CN102539745B - Early diagnosis and early warning kit for transplanted kidney rejection reaction and detection method - Google Patents
Early diagnosis and early warning kit for transplanted kidney rejection reaction and detection method Download PDFInfo
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Abstract
The invention relates to an early diagnosis kit and an early warning kit, in particular to an early diagnosis and early warning kit for transplanted kidney rejection reaction and a use method of the kit, and further to some proteins applied in the early warning of transplanted kidney acute rejection reaction or transplanted kidney rejection reaction. By applying a liquid protein chip (luminex) detection technique, the expression level of 96 cytokines/chemokines and receptors thereof in 266 serum samples from 142 patients having acute transplanted kidney rejection reaction and transplant recipients with stable function of transplanted kidneys are monitored and comprehensively analyzed. An early warning system for the transplanted kidney rejection reaction comprising at least one combined marker and an early diagnosis system for the transplanted kidney rejection reaction comprising at least one factor combined marker are established. The prediction and accuracy rate of the acute rejection achieve 98.6%, and the internal cross validation rates are 97.1% and 97.2%, respectively.
Description
Technical field
The present invention relates to a kind of early diagnosis kit and a kind of early warning kit, specifically, relate to the early diagnosis of a kind of transplanted kidney rejection and early warning kit and detection method, also relate to the protein of some application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
Background technology
Go the number of dialysing because of chronic renal failure and has been increased to 106.5 ten thousand people in 2000 by 42.6 ten thousand people of nineteen ninety in the whole world, expects 2010 and will reach more than 200 ten thousand people.The growth of this number causes global dialysis expense to increase rapidly, and developed country, developing country have all been caused huge financial burden.Approximately there are 1,000,000 uremic patients in China, and annual newly-increased 120,000 people approximately have 500,000 needs of patients kidney transplants every year.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.Repeatedly outbreak and the chronic rejection of acute rejection are the main causes that causes graft mistake merit, and the toxic and side effect that the long-term taking immunodepressant is brought such as renal toxicity, infection, induced tumor etc., also affects graft and receptor's long-term surviving.
The acute rejection of outbreak and chronic rejection are the major reasons that causes graft mistake merit repeatedly, but lack clinically effective dynamic monitoring and non-invasive diagnosis means.The transplanted kidney aspiration biopsy is present the most reliable diagnostic method, 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 observation.And biopsy extraction also has 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, in the urgent need to explore be fit to population of China, reliably, can satisfy the transplanted kidney rejection Noninvasive diagnosis method of dynamic observation needs.Finishing of this problem will assist the prolongation patient to transplant rear life cycle to a great extent, improve life quality, reducing country's medical treatment drops into, save family expense, have great clinical value and social and economic benefits, 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 dynamically to observe cytokine profiles 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) soluble 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, 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 is without marked change, and the monitoring of prompting IL-6 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. is studied and reports with the correlativity of transplanted kidney rejection in blood platelet/T cell activation antigen 1 (platelet and T cell activation antigen 1, PTA1, CD226) and the allogeneic mixed lymphocyte reaction (MLP).Yet, although numerous researcher is devoted to screen the specific biomarker of graft rejection, not yet filter out so far Noninvasive diagnosis and antidiastole that specific molecular is applied to rejection.The non-invasive diagnosis and the pre-alarming system that still lack in the world at present the transplanted kidney rejection.
Graft-rejection is a very complicated process, cellular immunity and humoral immunity mechanism all participate in this process, and, the difference of graft, the impact of operation, transplant the difference of receptor's individuality and clinically the participation of the many 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 large 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 known protein labeling, successfully set up by Leptin, prolactin (prolactin), osteopontin (osteopontin, OPN) and the serodiagnosis system of the early stage epithelial ovarian cancer that forms of insulin-like growth factor II (IGF-II), to diagnose susceptibility, specificity, positive predictive value (positive predictive value, PPV) and negative predictive value (negtive predictive value, NPV) be increased to respectively 95%, 95%, 95% and 94%.Therefore, when identifying the graft rejection Specific marker, 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 whole and coloured microballoon (color-codedmicrosphere or bead), laser technology, fluidics, high speed digital signal processor and computer algorithm, have 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.The 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 simultaneously a plurality of indexs, 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;
The 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 Protein S CF, sRAGE, CXCL7NAP2, CCL14 α-HCC1, sCD40L, GCSF, GRO, IL-1R α, MCP3, IL-20, MIP1d, IL-28A, IL-29IFN λ 1 application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
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, TNF alpha antibody, 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 (sTNFRSF 1B), anti-sVEGFR1 (sFlt-1), anti-sVEGFR2 (sFlk-1), anti-sVEGFR3 (sFlt-4), anti-sgp 130, 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, more preferably at least 4 kinds of antibody, most preferably at least 5 kinds of antibody.
