CN101251539A - Kit for early diagnosis of renal transplantation acute rejection - Google Patents
Kit for early diagnosis of renal transplantation acute rejection Download PDFInfo
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- CN101251539A CN101251539A CNA2008100062680A CN200810006268A CN101251539A CN 101251539 A CN101251539 A CN 101251539A CN A2008100062680 A CNA2008100062680 A CN A2008100062680A CN 200810006268 A CN200810006268 A CN 200810006268A CN 101251539 A CN101251539 A CN 101251539A
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Abstract
The invention discloses a reagent box for an early diagnosis of the renal transplantation acute rejection, comprising at least one detecting reagent and a catch reagent, wherein the catch reagent can particularly identify the alfa-1 antichymotrypsin and/or the zinc-alfa-2 glycoprotein. The detection reagent box of the invention utilizes a biochemical-chemical test to analyze distinguishing albumen markings in early urines of a patient receiving a renal transplantation operation, achieving an aim of the early diagnosis of the renal transplantation acute rejection; moreover, the detection method is unique and high in specificity, and has a brightening market prospect.
Description
Technical field
The present invention relates to biological technical field, be particularly related to three kinds of mark candidate albumens that occur in the early stage patient's urine of renal transplantation acute rejection, be α-1 antichymotrypsin (alpha-1-antichymotrypsin, AACT), zinc-α 2 glycoprotein (Chain A, Zn-Alpha-2-Glycoprotein, ZAG) and tumor rejection antigen gp96 (tumorrejection antigen gp96 also claims GRP94).Utilization can prepare the early diagnosis of renal transplantation acute rejection kit at the specific antibody of above-mentioned three kinds of albumen, and this kit can be used for clinical early diagnosis renal transplantation acute rejection.
Background technology
Renal transplantation acute rejection (being called for short suddenly row) remains the principal element that hinders the transplanted kidney survival rate, and the successful treatment when anxious row taken place requires early diagnosis and in time gives abundant treatment.Usually the index of diagnosis generation acute rejection has: patient's clinical manifestation, every biochemical indicator and biopsy.Preceding two is not cocksure, and the clinical experience that mainly depends on the clinician is judged.In the serum creatinine level increase a very effective index normally diagnosing transplanted kidney depletion (allograft dysfunction), but these index right and wrong are special, and can not reflect the change of early stage renal function.Being considered to diagnose " goldstandard " of kidney transplantation exclusion reaction is biopsy, but it is reported, among the patient who makes a definite diagnosis with biopsy, is only successfully saved less than 30%.And the biopsy cost is big, belongs to traumatic inspection and often follows complication, comprising: pain, blood urine, kidney hemotoncus, graft thrombosis, septicemia, shock and graft depletion etc.
Diagnosed the generation of renal transplantation acute rejection in recent years, always towards the detection side who seeks Noninvasive to effort.Mainly contain following aspect: 1, check the solubility adhesion molecule in the blood, cell factor, urokinase, plasminogen activated receptor, the cell factor that lymphocyte is expressed, perforin, granzyme B and FAS part; 2, the IL-2 in the detection urine, adhesion molecule, complement C4d, urokinase, plasminogen activated receptor; 3, other comprise and analyze granzyme B in the urine cell, perforin, and the expression of the mRNA of granulysin and CD103, also the method for useful SABC detects various T cell sign things.Yet these detection methods are because problem such as specificity and susceptibility and all not by wide clinical application.
The urine simple and convenient not damaged of originating, patient is acceptant, and no sense of discomfort does not have contraindication.Compare with biopsy, urine can be reacted the overall condition of kidney, can react the physiological status and the functional level of kidney more timely and accurately.In view of above reason, select urine as the research object of seeking the rejection mark, protein marker is used as the index that the early diagnosis acute rejection takes place in the searching renal transplant recipients urine, and then the kit of development early diagnosis acute rejection will produce great marketable value.
