CN102854305B - Prewarning kit for transplant kidney rejection and use method thereof - Google Patents
Prewarning kit for transplant kidney rejection and use method thereof Download PDFInfo
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Abstract
The invention relates to a prewarning kit, in particular to a prewarning kit for transplant kidney rejection and a use method thereof. Liquid protein chip luminex detecting technology is used for conducting monitoring and comprehensive analyzing on expressing level of 96 kinds of cell factors/chemotactic factors and receptors of the cell factors/chemotactic factors in 266 portions of serum of patients in 142 cases of acute transplant kidney rejection and transplantation receptors with a stable transplant kidney function. A transplant kidney rejection prewarning system containing at least one united marker is built. Prediction and accuracy of acute rejection is 98.6%, and internal cross validation rate is respectively 97.1% and 97.2%.
Description
The application is dividing an application of patented claim 201110438939.2.
Technical field
The present invention relates to a kind of early warning kit, specifically, relate to a kind of transplanted kidney rejection early warning kit and using method thereof.
Background technology
106.5 ten thousand people that the number of dialysing increased to by 42.6 ten thousand people of nineteen ninety 2000 gos because of chronic renal failure in the whole world, ten thousand people surplus expecting 2010 and will reaching 200.The growth of this number causes global dialysis expense to increase rapidly, and developed country, developing country have all been caused huge financial burden.China has 1,000,000 uremic patients approximately, and annual newly-increased 120,000 people have 500,000 needs of patients kidney transplants every year approximately.China totally carries out nearly 100,000 examples of various organ transplants at present, becomes the second in the world organ transplant big country, and wherein maximum is kidney transplant, and accumulative total reaches 80,000 many cases.It is the final goal that organ transplant is pursued that transplanting receptor and graft have the function long-term surviving.Along with the progress of surgical technic, the raising of tissue matching's technology and the development of immunodepressant, 1 annual survival rate improves greatly behind the renal transplantation, but long-term prognosis still remains to be improved.Outbreak repeatedly and the chronic rejection of acute rejection are the main causes that causes graft mistake merit, take the toxic and side effect that immunodepressant is brought for a long time, as renal toxicity, infection, induced tumor etc., also influence graft and receptor's long-term surviving.
Fa Zuo acute rejection and chronic rejection are the major reasons that causes graft mistake merit repeatedly, but lack effective dynamic monitoring and non-invasive diagnosis means clinically.The transplanted kidney aspiration biopsy is the most reliable diagnostic method at present, but has traumaticly, can cause that infection, hemorrhage even graft are lost etc., is difficult for being accepted by the patient and being unfavorable for dynamic observing.And biopsy is drawn materials also certain limitation, and according to the Banff standard, the patient that many clinical signs of suspected transplanted kidney repel may be able to not meet the diagnostic criteria of repulsion on pathomorphism.
Therefore, press for explore to be fit to population of China, reliably, can satisfy the non-invasive diagnostic method of transplanted kidney rejection that dynamic observes needs.Finishing of this problem will assist the prolongation patient to transplant back life cycle to a great extent, improve life quality, reducing country's medical treatment drops into, save family expense, have great clinical value and society, economic benefit, and help China to transplant the output that the boundary has the independent intellectual property right achievement of the leading level in the world.
The present Research of the non-invasive diagnostic techniques of transplanted kidney rejection: from the eighties in 20th century, transplant educational circles and begun to dynamic observe various kinds of cell factor level and variation in the various serum of transplanting receptor, explore mechanism and the diagnostic method thereof of graft-rejection.For example IL-6, solvable type IL-6 acceptor (IL-6R) solubility CD30 (sCD30), the liver cell regeneration factor (hepatocyte growth factor, HGF), IL-18, transforming growth factor-β (Transforming Growth Factor Beta, TGF-β), IL-17, granzyme B, perforin, FasL, granulysin, CD154, ICOS, CTLA4, PD-1 equimolecular are relevant with the transplanted kidney rejection.No.309 Hospital of PLA organ transplant center was at the dynamic measurement to 37 routine renal transplantation recipients and 55 normal human serum IL-6 levels in 1994, the result shows, transplanted kidney acute renal allograft rejection patients serum IL-6 level obviously raises, and chronic rejection patient IL-6 level does not have marked change, and prompting blood serum IL-6 level monitoring is one of important diagnostic index of transplanted kidney rejection; Subsequently, again respectively to sero-immunity cell inhibition acceptor (leukocyte-associated Ig-like receptor-1, LAIR-1), solvable type LAIR-2 molecule, HLA-G, (specific expressed p140 etc. studies with the correlativity of transplanted kidney rejection and reports blood platelet/T cell activation antigen 1 CD226) and in the allogeneic mixed lymphocyte reaction (MLP) for platelet and T cell activation antigen1, PTA1.Yet, although numerous researcher is devoted to screen the specific biomarker of graft rejection, do not filter out the non-invasive diagnosis and differential diagnosis that specific molecular is applied to rejection so far as yet.The non-invasive diagnosis and the pre-alarming system that still lack at present the transplanted kidney rejection in the world.
Disclose in the structure of acute rejection in renal related immune genetic chip and the preliminary discussion of clinical practice (Zheng Jianming, Medical University Of Tianjin, the 2008) literary composition and used the molecule relevant with acute rejection in renal transplantation of genetic chip screening to be CD40L.
Graft-rejection is a very complicated process, cellular immunity and humoral immunity mechanism all participate in this process, and, the difference of graft, the influence of operation, transplant the difference of receptor's individuality and clinically the participation of numerous factors such as application of different immunosuppressant schemes make the diagnosis of graft-rejection more complicated and difficult.In addition, previously studies show that by detecting the individual molecule graft-rejection of clarifying a diagnosis very big difficulty is arranged.The successful foundation of the serodiagnosis system of early stage epithelial ovarian cancer is for the non-invasive diagnosis of graft-rejection provides new research thinking.2005, the Gil of Yale University etc. are by the screening to the known protein mark, successfully set up by Leptin, prolactin (prolactin), osteopontin (osteopontin, OPN) and the serodiagnosis system of the early stage epithelial ovarian cancer formed of IGF-(IGF-II), to diagnose susceptibility, specificity, positive predictive value (positive predictive value, PPV) and negative predictive value (negtive predictive value NPV) is increased to 95%, 95%, 95% and 94% respectively.Therefore, when identifying graft rejection specificity marker thing, the biomarker relevant to known graft rejection screens and makes up, by the polymolecular analysis-by-synthesis, set up associating mark group diagnosis and the pre-alarming system of rejection, have stronger feasibility and the susceptibility of Geng Gao.