The 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-GC SF, 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) streptavidin-phycoerythrin (phycoerythrin-streptavidin) labelled antibody;
(8) pearl dilution;
(9) antibody of the albumen of detection transplanted kidney rejection claimed in claim 1;
(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 the 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, TNF alpha antibody, 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, again preferably at least 4 kinds of antibody, most preferably at least 5 kinds of antibody.
The 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-GC SF, 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 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 detection transplanted kidney rejection claimed in claim 10;
(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, freezing saving backup 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 respectively standard items, Quality Control contrast and the sample of gradient dilution;
(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.
The 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, after the packing-80.C Refrigerator store 2ml for subsequent use;
(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 respectively standard items, Quality Control contrast and the sample of 25 μ l gradient dilutions;
(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.
The second optimal technical scheme of the using method of kit of the present invention is, step (3) Plays product, Quality Control contrast and sample according to 1; 4～5 ratio stepwise dilution.The initial concentration of standard items and Quality Control contrast has adopted and Luminex detection kit Human 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), identical concentration among the Human Sepsis Panel I (HSEP-63K).
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 that MILLIPLEX Analyst analyzes.
The 4th goal of the invention of the present invention is to provide Protein S CF, sRAGE, CXCL7NAP2, CCL14 α-HCC1, sCD40L, GCSF, GRO, IL-1R α, MCP3, IL-20, MIP1d, IL-28A, IL-29IFN λ 1 application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
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; Then using the Luminex system 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, diagnosis and the pre-alarming system of the transplanted kidney rejection of foundation take the multiple-factor analysis-by-synthesis as 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 the stable group of transplanted kidney function 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 between stable group 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 altogether 35 label that significant difference is arranged: SCF after the analysis, 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 is there are no the report in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
The below is elaborated to content of the present invention:
The inventor use liquid protein-chip luminex detection technique to the stable transplanting receptor of 142 routine acute grafing kidney rejection patients and transplanted kidney function the expression of 96 kinds of cell factor/chemotactic factor (CF)s in totally 266 parts of serum and acceptor thereof carried out monitoring and analysis-by-synthesis, 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, the cross-validation 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, protein factor of the present invention contained in the described kit is more, and its accuracy is higher, 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, diagnosis and the pre-alarming system of the transplanted kidney rejection of foundation take the multiple-factor analysis-by-synthesis as feature.
Kit of the present invention is the Luminex detection kit HumanCytokine/Chemokine (MPXHCYTO-60K) in U.S. Millipore Corp. (Millipore), Human Cytokine/Chemokine Panel II (MPXHCYP2-62K), Human Cytokine/Chemokine Panel III (MPXHCYP3-63K), Human Soluble Cytokine Receptor Panel (HSCR-32K-PMX14), on the basis of Human Sepsis Panel I (HSEP-63K), the expression of 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 acceptor 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 the in early days concentration comparison diagram of each factor in serum in the diagnostic system of the stable group of transplanted kidney function and acute grafing kidney rejection group, and wherein 0 is stable group, and 1 is the repulsion group;
Fig. 6 is the stable group of transplanted kidney function and acute grafing kidney rejection group each factor concentration comparison diagram in serum in early warning system, and wherein 0 is stable group, and 1 is the repulsion group.
Following embodiment only makes further explanation content of the present invention, not to Composition of contents restriction of the present invention.
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 choose the front basic disease of art or few intercurrent disease, the Renal Transplant Recipients of severe complication does not occur 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 Refrigerator stores are for subsequent use after the packing.Collect at any time in addition the serum of acute rejection group patient after acute rejection is made a definite diagnosis.
