CN102539745A - 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
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.The 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, like renal toxicity, infection, induced tumor etc., also influence 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 effective dynamic monitoring and non-invasive diagnosis means clinically.The transplanted kidney aspiration biopsy is at present reliable diagnostic method, but has traumaticly, can cause that infection, hemorrhage even graft are lost etc., is difficult for being accepted and being unfavorable for dynamic observing by the patient.And biopsy is drawn materials also has certain limitation, and according to the Banff standard, the patient that many clinical signs of suspected transplanted kidney repel possibly 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.Life cycle after the completion of this problem will assist the prolongation patient to transplant 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 the 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, 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; Blood platelet/T cell activation antigen 1 (platelet and T cell activation antigen 1, PTA1, CD226) with the allogeneic mixed lymphocyte reaction (MLP) in specific expressed p140 etc. study with the correlativity of transplanted kidney rejection and report.Yet,, do not filter out the non-invasive diagnosis and differential diagnosis that specific molecular is applied to rejection so far as yet although numerous researcher is devoted to screen the specific biomarker of graft rejection.The non-invasive diagnosis and the pre-alarming system that still lack at present the transplanted kidney rejection in the world.
Graft-rejection is a very complicated process; Cellular immunity and humoral immunity mechanism are all participated in this process; And; The difference of graft, the influence of operation, transplant the individual difference of receptor and clinically the participation of numerous factors such as application of different immunosuppressant schemes make the diagnosis of graft-rejection complicated and difficult more.In addition, previously research shows, through 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 through the screening to the known protein mark; Prolactin (prolactin), osteopontin (osteopontin have successfully been set up by Leptin; OPN) and the serodiagnosis system of the early stage epithelial ovarian cancer formed 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) 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, through the polymolecular analysis-by-synthesis; Set up the associating mark crowd 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-codedmicrosphere or bead), laser technology, fluidics, high speed digital signal processor and computer algorithm, has high detection specificity and sensitivity.The color of microballoon obtains through 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 through carboxyl, therefore can accomplish 100 kinds of different biologicallies in a reacting hole.The Luminex technology is tested and appraised the color of microballoon and confirms reaction type, and is to accomplish through the reporter molecules on the target material to the quantitative test of reaction.Multiple advantages such as it possesses the multiparameter high throughput analysis, practice thrift 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, in 4 hours, can accomplish analysis, 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 method for application 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, the application of IL-29IFN λ 1 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, said kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and said 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 (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, 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: said kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and said 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, preferred 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: said kit also contains:
(1) 96 hole filter membrane plate and two shrouding films;
(2) standard items of protein antibodies in the said kit;
(3) Quality Control contrast;
(4) serum matrix;
(5) experiment liquid;
(6) washing lotion;
(7) streptavidin-phycoerythrin (phycoerythrin-strepto-affinity is plain) labelled antibody;
(8) pearl dilution;
(9) antibody of the albumen of the described detection transplanted kidney of claim 1 rejection;
(10) pearl of preparatory coated antibody.
The 4th optimal technical scheme of this technical scheme of the present invention is: in the said kit:
(1) said 96 hole filter membrane plates and two shrouding films are available from Millipore company, article No. MX-PLATE;
(2) said standard items are available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) said Quality Control contrast is available from Millipore company, and article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) said serum matrix is available from Millipore company, and article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) said experiment liquid is available from Millipore company, article No. L-AB;
(6) said washing lotion is available from Millipore company, article No. L-WB;
(7) said streptavidin-phycoerythrin labelled antibody is available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) said 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 said preparatory 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, said kit comprise the antibody of the albumen that detects the transplanted kidney rejection, and said 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: said kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and said 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, preferred 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 said kit also contains:
(1) 96 hole filter membrane plate and two shrouding films;
(2) standard items of protein antibodies in the said 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 preparatory coated antibody.
The 3rd optimal technical scheme of this technical scheme of the present invention is in the said kit:
(1) said 96 hole filter membrane plates and two shrouding films are available from Millipore company, article No. MX-PLATE;
(2) said standard items are available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) said Quality Control contrast is available from Millipore company, and article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) said serum matrix is available from Millipore company, and article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) said experiment liquid is available from Millipore company, article No. L-AB;
(6) said washing lotion is available from Millipore company, article No. L-WB;
(7) said streptavidin-phycoerythrin labelled antibody is available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) said 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 said preparatory 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 method of application 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 subsequent use 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) behind the shrouding with hatching 0.5～2h under 4 ℃ of incubated overnight of brassboard jolting or the room temperature; 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 relevant detection antibody; 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, and check and analysis obtain experimental result.
First optimal technical scheme of the method for application 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.The C refrigerator is preserved subsequent use 2ml;
(2) application 2 00 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 incubated overnight 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 method for application of kit of the present invention does, standard items, Quality Control contrast and sample according to 1 in step (3); 4～5 ratio stepwise dilution.The initial concentration of standard items and Quality Control contrast adopted with 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), Human Sepsis Panel I (HSEP-63K) in identical concentration.