The Luminex technology is a kind of multi-functional liquid-phase chip analysis platform.It is put in order and coloured microballoon (color-coded microsphere or bead), laser technology, fluidics, high speed digital signal processor and computer algorithm, has high detection specificity and sensitivity.The color of microballoon obtains by two kinds of fluorescent dyeings, and the ratio of regulating two kinds of fluorescent dyes can obtain the microballoon of 100 kinds of different colours, and the microballoon of every kind of color can carry a kind of bioprobe.Probe is attached to microsphere surface by carboxyl, therefore can finish 100 kinds of different biologicallies in a reacting hole.The Luminex technology is tested and appraised the color of microballoon and determines reaction type, and to the reaction quantitative test be to finish by the reporter molecules on the target material.Multiple advantages such as it possesses the multiparameter high throughput analysis, save reagent and sample, simple to operate.Adopt Luminex liquid chip systematic quantification to detect candidate molecules, only need the 30ul sample can detect a plurality of indexs simultaneously, can finish analysis in 4 hours, sensitivity can reach 3pg/ml.
Summary of the invention
Primary goal of the invention of the present invention is to provide a kind of transplanted kidney rejection early diagnosis kit;
Second goal of the invention of the present invention is to provide a kind of transplanted kidney rejection early warning kit;
The 3rd goal of the invention of the present invention is to provide the using method of above kit;
The 4th goal of the invention of the present invention is to provide anti-IL-1R Alpha antibodies, the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection of anti-sCD40L antibody and anti-IL-20 antibody.
In order to realize first goal of the invention of the present invention, the technical scheme of employing is:
The present invention relates to a kind of transplanted kidney rejection early diagnosis kit, described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and described antibody is selected from anti-EGF, anti-Eotaxin, anti-FGF-2, anti-Flt-3ligand, anti-Fractalkine, anti-G-CSF, anti-GM-CSF, anti-GRO, anti-IFN-α 2, anti-IFN-γ, anti-IL-10, anti-IL-12 (p40), anti-IL-12 (p70), anti-il-13, anti-IL-15, anti-IL-17, anti-IL-1ra, anti-IL-1 Alpha, anti-il-i-beta, anti-IL-2, anti-IL-3, anti-IL-4, anti-IL-5, anti-IL-6, anti-IL-7, anti-IL-8, anti-IL-9, anti-IP-10, anti-MCP-1, anti-MCP-3, anti-MDC (CCL22), anti-MIP-1 α, anti-MIP-1 β, anti-PDGF-AA, anti-PDGF-AB/BB, anti-RANTES, anti-TGF-α, anti-TNF-α, anti-TNF-β, anti-VEGF, anti-sCD40L, anti-sIL-2R α, anti-6Ckine, anti-BCA-1, anti-CTACK, anti-ENA-78, anti-Eotaxin-2, anti-Eotaxin-3, anti-I-309, anti-IL-16, anti-IL-20, anti-IL-21, anti-il-23, anti-IL-28a, anti-IL-33, anti-LIF, anti-MCP-2, anti-MCP-4, anti-MIP-1d, anti-SCF, anti-SDF-1A+ β, anti-TARC, anti-TPO, anti-TRAIL, anti-TSLP, anti-GCP2, anti-HCC-1, anti-I-TAC, anti-IL-11, anti-IL-29, anti-Lymphotactin, anti-M-CSF, anti-MIG, anti-MIP-3 α, anti-MIP-3 β, anti-NAP2, anti-sCD30 (sTNFRSF8), anti-sEGFR, anti-sIL-1RI (sCD121a), anti-sIL-1RII (sCD121b), anti-sIL-2R α (sCD25), anti-sIL-4R (sCD124), anti-sIL-6R (sCD126), anti-sRAGE, anti-sTNFRI (sTNFRSF1A), anti-sTNFRII (sTNFRSF1B), anti-sVEGFR1 (sFlt-1), anti-sVEGFR2 (sFlk-1), anti-sVEGFR3 (sFlt-4), anti-sgp130, anti-MIF, anti-PAI-1 (Total), anti-sFas, anti-sFasL, at least a in anti-sICAM-1 or the anti-sVCAM-1 antibody, preferred at least two kinds of antibody, more preferably at least 3 kinds of antibody, preferably at least 4 kinds of antibody, most preferably at least 5 kinds of antibody again.
First optimal technical scheme of this technical scheme of the present invention is: described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and described antibody is selected from anti-SCF, anti-sEGFR, anti-sIL-2R α, anti-sRAGE, anti-sTNFR1, anti-sTNFR2, anti-sVEGFR2, anti-sVCAM1, anti-sFas, anti-CCL14 α-HCC1, anti-sCD40L, anti-Eotaxin, anti-FGF2, anti-Flt3ligand, anti-Fracktalkine, anti-GCSF, anti-GRO, anti-IFN γ, anti-IL-1R α, anti-IL-3, anti-IL-4, anti-IL-6, anti-IL-12p70, anti-il-13, anti-IP10, anti-MIP1 α, anti-VEGF, anti-BCA-1, anti-IL-16, anti-MIP1d, anti-IL-20, anti-IL-28A, anti-CXCL9MIG, at least a antibody among anti-CXCL111TAC or the anti-CCL19MIP3 β, preferably at least two kinds of antibody, more preferably at least 3 kinds, preferably at least 4 kinds again, most preferably at least 5 kinds.
The 3rd optimal technical scheme of this technical scheme of the present invention is: described kit also contains:
(1) 96 hole filter membrane plate and two shrouding films;
(2) standard items of protein antibodies in the described kit;
(3) Quality Control contrast;
(4) serum matrix;
(5) experiment liquid;
(6) washing lotion;
(7) labelled antibody streptavidin-phycoerythrin(phycoerythrin-strepto-affinity element);
(8) pearl dilution;
(9) antibody of the albumen of the described detection transplanted kidney of claim 1 rejection;
(10) pearl of pre-coated antibody.
