CN1205081A - Method of determining or detecting donor organ demage following xenotransplantation based on donor organ-derived analytes - Google Patents
Method of determining or detecting donor organ demage following xenotransplantation based on donor organ-derived analytes Download PDFInfo
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Abstract
A method of determining or detecting the presence of a donor organ-derived analyte in a biological fluid indicative of donor organ damage following xenotransplantation and, thereby, enabling identification of xenograft organ damage in a recipient comprises capturing said analyte by an antibody which is: a) specific for the donor organ-derived analyte; or b) capable of cross-reacting with said donor organ-derived analyte and directly or indirectly determining the donor organ-derived analyte. This method enables one to distinguish between host-derived and donor-derived analytes and, thereby, to obtain an indication of xenotransplant status and possibly rejection, so that corrective therapy can be undertaken as appropriate as soon as such a donor organ-derived analyte is identified. The method applies to in vivo and ex vivo xenotransplantation.
Description
Invention field
The present invention relates to measure or detect the method for learning the analyte that donor organ that the indication donor organ that exists in the liquid damages derives at the heteroplastic transplantation artifact.
Background technology
Worldwide there is at present the donor organ that is used for allograft (liver for example, kidney and heart) shortage and the someone advise and prove by before the spare-part surgery operation of normal position, using the animal organ can overcome this problem, although be temporary transient.Chari, R.S. waits people (" new BMJ " (1994); The 33rd volume, the 4th phase, 234-237 page or leaf) with external pig liver perfusion liquid, subsequently with the decline of the liver transplant treatment liver of position often.A patient was stablized 10 days, successfully carried out the liver transplant of normal position subsequently.But not success is attempted also in former organ heteroplastic graft (heteroplastic transplantation) because after implanting acceptor in the several hrs the known phenomenon that is called hyperacute rejection make firm transplanted organ by host's immune system uniform and be ostracised.The progress that obtains in the molecular genetics field recently can have been developed transgenic animals (mainly being pig), its organ has carried out genetic engineering procedure, does not almost have immunogenicity (" portability information " (1995) when transferring to different kind (being people/non-human primates); The 5th volume, the 9th phase).In this report, suppose by expressing human in the transgenosis organ " decline speedup factor " gene and obtained immune tolerance, the described factor has and suppresses the host to complement component () activation for example, the Cb3 compound, and therefore make hyperacute rejection be reduced to minimum.But as the situation of the interior organ transplant (allograft) of kind, xenograft rejection remains arguement, and the transplanting doctor must and further be perplexed by the tangible fact with this arguement struggle: come from different types of tissue and be present in transplant recipient.
Conventional liver functional test can not be distinguished the analyte that donor and acceptor (for example pig and people) organ are derived.For example, the mensuration of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) is not recognized people and pig enzyme, and the trial that the source of back enzyme is transplanted in the identification that provides thus is invalid.Similarly situation is present in kidney heteroplastic transplantation analysis.In fact, the protein labeling that does not have common received kidney integrality at present.
Glutathione S-transferase (GSTs) comprises mainly by α (α GST), μ (μ GST), a plurality of gene families of the protein that π (π GST) and θ-class (θ GST) isotype (being defined by isoelectric point) are formed, and mainly by means of with the conjugation of glutathione, be responsible for the detoxification (Beckett of various xenobiotics, G.J. and Hayes, J.D. " clinical chemistry progress " (1993); The 30th phase, the 281-380 page or leaf).In the people, α GST the homogeneous of liver distribute with and high relatively liver level and be meant short plasma half-life (about 1 hour) as this enzyme of indicator of transplanting back liver situation and drug-induced liver damage sensitiveer than aminopherase.
EP-A 0640145 discloses and has helped the method that early diagnosis is repelled in the liver transplant acceptor, this method is included in blood plasma or serum transaminase had not before had or vicissitudinous situation under, measure the blood plasma of acceptor or the increase of serum α GST.
The π glutathione S-transferase is positioned the tenuigenin of the bile duct epithelial cell in the liver, and is considered to only exist with dimeric forms of the same race.The GST different isodynamic enzyme function in different liver areas has unique body of hint that distributes in the liver of this xenogenesis, the measurement that therefore can sum up the GST level of blood plasma or courage helps the monitoring for the liver situation of individuality.Also have similar unique distribution in renal tissue, wherein α GST is positioned the tubule zone of the near-end of the nephron, and π GST is positioned the distal tubule zone, and (" cancer " be (1991) (Philadelphia) for Campbell, people such as J.A.H.; The 67th phase, the 1608-1613 page or leaf).In nearest report, someone advises, because the locus specificity characteristic of histologic lesion separately, detect simultaneously that α/π GST can be used for distinguishing respectively cyclosporin A pnehrotoxicity and graft rejection (Sundberg in the people urine, A.G.M. wait the people, " nephron " (1994): the 67th phase, 308-316 page or leaf).