Summary of the invention
The inventor is through a large amount of experimental studies have found that, the renal transplantation acute rejection patient some specific proteinses can occur in the urine during acute rejection, but also the variation of distinctive amount occurs.Raising earlier appears in some protein content during acute rejection, reduces again, perhaps reduces earlier, returns to initial level then gradually, and the albumen of this variation tendency is arranged, and may be significant to the monitoring of rejection process.The inventor found through experiments, α-1 antichymotrypsin (AACT), and zinc-α 2 glycoprotein (ZAG) and tumor rejection antigen gp96 (GRP94) present above-mentioned trend in the renal transplantation acute rejection process.In view of above reason, the inventor has not only verified the feasibility of above-mentioned albumen as the protein marker in the renal transplantation acute rejection patient urine by experiment, and utilizes these protein markers further to develop the kit that is used for early diagnosis of renal transplantation acute rejection.
α-1 antichymotrypsin (AACT) belongs to the serpin superfamily, is a glycoprotein that is present in the human serum.It can suppress the proteinase of chymotrypsin-like and the cytotoxic T lymphocyte activity in the body in vivo, and this albumen has the activity of regulation and control immunity and inflammatory process, also can be used as tumor markers.
Zinc-α 2 glycoprotein (Zn-Alpha-2-Glycoprotein, ZAG) closely related with MHC antigen on amino acid sequence and domain, with histocompatbility 1 class antigen sequence homology is highly arranged.Evidence show that this albumen belongs to immunoglobulin gene superfamily.Zinc-α 2 glycoprotein may be the secretion MHC correlation molecules after being sheared, and may play a role in the immune response process.
Tumor rejection antigen gp96 claims that also GRP94 is heat shock protein (HSP) family member, has the function of antigen processing and submission, and it can play the molecular chaperones effect in conjunction with other new lives or abnormal protein molecule.
The purpose of this invention is to provide a kind of kit that is used for early diagnosis of renal transplantation acute rejection, use can the specific recognition renal transplantation acute rejection early stage protein marker that occurs of this kit, so the progress that can be used for following the tracks of the kidney transplant postoperative patient, and can the early diagnosis renal transplantation acute rejection.
Another object of the present invention provides above-mentioned protein marker and is used for the purposes of the specific antibody of early diagnosis of renal transplantation acute rejection in preparation, utilizes its specific antibody can prepare the kit that is used for early diagnosis of renal transplantation acute rejection.
Technical scheme of the present invention is as follows:
A kind of kit that is used for early diagnosis of renal transplantation acute rejection comprises at least a detectable and catches reagent, and described seizure reagent can specific recognition α-1 antichymotrypsin and/or zinc-α 2 glycoprotein.
The above-mentioned kit that is used for early diagnosis of renal transplantation acute rejection can also contain the seizure reagent of specific recognition tumor rejection antigen gp96.
Specific antibodies such as the preferred monoclonal antibody of above-mentioned seizure reagent, polyclonal antibody, recombinant antibodies or antibody fragment, described specific antibody can also can be at α-1 antichymotrypsin, zinc-α 2 glycoprotein and tumor rejection antigen gp96 at α-1 antichymotrypsin and/or zinc-α 2 glycoprotein.Above-mentioned antibody can obtain by commercial sources, can also finish by the conventional method of Antibody Preparation, as Hangzhoupro body by recombinant methods, as phage displaying antibody, isolated antibody from transgenic animals (as mouse), with the antibody that the recombinant expression carrier that changes host cell over to is expressed, isolated antibody from the reorganization combinatorial antibody library.In addition, also can, obtain with the antibody in the conventional method purified blood serum again to obtain immune serum with its immune animal by the marker protein in expression and the above-mentioned people of purifying source.
Above-mentioned seizure reagent can also be can and other chemical reagent of α-1 antichymotrypsin, zinc-α 2 glycoprotein and/or tumor rejection antigen gp96 specific recognition, should can pass through the above-mentioned marker protein of biochemical reaction specific recognition by these chemical reagent, and be not limited to the antigen-antibody recognition method.
Above-mentioned detectable comprises detectable mark composition, and described mark composition comprises enzyme, prothetic group, fluorescent material, luminescent substance, a kind of in bioluminescence material, the radioactivity material.Can qualitatively or quantitatively determine marker protein in the sample by above-mentioned detectable label composition.