The 4th optimal technical scheme of this technical scheme of the present invention is: in the described kit:
(1) described 96 hole filter membrane plates and two shrouding films are available from Millipore company, article No. MX-PLATE;
(2) described standard items are available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) described Quality Control contrast is available from Millipore company, and article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) described serum matrix is available from Millipore company, and article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) described experiment liquid is available from Millipore company, article No. L-AB;
(6) described washing lotion is available from Millipore company, article No. L-WB;
(7) described streptavidin-phycoerythrin labelled antibody is available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) described pearl dilution is available from Millipore company, article No. LBD;
(9) detect antibody available from Millipore company, article No. MXH1060, MXH1062, MXH1063, LHSP-1063, HSCR-1032;
(10) pearl of described pre-coated antibody is available from Millipore company, article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
For realizing second goal of the invention of the present invention, the technical scheme of employing is: a kind of transplanted kidney rejection early warning kit, described kit comprise the antibody of the albumen that detects the transplanted kidney rejection, described antibody is selected from anti-EGF, anti-Eotaxin, anti-FGF-2, anti-Flt-3ligand, anti-Fractalkine, anti-G-CSF, anti-GM-CSF, anti-GRO, anti-IFN-α 2, anti-IFN-γ, anti-IL-10, anti-IL-12 (p40), anti-IL-12 (p70), anti-il-13, anti-IL-15, anti-IL-17, anti-IL-1ra, anti-IL-1 Alpha, anti-il-i-beta, anti-IL-2, anti-IL-3, anti-IL-4, anti-IL-5, anti-IL-6, anti-IL-7, anti-IL-8, anti-IL-9, anti-IP-10, anti-MCP-1, anti-MCP-3, anti-MDC (CCL22), anti-MIP-1 α, anti-MIP-1 β, anti-PDGF-AA, anti-PDGF-AB/BB, anti-RANTES, anti-TGF-α, anti-TNF-α, anti-TNF-β, anti-VEGF, anti-sCD40L, anti-sIL-2R α, anti-6Ckine, anti-BCA-1, anti-CTACK, anti-ENA-78, anti-Eotaxin-2, anti-Eotaxin-3, anti-I-309, anti-IL-16, anti-IL-20, anti-IL-21, anti-il-23, anti-IL-28a, anti-IL-33, anti-LIF, anti-MCP-2, anti-MCP-4, anti-MIP-1d, anti-SCF, anti-SDF-1A+ β, anti-TARC, anti-TPO, anti-TRAIL, anti-TSLP, anti-GCP2, anti-HCC-1, anti-I-TAC, anti-IL-11, anti-IL-29, anti-Lymphotactin, anti-M-CSF, anti-MIG, anti-MIP-3 α, anti-MIP-3 β, anti-NAP2, anti-sCD30 (sTNFRSF8), anti-sEGFR, anti-sIL-1RI (sCD121a), anti-sIL-1RII (sCD121b), anti-sIL-2R α (sCD25), anti-sIL-4R (sCD124), anti-sIL-6R (sCD126), anti-sRAGE, anti-sTNFRI (sTNFRSF1A), anti-sTNFRII (sTNFRSF1B), anti-sVEGFR1 (sFlt-1), anti-sVEGFR2 (sFlk-1), anti-sVEGFR3 (sFlt-4), anti-sgp130, anti-MIF, anti-PAI-1 (Total), anti-sFas, anti-sFasL, at least a in anti-sICAM-1 or the anti-sVCAM-1 antibody, preferably at least two kinds of antibody, more preferably at least 3 kinds of antibody, preferably at least 4 kinds of antibody, most preferably at least 5 kinds of antibody again.
First optimal technical scheme of this technical scheme of the present invention is: described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and described antibody is selected from anti-SCF, anti-sEGFR, anti-sIL-2R α, anti-sRAGE, anti-sTNFR1, anti-sTNFR2, anti-sVEGFR2, anti-sVCAM1, anti-sFas, anti-CCL14 α-HCC1, anti-sCD40L, anti-Eotaxin, anti-FGF2, anti-Flt3ligand, anti-Fracktalkine, anti-GCSF, anti-GRO, anti-IFN γ, anti-IL-1R α, anti-IL-3, anti-IL-4, anti-IL-6, anti-IL-12p70, anti-il-13, anti-IP10, anti-MIP1 α, anti-VEGF, anti-BCA-1, anti-IL-16, anti-MIP1d, anti-IL-20, anti-IL-28A, anti-CXCL9MIG, at least a antibody among anti-CXCL111TAC or the anti-CCL19MIP3 β, preferably at least two kinds of antibody, more preferably at least 3 kinds, preferably at least 4 kinds again, most preferably at least 5 kinds.
Second optimal technical scheme of this technical scheme of the present invention is that described kit also contains:
(1) 96 hole filter membrane plate and two shrouding films;
(2) standard items of protein antibodies in the described kit;
(3) Quality Control contrast;
(4) serum matrix;
(5) experiment liquid;
(6) washing lotion;
(7) streptavidin-phycoerythrin labelled antibody;
(8) pearl dilution;
(9) antibody of the albumen of the described detection transplanted kidney of claim 10 rejection;
(10) pearl of pre-coated antibody.
The 3rd optimal technical scheme of this technical scheme of the present invention is in the described kit:
(1) described 96 hole filter membrane plates and two shrouding films are available from Millipore company, article No. MX-PLATE;
(2) described standard items are available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) described Quality Control contrast is available from Millipore company, and article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) described serum matrix is available from Millipore company, and article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) described experiment liquid is available from Millipore company, article No. L-AB;
(6) described washing lotion is available from Millipore company, article No. L-WB;
(7) described streptavidin-phycoerythrin labelled antibody is available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) described pearl dilution is available from Millipore company, article No. LBD;
(9) detect antibody available from Millipore company, article No. MXH1060, MXH1062, MXH1063, LHSP-1063, HSCR-1032;
(10) pearl of described pre-coated antibody is available from Millipore company, article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
In order to realize the 3rd goal of the invention of the present invention, the technical scheme of employing is:
The using method of kit of the present invention is: may further comprise the steps:
(1) gather whole blood, room temperature leaves standstill, and 1000rpm collected supernatant in centrifugal 5 ~ 15 minutes, and freezing preservation is standby after the packing;
(2) with experiment liquid or the cleansing solution brassboard of prewetting, placed the oscillator jolting 5 ~ 20 minutes; Siphon away the liquid in the experiment plate hole, blot at the bottom of the brassboard;
(3) respective aperture adds standard items, Quality Control contrast and the sample of gradient dilution respectively;
(4) in background hole, standard items and Quality Control control wells, add serum matrix;
(5) every hole adds pearl;
(6) will hatch 0.5 ~ 2h under 4 ℃ of overnight incubation of brassboard jolting or the room temperature behind the shrouding; Siphon away the liquid in the experiment plate hole, blot at the bottom of the brassboard; Utilize twice of washing lotion washing brassboard;
(7) every hole adds the corresponding antibody that detects; Jolting brassboard 0.5 ~ 2h under the room temperature;
(8) every hole adds the streptavidin-phycoerythrin labelled antibody; The jolting brassboard is hatched 20 ~ 40min under the room temperature; Wash with washing lotion and to pull twice;
(9) every hole adds sheath fluid, detects to analyze to obtain experimental result.