Therefore, need be as the difference host who detects the means that transplant organ damages and the method for the analyte that graft derives, or be used to distinguish the method that host and/or transplant organ damage really.Summary of the invention
The invention provides and measure or detect the method for learning the analyte that donor organ that the indication donor organ that exists in the liquid damages derives at the heteroplastic transplantation artifact, can discern the damage of heterograft organ in the described acceptor thus, described method comprises by the described analyte of antibody capture and directly or indirectly measures the analyte that donor organ is derived that described antibody is:
A) be specific to the analyte that donor organ is derived; Or
B) the analyte generation cross reaction that can derive with described donor organ.
Therefore, the analyte of deriving with donor that method of the present invention can be distinguished host derivation, thereby can indicate heterograft situation and possible repulsion, so that can carry out correct treatment as quickly as possible, the analyte that donor organ is derived in the biological fluid of acceptor is identified like this, as the situation of detailed proof more hereinafter.
Described herein biological fluid is meant for example body fluid such as bile, blood plasma, serum and urine and organize Supporting Media and perfusion liquid.Described herein biological fluid also relates to matrix usually.
Described herein donor organ is derived is meant that organ itself and tissue and cell, described tissue and cell are the ingredients of organ and are derived by it.
Described equally herein donor material is meant organ itself and tissue and cell, and described tissue and cell are the ingredients of organ and are derived by it.
Therefore, as concrete example, donor material can be a liver, and the part of liver is lobe of the liver or liver cell for example.Usually, for hepatocellular situation, use the liver cell cartridge case.
Usually, acceptor is the mankind or non-human primates.
Therefore, preferably donor material is to come from for example pig of non-human primates or animal, and described animal has and human similar physiology and biochemistry characteristic.
Donor animal can be transgenic animals.
Heteroplastic transplantation can be that body is interior or external.
What transplanted organ was suitable is heart, kidney or liver.
Preferably, the analyte that donor organ is derived is a protein, more particularly enzyme.
Preferably, the donor organ analyte of deriving will be a donor specific.Under the situation that has corresponding receptor assay thing, preferably receptor assay thing and the described donor organ analyte of deriving has low homology.But the analyte that donor organ is derived can have the homology (for example homology is greater than 80%) of height with corresponding receptor assay thing, and according to the present invention described below, the analyte that donor organ is derived also is detectable.
Preferred enzyme is a glutathione S-transferase, more particularly α GST.
Moreover, preferably, measure by the analyte that method of immunity is derived to the donor organ of catching.Suitable method of immunity comprises enzyme immunoassay (EIA).
Therefore,, adopt the IgG (anti--pig α GST) that directly or indirectly is attached to solid phase for the pig α GST in people's biological fluid, or another kind of optionally method, adopt the IgG (Anti-Human α GST) that shows cross reactivity can catch pig α GST.
We have prepared rabbit polyclonal IgG (anti--pig α GST), and it has the high degree of specificity with pig α GST.For for example in the presence of the people α of high concentration GST, this antibody also shows under the situation with the cross reactivity of the little degree (about 5-10%) of people α GST, can measure people α GST individually so, and according to hereinafter describing the amount of correct calculation pig α GST.
We have discerned with pig α GST has the almost monoclonal Anti-Human α GST of 100% cross reactivity, as hereinafter embodiment 3 descriptions.
Enzyme immunoassay (EIA) can be a kind of sandwich method or half competitiveness enzyme immunoassays, describes respectively as embodiment 1 and 2 hereinafter.
In one side more of the present invention, the viability of test donor material before heteroplastic transplantation.
Therefore, the viability that the present invention also can test donor material by the analyte that donor organ in the mensuration biological fluid is derived, by using method defined above, described biological fluid pours into this donor material.
Aspect of the present invention, can the check and analysis thing according to the known method except that the present invention.For example, if the analyte that organ is derived is an enzyme, can utilize enzymatic determination to detect the analyte that described organ is derived so based on the application of zymolyte.