As mentioned above, enzyme can be a horseradish peroxidase, alkaline phosphatase, beta-galactase or acetylcholine vinegar enzyme; The example of prothetic group comprises streptomycete avidin/biotin albumen and avidin/biotin albumen; The example of fluorescent material comprises umbelliferone, fluorescein, fluorescein isothiocyanic acid vinegar, rhodamine, dansyl Cl or phycoerythrin; The example of luminescent substance comprises luminol, and the bioluminescence examples of substances includes but not limited to luciferase, fluorescein and aequorin; The radioactivity material comprises 125I, 35S, 14C, 3H, or Mg52.
Above-mentioned detectable can with catch the reagent packing, also can with catch the reagent coupling.
Test sample of the present invention is the urine of kidney transplant postoperative patient; Above-mentioned detection kit also contains required dilution reagent or the buffer reagent of detection that is necessary.
Prepare detection kit of the present invention, can obtain the dilution buffer liquid of seizure reagent, detectable and necessity in the kit by the market business approach, what wherein the seizure reagent in the embodiments of the invention adopted is the specific antibody of commercial source, and the method for Antibody Preparation prepares routinely.Each component of mentioned reagent box only needs conventional the use, need not special operation, can form that the reagent operation instruction is carried out or according to the operation of normal experiment method, those skilled in the art can correctly use detection kit in conjunction with the characteristics of sample according to each.
The identification of above-mentioned marker protein detects the mode that carrier is not limited to kit, and other detects carrier such as protein chip, detection test paper etc. also can be used to detect above-mentioned protein marker.
Above-mentioned marker protein has preparation and is used for the purposes of renal transplantation acute rejection as the specific antibody of early diagnosis, kit, the protein chip that utilizes its specific antibody to prepare to be used for early diagnosis of renal transplantation acute rejection or detect testing product such as test paper.
Detection kit of the present invention is to utilize biochemical test to analyze differential protein mark in the early stage urine of kidney transplant postoperative patient, reach the purpose of early diagnosis renal transplantation acute rejection, the detection method uniqueness, therefore the specificity height has great market outlook.
Description of drawings
The three phases of acute rejection takes place for the renal transplant patient in Fig. 1, and 1 phase one-7 that refer to reaction were to-1 day among the figure, and 2 refer to the subordinate phase 0 to 13 day of reaction, and 3 refer to 14 to 21 days phase IIIs of reaction.
Fig. 2 is three phase urine 2-DE collection of illustrative plates before, during and after patient's CQ acute rejection.On behalf of CQ patient, CQ (7), CQ (2), CQ (1) acute rejection takes place preceding 7 days, preceding 2 days and preceding 1 day, and CQ6, CQ13, CQ20 represent respectively and send out CQ patient and gave birth to behind the acute rejection the 14th day and the 21st day the 7th day.Arrow refers to 16 protein sites that identify, and wherein is respectively AACT (upper left square frame), ZAG (lower-left square frame) and three kinds of albumen of GRP94 (upper right square frame) with little square frame mark.
Fig. 3 is three phase urine 2-DE collection of illustrative plates before, during and after patient's LYX acute rejection.On behalf of LYX patient, LYX (7), LYX (2), LYX (1) acute rejection takes place preceding 7 days, preceding 2 days and preceding 1 day, and on behalf of LYX patient, LYX6, LYX13, LYX20 took place behind the acute rejection the 14th day and the 21st day the 7th day respectively.Arrow refers to 16 protein sites that identify, and wherein is respectively AACT (upper left square frame), ZAG (lower-left square frame) and three kinds of albumen of GRP94 (upper right square frame) with little square frame mark.
Fig. 4 is an AACT opalescence variable density trend.According to three phases of acute rejection, utilize each protein site optical density of PDQuest software analysis in 2-DE figure accumulation optical density number percent and the changing trend diagram drawn.
Fig. 5 is a GRP94 opalescence variable density trend.According to three phases of acute rejection, utilize each protein site optical density of PDQuest software analysis in 2-DE figure accumulation optical density number percent and the changing trend diagram drawn.
Fig. 6 is a ZAG opalescence variable density trend.According to three phases of acute rejection, utilize each protein site optical density of PDQuest software analysis in 2-DE figure accumulation optical density number percent and the changing trend diagram drawn.
Fig. 7 detects AACT albumen for utilizing detection kit of the present invention.
Fig. 8 detects ZAG albumen for utilizing detection kit of the present invention.
Fig. 9 detects GRP94 albumen for utilizing detection kit of the present invention.