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 pulse treatment with methylprednisolone, will collect case and be divided into two groups: acute rejection group and transplanted kidney function are stable to be organized.Acute rejection group 33 examples, the stable group of transplanted kidney function 38 examples.
1.3 the acute rejection group enters the group standard:
1. the renal transplant recipients of kidney transplant surgical complications, infection and other severe complications does not occur;
2. the Biopsy pathological diagnosis is the case of acute rejection;
3. judge according to the validity of clinical diagnosis and pulse treatment with methylprednisolone without the case of Biopsy: renal transplant recipients serum creatinine＞120 μ mol/L, urea nitrogen＞8.3mmol/L, the transplanted kidney B ultrasonic shows that the increase of transplanted kidney volume, Oligemia, vascular resistence increase, 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 pulse treatment with methylprednisolone.
1.4 the stable group of transplanted kidney function enters the group standard:
1. the renal transplant recipients of kidney transplant surgical complications, infection and other severe complications does not occur;
2. rejection does not occur;
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 the serum sample of (behind the renal transplantation 7 days or 14 days) 32 parts of the serum collected when rear of totally 38 parts of acute rejection group generation rejections and making a definite diagnosis when choosing the stable group of transplanted kidney function patients serum creatinine and recovering normal;
Detect altogether 96 kinds of cell factors, cytokine receptor and chemotactic factor (CF)s expression in acute rejection group and the stable group of transplanted kidney function patients serum 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 the stable group of transplanted kidney function 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 stable group, 1 is 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 respectively 25 μ l gradient dilutions after paper handkerchief blots at the bottom of the brassboard, 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 again the streptavidin-phycoerythrin labelled antibody, 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, the diagnosis system of the transplanted kidney rejection of foundation take the multiple-factor analysis-by-synthesis as 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 between stable group 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 altogether 35 label that significant difference is arranged: SCF after the analysis, 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.
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, it is 91.4 that statistical analysis obtains the Predict masculine gender rate.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. 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. 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 choose the front basic disease of art or few intercurrent disease, the Renal Transplant Recipients of severe complication does not occur 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 Refrigerator stores are for subsequent use after the packing.Collect at any time in addition the serum of acute rejection group patient after acute rejection is made a definite diagnosis.
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 pulse treatment with methylprednisolone, will collect case and be divided into two groups: acute rejection group and transplanted kidney function are stable to be organized.Acute rejection group 33 examples, the stable group of transplanted kidney function 38 examples.
1.3 the acute rejection group enters the group standard:
1. the renal transplant recipients of kidney transplant surgical complications, infection and other severe complications does not occur.
2. the Biopsy pathological diagnosis is the case of acute rejection
3. judge according to the validity of clinical diagnosis and pulse treatment with methylprednisolone without the case of Biopsy: renal transplant recipients serum creatinine＞120 μ mol/L, urea nitrogen＞8.3mmol/L, the transplanted kidney B ultrasonic shows that the increase of transplanted kidney volume, Oligemia, vascular resistence increase, 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 pulse treatment with methylprednisolone;
1.4 the stable group of transplanted kidney function enters the group standard:
1. the renal transplant recipients of kidney transplant surgical complications, infection and other severe complications does not occur.
2. rejection does not occur
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 the serum sample of (behind the renal transplantation 7 days or 14 days) 32 parts of the serum collected when rear of totally 38 parts of acute rejection group generation rejections and making a definite diagnosis when choosing the stable group of transplanted kidney function patients serum creatinine and recovering normal
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 altogether 96 kinds of cell factors, cytokine receptor and chemotactic factor (CF)s expression in acute rejection group and the stable group of transplanted kidney function patients serum.Concrete experimentation is seen each kit instructions.Wherein, the stable group of transplanted kidney function 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 stable group, 1 is 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 respectively 25 μ l gradient dilutions after paper handkerchief blots at the bottom of the brassboard, 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 again the streptavidin-phycoerythrin labelled antibody, luminex200 goes up machine testing, MILLIPLEX Analyst software analysis after washing plate
3, statistical method filters out the relevant mark of graft-rejection, and these marks of use in conjunction group sets up the transplanted kidney rejection pre-alarming system take the multiple-factor analysis-by-synthesis as 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 between stable group 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 altogether 35 label that significant difference is arranged: SCF after the analysis, sEGFR, sIL-2R α, sRAGE, sTNFR1, sTNFR2, sVEGFR2, sVCAM1, sFas, CCL 14 α-HCC1, sCD40L, Eotaxin, FGF2, Flt3ligand, Fracktalkine, GC SF, 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, it is 91.4 that statistical analysis obtains the Predict masculine gender rate.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. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
Embodiment 6 transplanted kidney rejection early warning kits
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, it is that 98.6%, ROC diagnosis area under curve is 99.8% that statistical analysis obtains the Predict masculine gender rate., 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. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
Claims (17)
1. transplanted kidney rejection early diagnosis or reaction early warning kit is characterized in that, described kit comprises 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-IL-10, anti-IL-12 (p40), anti-IL-12 (p70), anti-il-13, anti-IL-15, anti-IL-17, anti-IL-3, anti-IL-5, 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-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-sEGFR, anti-sIL-1RI (sCD121a), anti-sIL-1RII (sCD121b), 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, at least a among anti-MIF or the anti-PAI-1.