The present invention also provides test kit or the pack that contains one or more compositions, is used to implement above-described method of the present invention.The accompanying drawing summary
In the accompanying drawings:
Fig. 1 is the synoptic diagram of the interlayer enzyme immunoassay (EIA) of embodiment 1;
Fig. 2 is at the absorption value at the 450/630nm place drafting figure with respect to the α GST concentration (micrograms per litre) in the enzyme immunoassay (EIA) at the interlayer immunizing dose of the pig α of embodiment 1 GST;
Fig. 3 is the synoptic diagram of the half competitiveness enzyme immunoassays of embodiment 2;
Fig. 4 is at the absorption value at the 450/630nm place drafting figure with respect to the α GST concentration (micrograms per litre) in the enzyme immunoassay (EIA) of measuring at the competitive immunization of the pig α of embodiment 1 GST;
Fig. 5 is the SDS-PAGE analysis chart of people and pig α GST;
Fig. 6 utilizes IgG (Anti-Human α GST) and goat IgG (anti--rabbit α GST)-people that the HRP conjugate carries out and immunoblotting assay figure of pig α GST;
Fig. 7 utilizes IgG (anti--pig α GST) and goat IgG (anti--rabbit α GST)-people that the HRP conjugate carries out and immunoblotting assay figure of pig α GST; With
Fig. 8 utilizes IgG (Anti-Human α GST) and goat IgG (anti--mouse α GST)-people that the HRP conjugate carries out and immunoblotting assay figure of pig α GST.
Implement pattern of the present invention
Further describe the present invention according to the following examples.
The preparation embodiment A
The purifying of pig α GST
By affinity chromatography and chromatofocusing (pH9.5-6.0) from pig liver purifying α GST.Accurate detailed purification scheme is as follows:
A. utilize Waring (Waring is trade (brand) name) bruiser, with the ratio of 3 portions of damping fluids of a liver, with pig livers homogenate 2 minutes in homogenate buffer of 30 grams.Homogenate buffer has following component:
10mM?Tris-HCl
250mM sucrose
5mM?EDTA?pH?7．8
2 mcg/ml leupeptins
2 mcg/ml pepstatins
B. with 10000g with centrifugal 60 minutes of liver homogenate liquid.
C. then with the supernatant load sample to previous in 10mM Tris pH9.1 on glutathione (the GSH)-Sepharose affinity column of balance.Again use the unconjugated protein of level pad wash-out.Utilize the GST of the 50mM Tris pH9.1 of the GST that contains 5mM at last from the affinity column elution of bound.
D. then the material of wash-out is dialysed in 25mM monoethanolamine pH9.5, and be applied to chromatofocusing post (by the PBE94 of Pharmacia supply).Elution buffer is the Polybuffer 96 (Polybuffer 96 is trade (brand) names) according to the explanation preparation of manufacturer.
The preparation Embodiment B
Antibody producing and purifying
According to hereinafter given timetable the pig α GST of purifying is expelled to New Zealand white rabbit from subcutaneous (s.c.), and the anti-α GST reactivity of assessment serum.Tire when enough high in case measure IgG (anti--pig α GST), give this animal bloodletting, collect serum by sxemiquantitative point engram analysis.From rabbit anteserum purifying total IgG, be used for bag by the a-protein affinity chromatography by droplet plate (half competitive EIA-embodiment 2) and biotinylation (interlayer EIA-embodiment 1).Obtain monoclonal IgG (Anti-Human α GST) from the medical university of Dutch Nijmegen, be not further purified as ascites fluid and before using.Immunity inoculation timetable (routine):
The 1st day: emit the blood sample of 5 milliliters pre-serum from the ear of rabbit, then 0.5 milliliter pig α GST antigen (100 microgram) is mixed with isopyknic Freund's complete adjuvant.With antigen and adjuvant homogenization to guarantee to obtain good emulsifying liquid.Then this mixed liquor is subcutaneously injected into a plurality of sites of the rabbit back of the body, described rabbit had before shaved off hair.
The 28th day: emit the blood sample of 5 milliliters of serum from the ear of rabbit, then 0.5 milliliter antigen (100 microgram) is mixed with isopyknic Freund's complete adjuvant.With antigen and adjuvant homogenization to guarantee to obtain good emulsifying liquid.Then this mixed liquor is subcutaneously injected into a plurality of sites of the rabbit back of the body.