First optimal technical scheme of the using method of kit of the present invention is may further comprise the steps:
(1) gather whole blood, after room temperature left standstill 1h, 1000rpm collected supernatant in centrifugal 10 minutes, and-80 ℃ of refrigerators are preserved standby 2ml after the packing;
(2) use 200 μ l experiment liquid or the cleansing solution brassboard of prewetting, placed the oscillator jolting 10 ~ 15 minutes; The application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard;
(3) respective aperture adds standard items, Quality Control contrast and the sample of 25 μ l gradient dilutions respectively;
(4) in background hole, standard items and Quality Control control wells, add serum matrix 25 μ l;
(5) every hole adds 25 μ l pearls;
(6) behind the shrouding brassboard placed under 4 ℃ of overnight incubation of oscillator jolting or the room temperature and hatch 1h; The application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard; Every hole adds 200 μ l washing lotions, and the application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard, washes and pulls twice;
(7) every hole adds 25 μ l detection antibody, oscillator jolting brassboard 1h under the room temperature;
(8) every hole adds 25 μ l streptavidin-phycoerythrin labelled antibodies, and oscillator jolting brassboard is hatched 30min under the room temperature;
Every hole adds 200 μ l washing lotions, and the application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard, washes and pulls twice;
(9) every hole adds 150 μ l sheath fluids, and the streaming dot matrix instrument detects, and software analysis obtains experimental result.
Second optimal technical scheme of the using method of kit of the present invention is the ratio stepwise dilution according to 1:4～5 of standard items, Quality Control contrast and sample in step (3).The initial concentration of standard items and Quality Control contrast has adopted the Cytokine/Chemokine(MPXHCYTO-60K with Luminex detection kit Human), Human Cytokine/Chemokine Panel II(MPXHCYP2-62K), Human Cytokine/Chemokine Panel III(MPXHCYP3-63K), Human Soluble Cytokine Receptor Panel(HSCR-32K-PMX14), Human Sepsis Panel I(HSEP-63K) in identical concentration.
The 3rd optimal technical scheme of the using method of kit of the present invention is that available from luminex company, article No. is #40-50000 at the sheath fluid described in the step (9).
The 4th optimal technical scheme of the using method of kit of the present invention is, is luminex200 at the streaming dot matrix instrument described in the step (9), and software is analyzed for MILLIPLEX Analyst.
The 4th goal of the invention of the present invention is to provide anti-IL-1R Alpha antibodies, the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection of anti-sCD40L antibody and anti-IL-20 antibody.
Wherein, the screening technique of described albumen is: at first, gather the serum of a plurality of time points of post-transplantation of renal transplantation recipients, and clinical case is analyzed and divided into groups; Using the Luminex system then detects the expression of 96 kinds of cell factors and chemotactic factor (CF) in each group serum; At last, statistical method filters out the relevant mark of graft-rejection, these marks of use in conjunction group, and setting up with the multiple-factor analysis-by-synthesis is diagnosis and the pre-alarming system of the transplanted kidney rejection of feature.
The present invention uses the Luminex detection kit of U.S. Millipore Corp. (Millipore): MPXHCYTO-60K, MPXHCYP2PMX23, MPXHCYP2-62K, MPXHCYP3-63K, HSCR-32K-PMX14, HSEP-63K-06, detect 96 kinds of cell factors, cytokine receptor and chemotactic factor (CF)s expression in acute rejection group and transplanted kidney function liptinite patients serum altogether.Concrete experimentation is seen each kit instructions.
After Statistics Application analysis software SPSS16.0 carries out test of normality and homogeneity test of variance to the detection data, the skewed distribution data is used minitab15.0 and is carried out data-switching, through after the data-switching for the application T check analysis acute rejection group of normal distribution and transplanted kidney function liptinite between expression the label of significant difference is arranged, the data of failing to be converted to normal distribution has the label of significant difference through the methods analyst expression of non-ginseng analysis.Obtain 35 label that significant difference is arranged: SCF after the analysis altogether, sEGFR, sIL-2R α, sRAGE, sTNFR1, sTNFR2, sVEGFR2, sVCAM1, sFas, CCL14 α-HCC1, sCD40L, Eotaxin, FGF2, Flt3ligand, Fracktalkine, GCSF, GRO, IFN γ, IL-1R α, IL-3, IL-4, IL-6, IL-12p70, IL-13, IP10, MIP1 α, VEGF, BCA-1, IL-16, MIP1d, IL-20, IL-28A, CXCL9MIG, CXCL111TAC, CCL19MIP3 β.Wherein, Protein S CF, 11sRAGE, CXCL7NAP2, CCL14 α-HCC1, sCD40L, GCSF, GRO, IL-1R α, MCP3, IL-20, MIP1d, IL-28A, IL-29IFN λ 1 do not see the report that has in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
Below content of the present invention is elaborated:
The inventor uses liquid protein-chip luminex detection technique, and 96 kinds of cell factor/chemotactic factor (CF)s in totally 266 parts of serum and receptor expression level thereof have been carried out monitoring and analysis-by-synthesis to the stable transplanting receptor of 142 routine acute grafing kidney rejection patients and transplanted kidney function, set up the transplanted kidney rejection early warning system that contains at least a associating mark and contained the transplanted kidney rejection early diagnosis system that at least a factor is united mark, to prediction and the rate of accuracy reached to 98.6% of acute rejection, internal chiasma checking rate is respectively 97.1% and 97.2%.Wherein, preferred, preferably contain at least 3 kinds of described protein antibodies in the transplanted kidney rejection early warning system, transplanted kidney rejection early diagnosis system contains at least 4 kinds of described protein antibodies.Wherein, the protein factor of the present invention that contains in the described kit is more many, and its accuracy is more high, but can increase the complicacy in the practical operation.