2. transplanted kidney rejection according to claim 1 early diagnosis or reaction early warning kit is characterized in that, described kit comprises 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-IL-10, anti-IL-12 (p40), anti-IL-12 (p70), anti-il-13, anti-IL-15, anti-IL-17, anti-IL-3, anti-IL-5, 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-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-sEGFR, anti-sIL-1RI (sCD121a), anti-sIL-1RII (sCD121b), 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, among anti-MIF or the anti-PAI-1 at least two kinds.
3. transplanted kidney rejection according to claim 1 early diagnosis or reaction early warning kit is characterized in that, described kit comprises 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-IL-10, anti-IL-12 (p40), anti-IL-12 (p70), anti-il-13, anti-IL-15, anti-IL-17, anti-IL-3, anti-IL-5, 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-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-sEGFR, anti-sIL-1RI (sCD121a), anti-sIL-1RII (sCD121b), 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, among anti-MIF or the anti-PAI-1 at least three kinds.
4. transplanted kidney rejection according to claim 1 early diagnosis or reaction early warning kit is characterized in that, described kit comprises 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-IL-10, anti-IL-12 (p40), anti-IL-12 (p70), anti-il-13, anti-IL-15, anti-IL-17, anti-IL-3, anti-IL-5, 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-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-sEGFR, anti-sIL-1RI (sCD121a), anti-sIL-1RII (sCD121b), 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, in anti-MIF or the anti-PAI-1 antibody at least 4 kinds.
5. transplanted kidney rejection according to claim 1 early diagnosis or reaction early warning kit is characterized in that, described kit comprises 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-IL-10, anti-IL-12 (p40), anti-IL-12 (p70), anti-il-13, anti-IL-15, anti-IL-17, anti-IL-3, anti-IL-5, 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-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-sEGFR, anti-sIL-1RI (sCD121a), anti-sIL-1RII (sCD121b), 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, among anti-MIF or the anti-PAI-1 at least 5 kinds.
6. transplanted kidney rejection according to claim 1 early diagnosis or reaction early warning kit is characterized in that, described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, described antibody is selected from anti-SCF, anti-sEGFR, anti-sIL-2R α, anti-sRAGE, anti-sTNFR1, anti-sTNFR2, anti-sVEGFR2, anti-sVCAM1, anti-CCL14 α-HCC1, anti-sCD40L, anti-Eotaxin, anti-FGF2, anti-Flt3ligand, anti-Fracktalkine, anti-GCSF, anti-GRO, 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 among anti-CXCL111TAC or the anti-CCL19MIP3 β.
7. transplanted kidney rejection early diagnosis kit according to claim 6 is characterized in that, described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, described antibody is selected from anti-SCF, anti-sEGFR, anti-sIL-2R α, anti-sRAGE, anti-sTNFR1, anti-sTNFR2, anti-sVEGFR2, anti-sVCAM 1, anti-CCL 14 α-HCC 1, anti-sCD40L, anti-Eotaxin, anti-FGF2, anti-Flt3ligand, anti-Fracktalkine, anti-GCSF, anti-GRO, 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, among anti-CXCL111TAC or the anti-CCL19MIP3 β at least two kinds.