Figure 10 is the optical density number percent column diagram that AACT, ZAG and three kinds of albumen of GRP94 are drawn according to the kit testing result at the renal transplantation acute rejection three phases.
Embodiment
Reach technological means and the beneficial effect that predetermined goal of the invention is taked for further setting forth the present invention, below in conjunction with accompanying drawing and preferred embodiment, describe the special marker protein that occurs in the renal transplantation acute rejection patient urine in detail, and the experimental result of using immuno-chemical method distinguishing mark thing, thereby better explain the technical scheme of detection kit of the protein marker identification of the sample that is used for the early stage patient of kidney transplant that the present invention proposes.
Select urine as the research object of seeking the rejection mark below by description of test, protein marker is used as the index that the early diagnosis acute rejection takes place in the searching renal transplant recipients urine.
One, experimental technique
1, case screening
Cooperate with Southwest Hospital, Chongqing City nephrology with Chongqing City's new bridge hospital kidney transplant center, leave and take urine specimen behind patient's renal transplantation.
Inclusion criteria: the therapeutic scheme of transplant patient postoperative is the same, 3 days strong dragon+ring phosphinylidyne ammonia of first of postoperative, second day afternoon or the 3rd day reach below 300 when patient's flesh edema due to dysfunction of the liver is flat, give CsA cyclosporine (Cyclosporin A)+MMF capsule+prednisone (pred) according to the gastrointestinal function situation.When acute rejection takes place patient, impacted 3-5 days with the strong dragon of first, if hormone impact invalid after, treat one-period with ATG.
Clinical manifestation: dashing forward has high fever, shiver with cold, and general malaise, agitation, transplanted kidney place distending pain, wound sepage or intraperitoneal drainage liquid increase, and the urine amount falls sharply or blood urine is arranged; The laboratory examination serum creatinine sharply raises and surpasses previous level 30%, and color ultrasound examination prompting transplanted kidney vascular resistence rising, cone are grown up, remove side by side block, surgery complication such as blood vessel embolism; With the remission of the strong imperial 0.5-1.0g/d impact treatment of heavy dose of first; The transplanted kidney aspiration biopsy turns out to be acute rejection.
Acute rejection same day takes place is 0 day to make a definite diagnosis patient in this experiment as shown in Figure 1, is divided into three phases: the phase one: 7 days before the anxious row take place; Subordinate phase: it was 0 day to the 13rd day the same day that anxious row is taken place; Phase III: the 14th day to the 21st day.
2, specimen collection
From renal transplant patient's postoperative first day, every morning 8:00 collected renal transplant patient's midstream urine 20-30ml, and under 4 ℃ of conditions, centrifugal 20 minutes of 3000rpm/min leaves and takes supernatant ,-80 ℃ of preservations.Collected 17 routine patients in 4 months altogether and surpassed 450 parts of samples, therefrom screened 2 examples totally 22 parts of samples according to inclusion criteria.
3, the processing of urine
This experiment extraction phase one (7 days),, the urine of subordinate phase the 6th, 13 days and phase III the 19th, 20,21 days is as experimental specimen.
Take out sample from-80 ℃, place 4 ℃ of thawings, get (4 ℃ of 20ml ultracentrifugation 2h, 200,000g), go precipitation, supernatant spends the night with the equal-volume acetone precipitation, sediment is through the centrifugal 30min of 15000rpm/min (4 ℃), acetone precipitation also repeats said process 2 times, leave and take the precipitation add 500ul IPG Lysis Buffer (7M urea (and ICN Biomedicals, USA), 2M thiourea (Fluka), 2% CHAPS (Sigma), 0.4% DTT (Sigam), 0.5%Pharmalyte 3-10 (Amershm Biosciences, cat NO.17-0456-01) and0.5% Pharmalyte 6-11 (Amersham Biosciences, catNO.17-6001-78) spend the night by 4 ℃ of joltings.
4,2D-PAGE electrophoretic analysis
The sample that jolting is spent the night is through the centrifugal 10min of 3000rpm/min (4 ℃), Bradford method protein quantification.