The screening process of label albumen of the present invention is:
(1) gathers before the transplantation of renal transplantation recipients and serum, urine sample and the PBMC of a plurality of time points of postoperative, and clinical case is analyzed and divided into groups.
(2) use the Luminex system to 96 kinds of cells in each group serum and the urine because of and the expression of chemotactic factor (CF) detect.
(3) statistical method (ANOVA) filters out the relevant mark of graft-rejection, these marks of use in conjunction group, and setting up with the multiple-factor analysis-by-synthesis is diagnosis and the pre-alarming system of the transplanted kidney rejection of feature.
Kit of the present invention is at the Luminex detection kit Human of U.S. Millipore Corp. (Millipore) Cytokine/Chemokine(MPXHCYTO-60K), Human Cytokine/Chemokine Panel II(MPXHCYP2-62K), Human Cytokine/Chemokine Panel III(MPXHCYP3-63K), Human Soluble Cytokine Receptor Panel(HSCR-32K-PMX14), Human Sepsis Panel I(HSEP-63K) on the basis, 96 kinds of cell factor/chemotactic factor (CF)s among the stable transplanting recipient's serum of acute grafing kidney rejection patient and transplanted kidney function and receptor expression level thereof carried out analyzing obtained, the major part of the reagent in the kit of the present invention is available from Millipore company, and the public can buy from the market by disclosed article No. among the present invention.The principle of Luminex detection kit is double antibody sandwich method.
The present invention has stronger feasibility and the susceptibility of Geng Gao by setting up associating mark group diagnosis and the pre-alarming system of rejection.
Description of drawings:
Fig. 1 is the diagnosis area under curve figure of transplanted kidney rejection early diagnosis kit among the embodiment 2;
Fig. 2 is the diagnosis area under curve figure of transplanted kidney rejection early diagnosis kit among the embodiment 3;
Fig. 3 is the diagnosis area under curve figure of transplanted kidney rejection early warning kit among the embodiment 5;
Fig. 4 is the diagnosis area under curve figure of transplanted kidney rejection early warning kit among the embodiment 6;
Fig. 5 is transplanted kidney function liptinite and the acute grafing kidney rejection group concentration comparison diagram of each factor in serum in the diagnostic system in early days, and wherein 0 is liptinite, and 1 is the repulsion group;
Fig. 6 is transplanted kidney function liptinite and acute grafing kidney rejection group each factor concentration comparison diagram in serum in early warning system, and wherein 0 is liptinite, and 1 is the repulsion group.
Following embodiment only makes further explanation content of the present invention, content of the present invention is not construed as limiting.
Embodiment
The screening of embodiment 1 transplanted kidney rejection early diagnosis kit significant difference label
1, gathers the serum of a plurality of time points of post-transplantation of renal transplantation recipients, and clinical case is analyzed and divided into groups.
1.1 it is few to choose the preceding basic disease of art or complication, the transplanted kidney receptor that severe complication does not take place in operation, gathered whole blood 2ml in 1 day, 7 days, 14 days, 21 days, 28 days, 2 months, 3 months ~ 12 months at the renal transplantation recipients post-transplantation respectively, after room temperature leaves standstill 1h, 1000rpm collected supernatant in centrifugal 10 minutes, and-80 ℃ of refrigerators are preserved standby after the packing.At any time collect the serum of acute rejection group patient after acute rejection is made a definite diagnosis in addition.
1.2 according to whether analysis-by-synthesis and the case screening such as effective of renal transplant recipients serum creatinine, urea nitrogen, transplanted kidney B ultrasonic, clinical manifestation, puncture pathological diagnosis result and hormone impact treatment, will collect case and be divided into two groups: acute rejection group and transplanted kidney function liptinite.Acute rejection group 33 examples, transplanted kidney function liptinite 38 examples.
1.3 the acute rejection group is gone into the group standard:
1. the renal transplant recipients of kidney transplant surgery complication, infection and other severe complications does not take place;
2. biopsy puncture pathological diagnosis is the case of acute rejection;
3. judge according to the validity of clinical diagnosis and hormone impact treatment without the case of biopsy puncture: the renal transplant recipients serum creatinine 120 μ mol/L, urea nitrogen〉8.3mmol/L, the transplanted kidney B ultrasonic shows that the transplanted kidney volume increases, blood flow reduces, vascular resistence increases, clinical manifestation is heating, accompany weak joint pain, body weight increase, blood pressure rising, hypourocrinia and transplanted kidney distending pain etc., puncture pathological diagnosis result is acute rejection, and serum creatinine descends more than 10% behind the hormone impact treatment.
1.4 the transplanted kidney function liptinite is gone into the group standard:
1. the renal transplant recipients of kidney transplant surgery complication, infection and other severe complications does not take place;
2. rejection does not take place;
3. transplanted kidney function recovers very fast (serum creatinine is down to normally (＜120 μ mol/L) in 14 days).
2, using the Luminex system detects the expression of 96 kinds of cell factors in each group serum and chemotactic factor (CF).
2.1 choose serum sample that transplanted kidney function liptinite patients serum creatinine recovers just often (behind the renal transplantation 7 days or 14 days) 32 parts of totally 38 parts of acute rejection group generation rejections and the serum collected when making a definite diagnosis the back;
Detect 96 kinds of cell factors, cytokine receptor and chemotactic factor (CF)s expression in acute rejection group and transplanted kidney function liptinite patients serum altogether 2.2 use the Luminex of U.S. Millipore Corp. (Millipore) detection kit (MPXHCYTO-60K, MPXHCYP2PMX23, MPXHCYP2-62K, MPXHCYP3-63K, HSCR-32K-PMX14, HSEP-63K-06); Concrete experimentation is seen each kit instructions; Wherein, in transplanted kidney function liptinite and the acute grafing kidney rejection group early diagnosis system concentration ratio of each factor in serum more as shown in Figure 5, wherein 0 is liptinite, 1 for the repulsion group;
Concise and to the point experimentation is: use the assay buffer200 μ l brassboard of prewetting, place the oscillator jolting after 10 minutes the application of negative pressure attractor siphon away the experiment plate hole liquid, the standard items that add 25 μ l gradient dilutions after paper handkerchief blots at the bottom of the brassboard respectively, contrast and sample, every hole adds 25 μ l pearls subsequently, behind the shrouding brassboard placed 4 ℃ of overnight incubation of oscillator jolting, add detection antibody after washing plate, room temperature oscillator jolting brassboard 1h, hatch 30min after adding the streptavidin-phycoerythrin labelled antibody again, luminex200 goes up machine testing, MILLIPLEX Analyst software analysis after washing plate;
3, statistical method filters out the relevant mark of graft-rejection, these marks of use in conjunction group, and setting up with the multiple-factor analysis-by-synthesis is the diagnosis system of the transplanted kidney rejection of feature.