The 3rd optimal technical scheme of the method for application 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 method for application of kit of the present invention does, 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 Protein S CF, sRAGE, CXCL7NAP2, CCL14 α-HCC1, sCD40L, GCSF, GRO, IL-1R α, MCP3, IL-20, MIP1d, IL-28A, the application of IL-29IFN λ 1 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; 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 Combined application crowd, and setting up with the multiple-factor analysis-by-synthesis is the diagnosis and the pre-alarming system of the transplanted kidney rejection of characteristic.
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 convert into 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.
Be elaborated in the face of content of the present invention down:
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; Transplanted kidney rejection early warning system that contains at least a associating mark and the transplanted kidney rejection early diagnosis system that contains at least a factor combined mark have been set up; To the 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 is contained in the said kit is many more, and its accuracy is high more, 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 Combined application crowd, and setting up with the multiple-factor analysis-by-synthesis is the diagnosis and the pre-alarming system of the transplanted kidney rejection of characteristic.
Kit of the present invention is on the basis of Luminex detection kit HumanCytokine/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) in U.S. Millipore Corp. (Millipore); 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 market through disclosed article No. among the present invention.The principle of Luminex detection kit is a double antibody sandwich method.
The present invention has stronger feasibility and the susceptibility of Geng Gao through setting up the associating mark crowd diagnosis and the pre-alarming system of rejection.
Description of drawings:
Fig. 1 is the diagnosis TG-AUC figure of transplanted kidney rejection early diagnosis kit among the embodiment 2;
Fig. 2 is the diagnosis TG-AUC figure of transplanted kidney rejection early diagnosis kit among the embodiment 3;
Fig. 3 is the diagnosis TG-AUC figure of transplanted kidney rejection early warning kit among the embodiment 5;
Fig. 4 is the diagnosis TG-AUC 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 is only done further explanation and explanation to content of the present invention, content of the present invention is not constituted restriction.
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 perhaps complication is few for the basis disease before choosing art; 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 left standstill 1h, 1000rpm collected supernatant in centrifugal 10 minutes, and-80 ℃ of refrigerators are preserved subsequent use 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, will collect case and be divided into two groups: acute rejection group and transplanted kidney function liptinite according to analysis-by-synthesis and case screenings such as whether renal transplant recipients serum creatinine, urea nitrogen, transplanted kidney B ultrasonic, clinical manifestation, puncture pathological diagnosis result and hormone impact treatment effective.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: 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, weight increase, elevation of blood pressure, hypourocrinia and transplanted kidney distending pain etc., puncture pathological diagnosis result is an 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 recovered faster (serum creatinine is reduced 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, the concentration ratio of each factor in serum is more as shown in Figure 5 in transplanted kidney function liptinite and the acute grafing kidney rejection group early diagnosis system, and wherein 0 is liptinite, and 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 liquid of experiment plate hole, add standard items, contrast and the sample of 25 μ l gradient dilutions after paper handkerchief blots at the bottom of the brassboard respectively, every subsequently hole adds 25 μ l pearls; Behind the shrouding brassboard is placed 4 ℃ of incubated overnight of oscillator jolting; Wash to add behind the plate and detect antibody, room temperature oscillator jolting brassboard 1h is hatched 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 Combined application crowd, and setting up with the multiple-factor analysis-by-synthesis is the diagnosis system of the transplanted kidney rejection of characteristic.
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 convert into 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 TG-AUC 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 said 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 preparatory 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 98.6% that statistical analysis obtains early diagnosis system prediction positive rate, and ROC diagnosis TG-AUC is 99.8%, 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 said 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 preparatory 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 perhaps complication is few for the basis disease before choosing art; 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 left standstill 1h, 1000rpm collected supernatant in centrifugal 10 minutes, and-80 ℃ of refrigerators are preserved subsequent use 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, will collect case and be divided into two groups: acute rejection group and transplanted kidney function liptinite according to analysis-by-synthesis and case screenings such as whether renal transplant recipients serum creatinine, urea nitrogen, transplanted kidney B ultrasonic, clinical manifestation, puncture pathological diagnosis result and hormone impact treatment effective.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: 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, weight increase, elevation of blood pressure, hypourocrinia and transplanted kidney distending pain etc., puncture pathological diagnosis result is an 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 recovered faster (serum creatinine is reduced 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 each factor concentration ratio in serum in early warning system are more as shown in Figure 6, and wherein 0 is liptinite, and 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 liquid of experiment plate hole, add standard items, contrast and the sample of 25 μ l gradient dilutions after paper handkerchief blots at the bottom of the brassboard respectively, every subsequently hole adds 25 μ l pearls; Behind the shrouding brassboard is placed 4 ℃ of incubated overnight of oscillator jolting; Wash to add behind the plate and detect antibody, room temperature oscillator jolting brassboard 1h is hatched 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 Combined application crowd, and setting up with the multiple-factor analysis-by-synthesis is the transplanted kidney rejection pre-alarming system of characteristic.