Each protein sample all uses curing PH adhesive tape (18cm, linear, PH4-7, Bio Rad) to analyze.The 200ug protein sample is at heavy swelling liquid (7M/L urea (ICN Biomedicals, USA), 2mol/L thiourea (ICN Biomedicals, USA), 4% CHAPS (Sigma), 30mmol/LDTT, 0.5% IPG Lysis Buffer, the trace bromophenol blue) resuspended in, passive aquation spend the night (12-16h).(one to IEF to carry out dielectrophoresis afterwards; Two to: the 12%SDS-PAGE electrophoresis), carry out according to Bio-Rad dielectrophoresis experiment guide.Equilibration buffer I(6M urea，2％ SDS(Serva)，30 ％Glycerol(Sigma)，50mM Tris-HCl(Sigma)pH 8.8，1％ DTT(Sigma)。Equilibration buffer II(4％ SDS(Serva)，10％v/v β-mercapthoethanol，23％ v/v Glycerol，24％ v/v Tris buffer(76.7g Tris-Base(Sigma)and 1.6g tris-Base(Sigma)。Runningbuffer：0.67％ Tris base(Sigma)，1.44％ glycine(Sigma)，0.1％SDS(Serva).Polyacrylamide gels：(30％ Acrylamide(GenomicSolution)，0.15％ Bis N，N′-Methylene-bis-acrylamide(Bio Rad)，14.532％ Tris base(Sigma)，4.725％ Tris HCl(Sigma)，pH＝8.8buffer，TEMED(BioRad)，10％ solution of ammonium persulphate(BioRad))。(USA) image scanning is carried out in dyeing then for Molecular Probes, Oregon, and the PDQest7.20 graphical analysis is than part analysis, searching discrepancy with SyproRuby .
5, identification of proteins (MALDI-TOF-TOF and database retrieval)
Use the MALDI-TOF-TOF spectrometer analysis.In brief, earlier the protein of interest particle is carried out (the Trypsin or Lysin-C solution:50mMNH4HCO3 of trypsinization in the glue, Lysin-Cat4 ℃ of 5mM CaCl2 and 12.5ng/ μ l of Trypsin or)., obtain peptide finger-print (Peptide MassFingerprint with MALDI-TOF-TOF then, PMF), these data are retrieved (http://www.matrixscience.com) with MASCOT MS/MS ion at ncbi database.Search argument is used: mammals, trypsin digestion, oxidation of methionines possible, onemissed cleavage allowed, no protein mass and pI restrictions, peptides mass tolerance: ± 50-70ppm, peptide charge:+1, Monoisotopic. think that when Mascot Score has statistical significance (p＜0.05) this albumen obtains to identify.
6, detection kit analysis verification
In order to verify 3 marker proteins that screen among the present invention, random choose 2 routine acute rejection patients (Z and D), stable (W2 and W3), hepatology postoperative 2 examples (G2 and G3) and 2 routine normal person's urine specimen T and the J of transplanting of 2 examples.Urine sample is handled the same, with the urine protein of handling well, behind Bradford method protein quantification, with protein concentration furnishing unanimity.10%SDS-PAGE separates, and then the albumen on the glue is transferred to polyvinylidene fluoride pvdf membrane (U.S. Milipore company).Spend the night with 5% skim milk sealing then.
Adopt rabbit to resist-AACT (SANTACRUZ BIOTECHNOLOGY as the seizure reagent in the detection kit of the present invention, INC.), rabbit is anti--ZAG (SANTA CRUZ BIOTECHNOLOGY, INC.) and rabbit anti--GRP94 (CHEMICON International, Inc.).Carry out antibody dilution according to the antibody operation instruction, added behind the above-mentioned antibody incubated at room one hour, TBST wash 4 times each 15 minutes.
As detectable of the present invention be horseradish peroxidase-labeled mouse-anti rabbit igg-HRP (SANTA CRUZ BIOTECHNOLOGY, INC.), with SANTA company colour developing liquid exposure image in the gel imaging instrument.
Two, experimental result:
1, three phase urine 2-DE collection of illustrative plates before, during and after the acute rejection.
As shown in Figures 2 and 3, analyze, screened 50 discrepancys, 16 protein sites having identified through the PDQest7.20 image analysis software.CQ (7)～CQ21, LYX (7)～LYX21 is respectively the urine 2-DE collection of illustrative plates of two patients in 3 phases of acute rejection.CQ, LYX are patient's code name, and CQ (7), LYX (7) representative is made a definite diagnosis and acute rejection taken place preceding 7 days, and CQ1, LYX1 representative is made a definite diagnosis and acute rejection taken place first day, and the like.Wherein be respectively AACT (upper left square frame), ZAG (lower-left square frame) and three kinds of albumen of GRP94 (upper right square frame) with little square frame mark.