8. transplanted kidney rejection according to claim 6 early diagnosis or reaction early warning kit is characterized in that, described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, described antibody is selected from anti-SCF, anti-sEGFR, anti-sIL-2R α, anti-sRAGE, anti-sTNFR1, anti-sTNFR2, anti-sVEGFR2, anti-sVCAM1, anti-CCL14 α-HCC1, anti-sCD40L, anti-Eotaxin, anti-FGF2, anti-Flt3ligand, anti-Fracktalkine, anti-GCSF, anti-GRO, 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, among anti-CXCL111TAC or the anti-CCL19MIP3 β at least 3 kinds.
9. transplanted kidney rejection according to claim 6 early diagnosis or reaction early warning kit is characterized in that, described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, described antibody is selected from anti-SCF, anti-sEGFR, anti-sIL-2R α, anti-sRAGE, anti-sTNFR1, anti-sTNFR2, anti-sVEGFR2, anti-sVCAM1, anti-CCL14 α-HCC1, anti-sCD40L, anti-Eotaxin, anti-FGF2, anti-Flt3ligand, anti-Fracktalkine, anti-GCSF, anti-GRO, 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, among anti-CXCL111TAC or the anti-CCL19MIP3 β at least 4 kinds.
10. transplanted kidney rejection according to claim 6 early diagnosis or reaction early warning kit is characterized in that, described kit comprises the antibody of the albumen that detects the transplanted kidney rejection, described antibody is selected from anti-SCF, anti-sEGFR, anti-sIL-2R α, anti-sRAGE, anti-sTNFR1, anti-sTNFR2, anti-sVEGFR2, anti-sVCAM1, anti-CCL14 α-HCC1, anti-sCD40L, anti-Eotaxin, anti-FGF2, anti-Flt3ligand, anti-Fracktalkine, anti-GCSF, anti-GRO, 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, among anti-CXCL111TAC or the anti-CCL19MIP3 β at least 5 kinds.
11. the described transplanted kidney rejection early diagnosis of arbitrary claim or reaction early warning kit is characterized in that according to claim 1～10, 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) phycoerythrin-streptavidin labelled antibody;
(8) pearl dilution;
(9) antibody of the albumen of detection transplanted kidney rejection claimed in claim 1;
(10) pearl of pre-coated antibody.
12. transplanted kidney rejection according to claim 11 early diagnosis or reaction early warning kit 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) described phycoerythrin-streptavidin 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.
13. the using method of kit as claimed in claim 1 is characterized in that:
(1) gather whole blood, room temperature leaves standstill, and 1000rpm collected supernatant in centrifugal 5～15 minutes, freezing saving backup 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 respectively standard items, Quality Control contrast and the sample of gradient dilution;
(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 phycoerythrin-streptavidin 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.
14. the using method of kit according to claim 13 is characterized in that:
(1) gather whole blood, after room temperature left standstill 1h, 1000rpm collected supernatant in centrifugal 10 minutes ,-80 ℃ of Refrigerator store 2ml for subsequent use 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 respectively standard items, Quality Control contrast and the sample of 25 μ l gradient dilutions;
(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 phycoerythrin-streptavidin 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.
15. according to claim 13 or 14 described using method, it is characterized in that, at the ratio stepwise dilution according to 1:4～5 of step (3) Plays product, Quality Control contrast and sample.
16. according to claim 13 or 14 described using method, it is characterized in that, available from luminex company, article No. is #40-50000 at the sheath fluid described in the step (9).
17. according to claim 13 or 14 described using method, it is characterized in that, is luminex200 at the streaming dot matrix instrument described in the step (9), and software is that MILLIPLEX Analyst analyzes.
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| CN 201210274365 Division CN102854306B (en) | 2011-12-23 | 2011-12-23 | Kit for early warning rejection reaction after kidney transplant and use method of kit |
| CN 201210274207 Division CN102854305B (en) | 2011-12-23 | 2011-12-23 | Prewarning kit for transplant kidney rejection and use method thereof |
| CN 201210274357 Division CN102854323B (en) | 2011-12-23 | 2011-12-23 | Transplant kidney rejection reaction early diagnosis kit and use method of kit |
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| EP2899545B1 (en) * | 2010-06-23 | 2018-08-22 | Astute Medical, Inc. | Methods for diagnosis and prognosis of renal injury and renal failure |
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