescence immunity analyzer parameter
After fluorescence immunity analyzer sets test card technological parameter, take the above-mentioned test card assembled, respectively with 0,25,50,100,200,400, phosphatidylinositol protein polysaccharide 3 standard substance of 800ng/mL, it is measured with test card, obtain the fluorescence intensity level of each standard substance, result is input in the parameter of analyser, completes the setting of the parameter of analyser.
Embodiment 2: sample accuracy test
1, main material: clinical sample is obtained at relevant hospital by our company, and totally 200 parts of ELISA definite value samples, wherein serum sample 100 parts, whole blood sample 100 parts, phosphatidylinositol protein polysaccharide 3 content distribution interval is between 0-800ng/mL.
2, test method:
1.1, detection method:
1) detectable and sample are balanced to room temperature, take out test card, keep flat；2) accurately drawing 100 μ l serum samples, 150 μ l samples drawn by sample when being whole blood, join in sample aperture, quantitatively judge result with fluorescence immune chromatography analyser in 25 minutes；3) after setting instrument relevant parameter, test card being put into storehouse to detect, instrument would indicate that the quantified results of sample concentration.
1.2, test result analysis:
After prepared by clinical sample detectable, by detection method, all clinical samples are detected, and analyze testing result.
3, result of the test
As it is shown in figure 1, clinical sample detect to 200 parts of clinical definite value pattern detection, sample mean deviation value is respectively less than 10%, maximum deviation less than 20%, R2 > 0.98, consistency coefficient > 0.95.Testing result shows that the detection kit of preparation is functional, is suitable for Clinical detection, meets the differentiation needs of different client's difference detection occasion.
Claims (9)
1. the test kit detecting phosphatidylinositol protein polysaccharide 3 with fluorescence immune chromatography standard measure, it is characterised in that: include test card and fluorescence immune chromatography analyser；Described test card is sequentially mutually overlap treated sample pad in PVC board, be adsorbed with the pad of the antibody of rare-earth fluorescence labeling, the nitrocellulose filter being coated with detection line and nature controlling line and adsorptive pads, reagent paper is formed after assembling, it is then cut into the wide test strips of 4mm, test strips is loaded formation test card in plastic clip；Described fluorescence immune chromatography analyser is a kind of Systems for optical inspection, it is possible to for testing result is quantitatively judged, the detection range to phosphatidylinositol protein polysaccharide 3 is 0-800ng/mL.
2. test kit according to claim 1, it is characterized in that: above described pad, be adsorbed with fluorescent microsphere traget antibody, described traget antibody is that rare-earth fluorescent microsphere adopts chemical crosslink technique to be attached the conjugate obtained with phosphatidylinositol protein polysaccharide 3 monoclonal antibody specific, and concrete preparation process is:
Step 1), the acquisition of cell strain of monoclonal antibody: with reference to phosphatidylinositol protein polysaccharide 3 aminoacid sequence, select the peptide sequence about 18 aminoacid of site synthetic that antigenicity is strong, it is linked on KLH, adopts the method for preparing monoclonal antibody of standard to prepare the cell strain of monoclonal antibody of specificity high-affinity；
Step 2), the preparation of monoclonal antibody: adopt the preparation of ascites production technology purification phosphatidylinositol protein polysaccharide 3 monoclonal antibody of standard, be stored in after subpackage-20 DEG C standby；
Step 3), the preparation of fluorescent microsphere traget antibody: the carbonate buffer solution taking rare-earth fluorescent microsphere pH9.5 washs, and the glucosan adding aldehyde radical reacts 3 hours, adds said monoclonal antibody mixing, 4 DEG C of reverse mixing overnight；Adding sodium borohydride solution and continue reaction 6 hours, ultrafiltration removes unconjugated material, and 4 DEG C store for future use.
3. test kit according to claim 1, it is characterized in that: above described nitrocellulose filter, be coated with detection line and nature controlling line, detection line is phosphatidylinositol protein polysaccharide 3 monoclonal antibody specific, and nature controlling line is goat anti-mouse igg antibody, and concrete preparation process is:
Step 1), adopt and the different sequence site of phosphatidylinositol protein polysaccharide 3 monoclonal antibody on acquisition pad, synthetic peptide sequence, with reference to said monoclonal antibody preparation flow obtain phosphatidylinositol protein polysaccharide 3 monoclonal antibody, be stored in-20 DEG C standby；
Step 2), phosphatidylinositol protein polysaccharide 3 monoclonal antibody and goat anti-mouse igg antibody are adjusted concentration to 8mg/ml with being coated diluent respectively, film liquid measure is 1.5 μ l/cm, using them, as detecting, line is parallel with nature controlling line to be sprayed on nitrocellulose filter and is coated, detection line and nature controlling line are spaced apart 4.5mm, it is subsequently placed in baking oven, dries 4 hours for 37 DEG C.
4. test kit according to claim 1, it is characterized in that: the concrete preparation process of described sample pad is: glass fibre membrane is soaked in 0.2MTris buffer (pH7.5), 1.5%TritonX-100, in the treatment fluid of 2%BSA, soak 3 hours in 4 DEG C, it is subsequently placed in baking oven, dries 4 hours for 37 DEG C.
5. test kit according to claim 1, it is characterized in that: described rare-earth fluorescent microsphere is doped with rare earth lanthanide, including any one or a few in europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd).
6. test kit according to claim 1, it is characterised in that: described rare-earth fluorescent microsphere excitation wavelength range is 320-360nm, and emission wavelength ranges is 540-580nm.
7. test kit according to claim 1, it is characterized in that: the monoclonal antibody used on pad and nitrocellulose filter in described test card is that phosphatidylinositol protein polysaccharide 3 matches antibody, identifying the different epi-positions of phosphatidylinositol protein polysaccharide 3 respectively, antibody is the monoclonal antibody of peptide affinity purification.
8. test kit according to claim 1, it is characterised in that: each several part of described test card does not contain fluorescent agent, and testing result is not affected.
9. the method detecting phosphatidylinositol protein polysaccharide 3 with fluorescence immune chromatography standard measure, utilizes rare-earth fluorescent micro-ball immune chromatography technology to realize the detection by quantitative to phosphatidylinositol protein polysaccharide 3, specifically includes following steps:
1) detectable and sample are balanced to room temperature, take out test card, keep flat；
2) accurately drawing 100 μ l serum samples, 150 μ l samples drawn by sample when being whole blood, join in sample aperture, quantitatively judge result with fluorescence immune chromatography analyser in 15-30 minute；
3) after setting instrument relevant parameter, test card being put into storehouse to detect, instrument would indicate that the quantified results of sample concentration.
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