2, mass spectrum and database retrieval
According to the PDQuest analysis result, there is the optical density difference more than 2 times in the same protein site in the sample.Then be considered as having the difference (p＜0.01) of expression.This experiment screening 50 discrepancys, identified 16 protein sites, adhere to 13 albumen (seeing Table 1) separately.According to the protein function retrieval, therefrom filter out AACT, ZAG and GRP94 totally 3 albumen, and draw the changing trend diagram of its optical density in three phase of acute rejection collection of illustrative plates, see Fig. 4,5,6.
This experiment shows, in the table 1 105
#Albumen alpha-1-antichymotrypsin (AACT) α-1 antichymotrypsin content in the three phases before, during and after anxious row reduces (see figure 4) on the whole gradually, about 7 days to subordinate phase is clinical definite the previous day, in 2-DE figure, almost disappear (as Fig. 2, Fig. 3), therefrom we raise at the content of early stage this albumen of acute rejection generation as can be seen, along with the application of anti-rejection medicine, the content of this albumen reduces gradually.
This experiment shows, in the table 1 285
#((Zn-Alpha-2-Glycoprotein ZAG) in the rejection process, shows as the trend that raises and afterwards reduce earlier to 2 glycoprotein to zinc albuminate-α generally, reaches the highest at the 14th day, reduces (see figure 6) afterwards gradually.
This experiment shows, in the table 1 154
#Albumen tumorrejection antigen gp96 also claims GRP94 in that anxious row rising gradually in preceding 7 days takes place, and after the clinical definite treatment, reduces gradually.
3, detection kit checking result
Detection kit checking result shows AACT and ZAG (Fig. 7,8,10) these two albumen only occur in patient's urine of generation acute rejection behind renal transplantation, and take place anxious ranked first the stage (D-5, D-3) and subordinate phase (D3, D9) content is apparently higher than phase III (D21) and other groups (W2, G3, T), and stable the transplanting (W2, W3), liver sausage anastomosis (G3) back and the almost not appearance of these 2 albumen of normal person (T).This albumen of GRP94 (Fig. 9,10) all occurs in 4 groups of crowds, is suddenly arranging patient's subordinate phase content apparently higher than first, phase III and other groups, though this albumen specificity is not strong, its content during acute rejection is obvious, and it the meaning of auxiliary diagnosis occurs having.
According to this experimental result, 3 stages of renal transplantation acute rejection, it is acute rejection early stage, emergence period and convalescence, variation tendency and detection kit checking result according to AACT and these two albumen of ZAG think that these two albumen can be used as the marker protein of the early detection that detects the acute rejection generation.To be used as at the specific antibody of above-mentioned two kinds of albumen and catch reagent, can qualitative or these two kinds of marker proteins of detection by quantitative in conjunction with suitable detectable.Can use separately during detection or the specific antibody of these two albumen is used simultaneously, antibody can use with the detectable coupling, to improve the specificity of detection efficiency and diagnosis.