After Statistics Application analysis software SPSS16.0 carries out test of normality and homogeneity test of variance to the detection data, the skewed distribution data is used minitab15.0 and is carried out data-switching, through after the data-switching for the application T check analysis acute rejection group of normal distribution and transplanted kidney function liptinite between expression the label of significant difference is arranged, the data of failing to be converted to normal distribution has the label of significant difference through the methods analyst expression of non-ginseng analysis.Obtain 35 label that significant difference is arranged: SCF after the analysis altogether, sEGFR, sIL-2R α, sRAGE, sTNFR1, sTNFR2, sVEGFR2, sVCAM1, sFas, CCL14 α-HCC1, sCD40L, Eotaxin, FGF2, Flt3ligand, Fracktalkine, GCSF, GRO, IFN γ, IL-1R α, IL-3, IL-4, IL-6, IL-12p70, IL-13, IP10, MIP1 α, VEGF, BCA-1, IL-16, MIP1d, IL-20, IL-28A, CXCL9MIG, CXCL111TAC, CCL19MIP3 β.
Embodiment 2
35 that embodiment 1 is obtained have the label of significant difference to analyze, have the label of significant difference to carry out obtaining after the logstic regretional analysis early warning diagnostic system of the acute rejection in renal transplantation that 4 factor: SCF, sEGFR, sIL2Ralpha and Eotaxin form to these expressions, statistical analysis obtains predicting that positive rate is 91.4.ROC diagnosis area under curve is 97.5%, sees accompanying drawing 1.
Wherein, specifically consisting of of kit:
(1) 96 hole filter membrane plate and two shrouding films; Available from Millipore company, article No. MX-PLATE;
(2) standard items of protein antibodies in the described kit; Available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) Quality Control contrast; Available from Millipore company, article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) serum matrix; Available from Millipore company, article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) experiment liquid; Available from Millipore company, article No. L-AB;
(6) washing lotion; Available from Millipore company, article No. L-WB;
(7) streptavidin-phycoerythrin labelled antibody; Available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) pearl dilution; Liquid is available from Millipore company, article No. LBD;
(9) antibody of the antibody of the antibody of the antibody of anti-SCF, anti-sEGFR, anti-sIL2Ralpha and anti-Eotaxin; Available from Millipore company, article No. MXH1060, MXH1062, MXH1063, LHSP-1063, HSCR-1032;
(10) pearl of pre-coated antibody, available from Millipore company, article No. L-AB; Article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
Embodiment 3: the composition of acute rejection in renal transplantation early diagnosis kit
35 that embodiment 1 is obtained have the label of significant difference to analyze, have the label of significant difference to carry out obtaining 14 factor: sTNFR2, Flt3Ligand, Fractalkine, IL1ra, IL2, MDC, MIP1alpha, SDF1alphabeta, TARC, TRAIL, SCF, CCL20, MIP3alpha and XCL1Lymphotactin after the logstic regretional analysis to these expressions; It is that 98.6%, ROC diagnosis area under curve is 99.8% that statistical analysis obtains early diagnosis system prediction positive rate, sees accompanying drawing 2.
Wherein, specifically consisting of of kit:
(1) 96 hole filter membrane plate and two shrouding films; Available from Millipore company, article No. MX-PLATE;
(2) standard items of protein antibodies in the described kit; Available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) Quality Control contrast; Available from Millipore company, article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) serum matrix; Available from Millipore company, article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) experiment liquid; Available from Millipore company, article No. L-AB;
(6) washing lotion; Available from Millipore company, article No. L-WB;
(7) streptavidin-phycoerythrin labelled antibody; Available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) pearl dilution; Liquid is available from Millipore company, article No. LBD;
(9) antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of anti-sTNFR2, anti-Flt3Ligand, anti-Fractalkine, anti-IL1ra, anti-IL2, anti-MDC, anti-MIP1alpha, anti-SDF1alphabeta, anti-TARC, anti-TRAIL, anti-SCF, anti-CCL20, anti-MIP3alpha and anti-XCL1Lymphotactin; Available from Millipore company, article No. MXH1060, MXH1062, MXH1063, LHSP-1063, HSCR-1032;
(10) pearl of pre-coated antibody, available from Millipore company, article No. L-AB; Article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
The screening of the significant difference label of embodiment 4 transplanted kidney rejection early warning kits
1, gathers the serum of a plurality of time points of post-transplantation of renal transplantation recipients, and clinical case is analyzed and divided into groups.
1.1 it is few to choose the preceding basic disease of art or complication, the transplanted kidney receptor that severe complication does not take place in operation, respectively renal transplantation recipients post-transplantation 1 day, 7 days, 14 days, 21 days, 28 days, 2 months, 3 months ... gathered whole blood 2ml in 12 months, after room temperature leaves standstill 1h, 1000rpm collected supernatant in centrifugal 10 minutes, and-80 ℃ of refrigerators are preserved standby after the packing.At any time collect the serum of acute rejection group patient after acute rejection is made a definite diagnosis in addition.
1.2 according to whether analysis-by-synthesis and the case screening such as effective of renal transplant recipients serum creatinine, urea nitrogen, transplanted kidney B ultrasonic, clinical manifestation, puncture pathological diagnosis result and hormone impact treatment, will collect case and be divided into two groups: acute rejection group and transplanted kidney function liptinite.Acute rejection group 33 examples, transplanted kidney function liptinite 38 examples.
1.3 the acute rejection group is gone into the group standard:
1. the renal transplant recipients of kidney transplant surgery complication, infection and other severe complications does not take place.