The 42nd day: the test blood that obtains 10 milliliters of blood from the ear of rabbit.
The 56th day: the description during according to the 28th day, carry out the second time to rabbit and strengthen.
The 70th day: the test blood sample that obtains 10 milliliters of blood from the ear of rabbit.When tiring when enough high, kill rabbit, collect blood as much as possible.
The preparation Embodiment C
Western blot
Making up respectively by means of following Western blotting, detection is used for all polyclones of following embodiment and reactivity and the cross reactivity of monoclonal IgGs and people and pig α GST:
(a) rabbit IgG (Anti-Human α GST) is used to survey the people of containing immunofixationization and the nitrocellulose filter of pig α GST.
(b) rabbit IgG (anti--pig α GST) is used to survey the people of containing immunofixationization and the nitrocellulose filter of pig α GST.
(c) mouse IgG (Anti-Human α GST) is used to survey the people of containing immunofixationization and the nitrocellulose filter of pig α GST.
The method that is used for the Western blotting detection is as follows:
1. the electrophoresis on 15%SDS-PAGE with people and pig α GST (0.5 microgram/swimming lane), and comprise the molecular weight marker thing.
2. after electrophoresis, downcut polyacrylamide gel, a semigel is carried out protein staining, and remaining is used for electrophoretic transfer to nitrocellulose filter.
3. after electrophoretic transfer, in the salt solution of the phosphoric acid buffer that contains 0.05% (w/v) Tween-20 (PBST) sealing damping fluid, sealed nitrocellulose filter 1 hour with 5% (w/v) Marvel (Marvel is trade (brand) name).
4. the solution below preparing then:
(i) be dissolved in rabbit IgG (Anti-Human α GST) among 1% (w/v) Marvel of PBST.
(ii) be dissolved in the rabbit IgG (anti--pig α GST) among 1% (w/v) Marvel of PBST.
(iii) be dissolved in the mouse IgG (Anti-Human α GST) among 1% (w/v) Marvel of PBST.In case and decantation sealing damping fluid, this solution is joined described film.
5. in PBST, clean nitrocellulose filter (2 times, each 5 minutes) then.
6. preparation anti-rabbit IgG-HRP conjugate (1/1000) and join above-mentioned 4 (i) and (ii) in being dissolved in 1% (w/v) Marvel of PBST then.Also prepare anti-mouse IgG-HRP conjugate (1/1000) and join above-mentioned 4 (iii).Obtain to be used for anti--kind (rabbit/mouse) conjugate of immune detection from Bio-Rad laboratory company limited.
7. after with anti--kind conjugate insulation 1 hour, decantation reactant and as cleaning film as described in above-mentioned 5.
8. prepare the diaminobenzidine substrate then and join described film.Brown precipitate indication positive reaction on nitrocellulose filter.
Preparation embodiment D
Synthesizing of pig α GST-horseradish peroxidase (HRP) conjugate:
Utilize thioether conjugation method to synthesize α GST-HRP conjugate.Utilize SMCC (succinimide 4-(N-maleimide methyl) cyclohexane-1-carboxylate) reactive maleimide base group to be imported to α GST molecule and the mercapto groups of covering up is connected to HRP (Duncan, R.J.S. wait people (1983); " biochemical analysis " 132 phases, the 68-73 page or leaf).Cover up step with after producing reactive mercapto groups taking off, α GST that maleimide is activated and HRP-SH-rise and mix and allow reaction 4.5 hours.To place 50% (v/v) glycerine by the last α GST-HRP conjugate that the thioether covalent bond forms and be stored in-20 ℃, to be used for the half competitive EIA of embodiment 2.
The preparation embodiment E
The biotinylation of IgG (anti--pig α GST)
By under alkali condition, the polyclone IgG of purifying (anti--pig α GST) is coupled to N-hydroxy-succinamide-biotin (NHS-biotin) prepares biotinylated IgG (anti--pig α GST), wherein polyclone IgG (anti--pig α GST) is previous by (as describing in preparing Embodiment B) with the α GST immunity inoculation rabbit generation of purifying.Remove unreacted NHS-biotin by dialysis widely then and with biotinylated IgG equal portions and refrigerated storage in-20 ℃, when needs use.
The interlayer enzyme immunoassay (EIA)
Employed program diagram is described in accompanying drawing 1.