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 convert into 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; 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, statistical analysis obtains predicting that positive rate is 91.4.ROC diagnosis TG-AUC 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 said 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 preparatory 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, SICAM-1 after the logstic regretional analysis to these expressions; 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 98.6%, ROC diagnosis TG-AUC 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 said 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 preparatory coated antibody, available from Millipore company, article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
Claims (26)
1. a transplanted kidney rejection early diagnosis kit is characterized in that, said kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and said 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.
2. transplanted kidney rejection early diagnosis kit according to claim 1 is characterized in that, said kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and said 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, 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.
3. transplanted kidney rejection early diagnosis kit according to claim 1 is characterized in that said kit also contains
(1) 96 hole filter membrane plate and two shrouding films;
(2) standard items of protein antibodies in the said 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 preparatory coated antibody.
4. transplanted kidney rejection early diagnosis kit according to claim 3 is characterized in that, in the said kit:
(1) said 96 hole filter membrane plates and two shrouding films are available from Millipore company, article No. MX-PLATE;
(2) said standard items are available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) said Quality Control contrast is available from Millipore company, and article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) said serum matrix is available from Millipore company, and article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) said experiment liquid is available from Millipore company, article No. L-AB;
(6) said washing lotion is available from Millipore company, article No. L-WB;
(7) the plain labelled antibody of said phycoerythrin-strepto-affinity is available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) said 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 said preparatory coated antibody is available from Millipore company, article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
5. a transplanted kidney rejection early warning kit is characterized in that, said kit comprises the antibody of the albumen that detects the transplanted kidney rejection, and said 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.
6. transplanted kidney rejection early warning kit according to claim 5 is characterized in that said 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, 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.
7. transplanted kidney rejection early warning kit according to claim 5 is characterized in that said kit also contains:
(1) 96 hole filter membrane plate and two shrouding films;
(2) standard items of protein antibodies in the said 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 6 rejection;
(10) pearl of preparatory coated antibody.
8. transplanted kidney rejection early warning kit according to claim 5 is characterized in that, in the said kit:
(1) said 96 hole filter membrane plates and two shrouding films are available from Millipore company, article No. MX-PLATE;
(2) said standard items are available from Millipore company, article No. MXH8060, MXH8062, MXH8063, LHSP-8063 and HSCR-8032;
(3) said Quality Control contrast is available from Millipore company, and article No. is: MXH6060, MXH6062, MXH6063, LHSP-6063 and HSCR-6032;
(4) said serum matrix is available from Millipore company, and article No. is: MXHSM, LHSP-SM, HSCR-SM;
(5) said experiment liquid is available from Millipore company, article No. L-AB;
(6) said washing lotion is available from Millipore company, article No. L-WB;
(7) the plain labelled antibody of said phycoerythrin-strepto-affinity is available from Millipore company, article No. L-SAPE9, L-SAPE3, L-SAPE10, L-SAPE11, L-SAPE6;
(8) said 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 said preparatory coated antibody is available from Millipore company, article No. MXHPMX39, MXHP2PMX23, HSP, HASP-PAI1, MXH3PMX9, HSCRPMX14.
9. the method for application of claim 1 or 5 said kits 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 subsequent use 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) behind the shrouding with hatching 0.5～2h under 4 ℃ of incubated overnight of brassboard jolting or the room temperature; 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 relevant detection antibody; 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, and check and analysis obtain experimental result.
10. the method for application of kit according to claim 9 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 subsequent use 2ml after the packing;
(2) application 2 00 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 incubated overnight 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.
11., it is characterized in that standard items, Quality Control contrast and sample in step (3) according to claim 9 or 10 described practical approaches according to 1: 4～5 ratio stepwise dilution.
12., it is characterized in that available from luminex company, article No. is #40-50000 at the sheath fluid described in the step (9) according to claim 9 or 10 described practical approaches.
13., it is characterized in that according to claim 9 or 10 described practical approaches, be luminex200 at the streaming dot matrix instrument described in the step (9), software is analyzed for MILLIPLEX Analyst.
14. Protein S CF and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
15. albumen sRAGE and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
16. PROTEIN C XCL7NAP2 and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
17. PROTEIN C CL14 α-HCC1 and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
18. albumen sCD40L and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
19. Protein G CSF and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
20. Protein G RO and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
21. protein I L-1R α and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
22. albumen MCP3 and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
23. protein I L-20 and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
24. albumen MIP1d and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
25. protein I L-28A and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
26. protein I L-29IFN λ 1 and antibody thereof the application in acute rejection in renal transplantation or the early warning of transplanted kidney rejection.
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| 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 |
| 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 |
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