2. biopsy puncture pathological diagnosis is the case of acute rejection
3. judge according to the validity of clinical diagnosis and hormone impact treatment without the case of biopsy puncture: the renal transplant recipients serum creatinine 120 μ mol/L, urea nitrogen〉8.3mmol/L, the transplanted kidney B ultrasonic shows that the transplanted kidney volume increases, blood flow reduces, vascular resistence increases, clinical manifestation is heating, accompany weak joint pain, body weight increase, blood pressure rising, hypourocrinia and transplanted kidney distending pain etc., puncture pathological diagnosis result is acute rejection, and serum creatinine descends more than 10% behind the hormone impact treatment;
1.4 the transplanted kidney function liptinite is gone into the group standard:
1. the renal transplant recipients of kidney transplant surgery complication, infection and other severe complications does not take place.
2. rejection does not take place
3. transplanted kidney function recovers very fast (serum creatinine is down to normally (＜120 μ mol/L) in 14 days)
2, using the Luminex system detects the expression of 96 kinds of cell factors in each group serum and chemotactic factor (CF).
2.1 choose serum sample that transplanted kidney function liptinite patients serum creatinine recovers just often (behind the renal transplantation 7 days or 14 days) 32 parts of totally 38 parts of acute rejection group generation rejections and the serum collected when making a definite diagnosis the back
2.2 use the Luminex of U.S. Millipore Corp. (Millipore) detection kit: MPXHCYTO-60K, MPXHCYP2PMX23, MPXHCYP2-62K, MPXHCYP3-63K, HSCR-32K-PMX14, HSEP-63K-06; Detect 96 kinds of cell factors, cytokine receptor and chemotactic factor (CF)s expression in acute rejection group and transplanted kidney function liptinite patients serum altogether.Concrete experimentation is seen each kit instructions.Wherein, transplanted kidney function liptinite and acute grafing kidney rejection group in early warning system the concentration ratio of each factor in serum more as shown in Figure 6, wherein 0 is liptinite, 1 for the repulsion group;
Concise and to the point experimentation: use the assay buffer200 μ l brassboard of prewetting, place the oscillator jolting after 10 minutes the application of negative pressure attractor siphon away the experiment plate hole liquid, the standard items that add 25 μ l gradient dilutions after paper handkerchief blots at the bottom of the brassboard respectively, contrast and sample, every hole adds 25 μ l pearls subsequently, behind the shrouding brassboard placed 4 ℃ of overnight incubation of oscillator jolting, add detection antibody after washing plate, room temperature oscillator jolting brassboard 1h, hatch 30min after adding the streptavidin-phycoerythrin labelled antibody again, luminex200 goes up machine testing, MILLIPLEX Analyst software analysis after washing plate
3, statistical method filters out the relevant mark of graft-rejection, these marks of use in conjunction group, and setting up with the multiple-factor analysis-by-synthesis is the transplanted kidney rejection pre-alarming system of feature.
After Statistics Application analysis software SPSS16.0 carries out test of normality and homogeneity test of variance to the detection data, the skewed distribution data is used minitab15.0 and is carried out data-switching, through after the data-switching for the application T check analysis acute rejection group of normal distribution and transplanted kidney function liptinite between expression the label of significant difference is arranged, the data of failing to be converted to normal distribution has the label of significant difference through the methods analyst expression of non-ginseng analysis.Obtain 35 label that significant difference is arranged: SCF after the analysis altogether, sEGFR, sIL-2R α, sRAGE, sTNFR1, sTNFR2, sVEGFR2, sVCAM1, sFas, CCL14 α-HCC1, sCD40L, Eotaxin, FGF2, Flt3ligand, Fracktalkine, GCSF, GRO, IFN γ, IL-1R α, IL-3, IL-4, IL-6, IL-12p70, IL-13, IP10, MIP1 α, VEGF, BCA-1, IL-16, MIP1d, IL-20, IL-28A, CXCL9MIG, CXCL111TAC, CCL19MIP3 β.
Embodiment 5 transplanted kidney rejection early warning kits
35 that embodiment 4 is obtained have the label of significant difference to analyze, there is the label of significant difference to carry out obtaining after the logstic regretional analysis 3 factors to these expressions: IL-1R α, the early warning diagnostic system of the acute rejection in renal transplantation that sCD40L and IL-20 form, statistical analysis obtains predicting that positive rate is 91.4.ROC diagnosis area under curve is 97.5%, sees accompanying drawing 3.
Wherein, specifically consisting of of kit:
(1) 96 hole filter membrane plate and two shrouding films; Available from Millipore company, article No. MX-PLATE;
(2) standard items of protein antibodies in the described kit; Available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) Quality Control contrast; Available from Millipore company, article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) serum matrix; Available from Millipore company, article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) experiment liquid; Available from Millipore company, article No. L-AB;
(6) washing lotion; Available from Millipore company, article No. L-WB;
(7) streptavidin-phycoerythrin labelled antibody; Available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) pearl dilution; Liquid is available from Millipore company, article No. LBD;
(9) antibody of anti-IL-1R α, the antibody of anti-sCD40L and the antibody of anti-IL-20; Available from Millipore company, article No. MXH1060, MXH1062, MXH1063, LHSP-1063, HSCR-1032;
(10) pearl of pre-coated antibody, available from Millipore company, article No. L-AB; Article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
35 that embodiment 4 is obtained have the label of significant difference to analyze, have the label of significant difference to carry out obtaining 11 factor: sIL-1R1, sVCAM1 after the logstic regretional analysis to these expressions, SICAM-1, Fracktalkine, IL-1R α, IP10, MDC, MIP1 α, SDF α β, the early warning diagnostic system of the acute rejection in renal transplantation that TRAIL and SCF form, statistical analysis obtains predicting that positive rate is that 98.6%, ROC diagnosis area under curve is 99.8%., see accompanying drawing 4.