A. bag is by NuncMaxisorp (Nunc Maxisorp is a trade name) droplet plate to use mouse monoclonal IgG (Anti-Human α GST) (with reference to the preparation Embodiment B), and described monoclonal antibody is by means of goat F (ab)
2Fragment (anti--mouse IgG) is fixing.Described antibody sandwich method have definite Mab binding site the position effect and also improved the sensitivity of measuring by the sex change that absorption is reduced to minimum-induce capture antibody.With protein/sucrose solution sealing droplet plate.
B. from describing the embodiment A as preparation, the pig α GST of the liver purifying that obtains immediately after death and be stored in 2-8 ℃ or-20 ℃ is used as the corrector of determination test.
C. biotinylated IgG (anti--pig α GST) conjugate is used for aided capture/detection of immobilized α GST.
D. be used in combination tetramethyl benzidine substrate (TMB) then, streptavidin-horseradish peroxidase (HRP) conjugate is used to detect complete antigen intercalation compound.
E. stop enzyme reaction by adding 1N sulfuric acid, utilize 630nn to measure absorption value at the 450nm place as the reference wavelength.Color intensity and α GST concentration are in direct ratio, are drawing A
450/630nmAfter concentration (micrograms per litre) figure, measure the concentration (referring to accompanying drawing 2) of unknown sample.Total test duration is lower than 3.5 hours.
Half competitiveness enzyme immunoassays
Employed program diagram is described in accompanying drawing 3.
A. in the present example, use by means of the fixing polyclone IgG of a-protein (anti--pig α GST) bag by Nunc Maxisorp droplet plate, can not promote catching of α GST because find the direct coated of polyclone IgG, may be owing to be attached to the sex change of the IgG on the droplet plate.
B. will be used as the conjugate of this example with the α GST of HRP mark, and join the unlabelled corrector/sample in the micropore subsequently.By this way, the state of conflict reaction is set between the α GST HRP-mark and unlabelled.
C. between suitable soak, after common 60 minutes, clean the droplet plate and add tmb substrate.
D. stop enzyme reaction by adding 1N sulfuric acid, utilize 630nm to measure absorption value at the 450nm place as the reference wavelength.Color intensity and pig α GST concentration are inversely proportional to, and are drawing A
450/630nmAfter concentration (micrograms per litre) figure, obtained quantitative measurement (referring to accompanying drawing 4) to unknown sample.
The assessment of the purity of immunoassay reaction agent
Carry out Western blot according to the program of in the preparation Embodiment C, describing.The results are described in accompanying drawing 5-8.
The main points of accompanying drawing 5-8 swimming lane:
Swimming lane 1: molecular weight marker thing.
Swimming lane 2: people α GST (0.5 microgram)
Swimming lane 3: pig α GST (0.5 microgram)
Accompanying drawing 5 has been described in the purity of people before the immunity inoculation rabbit and pig α GST (by the single bands of a spectrum under various situations indications) and has been confirmed to exist without any other people or the pig protein of deriving, and described protein may cause the mensuration specificity of reduction.The immunoblotting assay of antibody response shows that IgG (Anti-Human α GST) and people α GST have high degree of specificity, and do not show and have significant cross reactivity (accompanying drawing 6) with pig α GST, as the weak signal strength with respect to pig α GST, people α GST has strong signal intensity indication.Do not show that according to pig α GST (α GST) p reactivity supported this conclusion in the immunoassays of people α GST (α GST) h-enzyme-specific, the immunoassays of described people α GST (α GST) h-enzyme-specific are by Biotrin InternationalLimited-Mount Merrion, the name that County Dublin, Ireland sell is called HEPKIT.The results are shown in table 1, it has represented the reactive assessment of α GSTp in Biotrin HEPKIT.Cross reactivity does not obviously appear when analyzing α GSTp in HEPKIT.
Table 1 (α GST) h (micrograms per litre) A
450/630nm
0．00???????????????0．069
1．25???????????????0．156
2．50???????????????0．332
5．00???????????????0．627
10．0???????????????1．088
20．0???????????????1．495
40．0???????????????1．762
PC 0.839 (6.9 micrograms per litre) (α GST) p (micrograms per litre) A
450/630nm
8?????????????????????0．012
40????????????????????0．010
200???????????????????0．012
1000 0.014PC=positive controls.