Table 1 differential protein particle mass spectrum qualification result
| Spot Lable | Sequenc e Coverag e(％) | Expect | Accessio n Number(g i/) | Calculat ed Mass (Da) | Calculat ed pI | Masco | Protein | |
| 105 143 154 285 310 | 58％ 27％ 28％ 24％ 32％ | 9.3e-32 3.9e-05 6.2e-12 1.9e-10 3.8e-05 | gi|17793 3 gi|22300 2 gi|58865 966 gi|58176 768 gi|58176 768 | 45453 50731 74162 32126 32126 | 5.32 7.95 5.02 5.71 5.71 | 375 109 177 163 110 | alpha-1-antichym otrypsin precursor fibrin beta tumor rejection antigen gp96 (predicted) Chain A， Zn-Alpha-2-Glyco protein；Cho-Zag Peg 200 Chain A， Zn-Alpha-2-Glyco protein；Cho-Zag Peg 200 |
| 383 561 574 747 748 758 779 1003 1031 1063 1177 | 43％ 14％ 13％ 39％ 34％ 34％ 17％ 20％ 18％ 6％ 43％ | 2e-14 0.00038 0.00052 9.4e-19 2.9e-21 1.5e-13 7.8e-13 7.4e-07 1.4e-13 1.5e-06 1.5e-25 | gi|18952 6 gi|17389 346 gi|29820 81 gi|19426 29 gi|22317 0 gi|19426 29 gi|72059 gi|17389 346 gi|76343 1 gi|77701 49 gi|19426 29 | 25786 27070 16178 44223 46252 44223 34325 27070 52048 70909 44223 | 7.26 7.0 5.87 5.37 5.54 5.37 5.66 7.0 5.69 5.43 5.37 | 202 100 98 245 270 193 186 126 185 123 314 | prostate specific antigen precursor Secreted and transmembrane 1 precusor[ Chain B，Familial Als Mutant G37r Cuznsod Alphal-Antitryps in fibrinogen gamma Alphal-Antitryps in leucine-rich alpha-2-glycopro tein-human Secreted and transmembrane 1 precusor Swiss-Prot Accession Number P02768 PRO1851 Alphal-Antitryps in |
The above, it only is better embodiment of the present invention, be not that the present invention is done any pro forma restriction, therefore be not in order to limit the present invention, any those skilled in the art, in not breaking away from the technical solution of the present invention scope, to any simple modification, equivalent variations and modification that above embodiment did, all still belong in the scope of technical solution of the present invention according to technical spirit of the present invention.
Claims (10)
1, a kind of kit that is used for early diagnosis of renal transplantation acute rejection comprises at least a detectable and catches reagent, and described seizure reagent can specific recognition α-1 antichymotrypsin and/or zinc-α 2 glycoprotein.
2, the described kit of claim 1 is characterized in that also containing the seizure reagent of specific recognition tumor rejection antigen gp96.
3, the described kit of claim 1 is characterized in that described seizure reagent comprises the specific antibody at α-1 antichymotrypsin and/or zinc-α 2 glycoprotein.
4, the described kit of claim 2 is characterized in that described seizure reagent comprises the specific antibody at α-1 antichymotrypsin, zinc-α 2 glycoprotein and tumor rejection antigen gp96.
5, claim 1 or 2 described kits is characterized in that described detectable comprises detectable mark composition, and described mark composition comprises a kind of in enzyme, prothetic group, fluorescent material, luminescent substance, bioluminescence material or the radioactivity material.
6, the described kit of claim 5, it is characterized in that described detectable with catch the reagent packing or with catch the reagent coupling.
7, claim 1 or 2 described kits is characterized in that the test sample of using is the urine of kidney transplant postoperative patient.
8, α-1 antichymotrypsin, zinc-α 2 glycoprotein and tumor rejection antigen gp96 are used for the purposes of the specific antibody of early diagnosis of renal transplantation acute rejection in preparation.
9, the described purposes of claim 8 is characterized in that described specific antibody is monoclonal antibody, polyclonal antibody, recombinant antibodies or antibody fragment.
10, the described purposes of claim 8, the detection carrier that it is characterized in that described specific antibody are kit, protein chip or detect test paper.
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| CN102207505A (en) * | 2010-03-29 | 2011-10-05 | 上海友科生物科技有限公司 | Method for in vitro detection of zinc-alpha2-glycoprotein, and kit thereof |
| CN102539745A (en) * | 2010-12-31 | 2012-07-04 | 中国人民解放军第三〇九医院 | Early diagnosis and early warning kit for transplanted kidney rejection reaction and detection method |
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| CN102854306A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Kit for early warning rejection reaction after kidney transplant and use method of kit |
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| CN104330570A (en) * | 2014-10-11 | 2015-02-04 | 中国科学院微生物研究所 | Application of human heat shock protein gp96 to prepare products screening hepatopathy |
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| CN102539745A (en) * | 2010-12-31 | 2012-07-04 | 中国人民解放军第三〇九医院 | Early diagnosis and early warning kit for transplanted kidney rejection reaction and detection method |
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| CN102854305B (en) * | 2011-12-23 | 2013-08-07 | 中国人民解放军第三〇九医院 | Prewarning kit for transplant kidney rejection and use method thereof |
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