Wherein, specifically consisting of of kit:
(1) 96 hole filter membrane plate and two shrouding films; Available from Millipore company, article No. MX-PLATE;
(2) standard items of protein antibodies in the described kit; Available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) Quality Control contrast; Available from Millipore company, article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) serum matrix; Available from Millipore company, article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) experiment liquid; Available from Millipore company, article No. L-AB;
(6) washing lotion; Available from Millipore company, article No. L-WB;
(7) streptavidin-phycoerythrin labelled antibody; Available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) pearl dilution; Liquid is available from Millipore company, article No. LBD;
(9) antibody of anti-sIL-1R1, the antibody of anti-sVCAM1, the antibody of anti-SICAM-1, the antibody of anti-Fracktalkine, the antibody of anti-IL-1R α, the antibody of anti-IP10, the antibody of anti-MDC, the antibody of anti-MIP1 α, the antibody of anti-SDF α β, the antibody of anti-TRAIL and the antibody of anti-SCF; Available from Millipore company, article No. MXH1060, MXH1062, MXH1063, LHSP-1063, HSCR-1032;
(10) pearl of pre-coated antibody, available from Millipore company, article No. L-AB; Article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
Claims (8)
1. a transplanted kidney rejection early warning kit is characterized in that, described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and described antibody is anti-IL-1R Alpha antibodies, anti-sCD40L antibody and anti-IL-20 antibody.
2. transplanted kidney rejection early warning kit according to claim 1 is characterized in that described kit also contains:
(1) 96 hole filter membrane plate and two shrouding films;
(2) standard items of protein antibodies in the described kit;
(3) Quality Control contrast;
(4) serum matrix;
(5) experiment liquid;
(6) washing lotion;
(7) the plain labelled antibody of phycoerythrin-strepto-affinity;
(8) pearl dilution;
(9) antibody of the albumen of the described detection transplanted kidney of claim 1 rejection;
(10) pearl of pre-coated antibody.
3. transplanted kidney rejection early warning kit according to claim 2 is characterized in that, in the described kit:
(1) described 96 hole filter membrane plates and two shrouding films are available from Millipore company, article No. MX-PLATE;
(2) described standard items are available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) described Quality Control contrast is available from Millipore company, and article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) described serum matrix is available from Millipore company, and article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) described experiment liquid is available from Millipore company, article No. L-AB;
(6) described washing lotion is available from Millipore company, article No. L-WB;
(7) the plain labelled antibody of described phycoerythrin-strepto-affinity is available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) described pearl dilution is available from Millipore company, article No. LBD;
(9) detect antibody available from Millipore company, article No. MXH1060, MXH1062, MXH1063, LHSP-1063, HSCR-1032;
(10) pearl of described pre-coated antibody is available from Millipore company, article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
4. the using method of the described kit of claim 1 is characterized in that:
(1) gather whole blood, room temperature leaves standstill, and 1000rpm collected supernatant in centrifugal 5～15 minutes, and freezing preservation is standby after the packing;
(2) with experiment liquid or the cleansing solution brassboard of prewetting, placed the oscillator jolting 5～20 minutes; Siphon away the liquid in the experiment plate hole, blot at the bottom of the brassboard;
(3) respective aperture adds standard items, Quality Control contrast and the sample of gradient dilution respectively;
(4) in background hole, standard items and Quality Control control wells, add serum matrix;
(5) every hole adds pearl;
(6) will hatch 0.5～2h under 4 ℃ of overnight incubation of brassboard jolting or the room temperature behind the shrouding; Siphon away the liquid in the experiment plate hole, blot at the bottom of the brassboard; Utilize twice of washing lotion washing brassboard;
(7) every hole adds the corresponding antibody that detects; Jolting brassboard 0.5～2h under the room temperature;
(8) every hole adds the plain labelled antibody of phycoerythrin-strepto-affinity; The jolting brassboard is hatched 20～40min under the room temperature; Wash with washing lotion and to pull twice;
(9) every hole adds sheath fluid, detects to analyze to obtain experimental result.
5. the using method of kit according to claim 4 is characterized in that:
(1) gather whole blood, after room temperature left standstill 1h, 1000rpm collected supernatant in centrifugal 10 minutes, and-80 ℃ of refrigerators are preserved standby 2ml after the packing;
(2) use 200 μ l experiment liquid or the cleansing solution brassboard of prewetting, placed the oscillator jolting 10～15 minutes; The application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard;
(3) respective aperture adds standard items, Quality Control contrast and the sample of 25 μ l gradient dilutions respectively;
(4) in background hole, standard items and Quality Control control wells, add serum matrix 25 μ l;
(5) every hole adds 25 μ l pearls;
(6) behind the shrouding brassboard placed under 4 ℃ of overnight incubation of oscillator jolting or the room temperature and hatch 1h; The application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard; Every hole adds 200 μ l washing lotions, and the application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard, washes and pulls twice;
(7) every hole adds 25 μ l detection antibody, oscillator jolting brassboard 1h under the room temperature;
(8) every hole adds the plain labelled antibody of 25 μ l phycoerythrin-strepto-affinity, and oscillator jolting brassboard is hatched 30min under the room temperature; Every hole adds 200 μ l washing lotions, and the application of negative pressure attractor siphons away the liquid in the experiment plate hole, and paper handkerchief blots at the bottom of the brassboard, washes and pulls twice;
(9) every hole adds 150 μ l sheath fluids, and the streaming dot matrix instrument detects, and software analysis obtains experimental result.
6. using method according to claim 4 is characterized in that, the ratio stepwise dilution according to 1:4～5 of standard items, Quality Control contrast and sample in step (3).
7. using method according to claim 4 is characterized in that, available from luminex company, article No. is #40-50000 at the sheath fluid described in the step (9).
8. using method according to claim 4 is characterized in that, is luminex200 at the streaming dot matrix instrument described in the step (9), and software is analyzed for MILLIPLEX Analyst.
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| CN103954755B (en) * | 2014-04-30 | 2017-04-05 | 广东省结核病控制中心 | A kind of diagnostic kit of mycobacterium tuberculosis latent infection |
| CA2949959A1 (en) * | 2014-05-22 | 2015-11-26 | Northwestern University | Gene expression profiles associated with sub-clinical kidney transplant rejection |
| CN107389910B (en) * | 2017-06-28 | 2019-07-19 | 中国人民解放军总医院 | A kind of kit for evaluating renal function |
| CN109655624A (en) * | 2019-02-11 | 2019-04-19 | 臻和（北京）科技有限公司 | A kind of preparation method of marker and its application for predicting cancer immunotherapy effect, kit and kit |
| CN113238059A (en) * | 2021-04-30 | 2021-08-10 | 复旦大学附属中山医院 | Method for prognosis evaluation of infected patient and multi-cytokine detection kit |
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