This true meaning is epochmaking, has specificity because mean enzyme immunoassay (EIA) and people α GST detection that people α GST is quantitative.Therefore, can detect anyone the α GST that is present in the sample specifically, from the cross pollution of pig antigen, the pig α GST in the described sample can be not determined yet.The result who in people α GST enzyme immunoassay (EIA), does not have cross reactivity and between IgG (anti--pig α GST) and people α GST, show the cross reactivity (accompanying drawing 7) of the 5-10% that has an appointment, as by strong signal intensity with respect to pig α GST, people α GST has weak signal strength indication, mean can use in the HEPKIT enzyme immunoassay (EIA) people α GST and pig (in the α GST enzyme immunoassay (EIA) in the pig α GST quantitative measurement to proofread and correct the distribution of people α GST in the pig α GST immunoassays.
For example, if sample provides following reading:
Measure α GST (micrograms per litre)
Pig EIA (pig α GST) 10000
Hepkit EIA (people α GST) 1000 guarantees in pig EIA that so about 7% the cross reactivity of people α GST means that people α GST provides for the reading of pig α GST
Micrograms per litre.
Therefore, the pig α GST that has the 10000-70=9930 micrograms per litre.
In addition, As mentioned above, in accompanying drawing 8, show the cross reactivity widely between mouse IgG (Anti-Human α GST) and the pig α GST significantly, as what indicate by the suitable intensity of corresponding bands of a spectrum/signal.The advantage of this phenomenon is the antibody that this antibody of the interlayer immunoassays that are used for pig α GST of embodiment 1 is used as " catching " or bag quilt flat board.
Embodiment 4
Measure specificity
Because being mainly used in of the mensuration of embodiment 1 and 2 detects pig α GST in the non-pig biological fluid, it is necessary therefore utilizing the sensitivity and the specificity that are present in the pig α GST assess and determine in the following matrix:
-people urine
-human serum
-human plasma
-human bile
-organize Supporting Media
In order to finish described analysis, pig α GST is fixed to all above-mentioned matrix and in the immunoassays of embodiment 1 and 2, tests subsequently.Except quantitative α GST detects, also the linearity (parallelism) of assessment test is as measurement sensitivity.Moreover the test specificity helps only to detect pig α GST and anyone α GST does not cause that in immunoassays significant interference also is necessary.In order to arrive this purpose, by the people α GST with different amounts mix contain pig α GST sample to assess potential cross reactivity, the detection reaction of investigator α GST.Suppose to have so significant sequence homology (this is not a problem that often occurs) between two isodynamic enzymes, only can overcome by analyzing the antiserum (anti--pig α GST) that has a potential cross reactivity with people α GST widely.
As what determine by interlayer and half competitiveness enzyme immunoassays, pig α GST quantitative measurement the results are shown in table 2 in the matrix of various samples.
The recovery of table 2 fixing sample in interlayer and half competitive pig α GST mensuration:
| Expectation value (μ g/L) | The sample (μ g/L) of dilution | Blood plasma-people (μ g/L) | MEM (fully) (μ g/L) | TC-100 (fully) (μ g/L) | Urine-people (μ g/L) | |||||
| sand. | comp. | sand. | comp. | sand. | comp. | sand. | comp. | sand. | comp. | |
| ????0 ?4000 ?2000 ?1000 ?500 ?250 ?125 | ?61.4 ?4530 ?2980 ?507 ?279 ?128 | ?19.4 ?3853 ?2699 ?446 ?260 ?140 | ?0 ?4299 ?2500 ?1041 ?249 ?85 ?0 | Excessively | ????0 ?4135 ?2262 ?1180 ?512 ?290 ?136 | ?46.8 ?3643 ?2420 ?1247 ?485 ?211 ?188 | ????0 ?4754 ?2059 ?983 ?540 ?273 ?197 | ?73 ?3889 ?2071 ?953 ?562 ?266 ?163 | ????0 ?4189.5 ?2165.2 ?844.5 ?392.8 ?151.9 ?17.9 | ?84 ?3936 ?1912 ?857 ?454 ?218 ?126 |
Main points:
1.Sand: the interlayer enzyme immunoassay (EIA).
2.Comp: half competitiveness enzyme immunoassays.
3. be used for the sample scope (0-1000 micrograms per litre) of the dilution 1/5 of competitive assay, further be diluted to 1/100 then, be used for interlayer test (scope 0-40 micrograms per litre).
4.MEM and TC-100: organize Supporting Media.
Can be clear that from table 2, can quantitative recovery pig α GST in the matrix of all tests.Obviously, it is necessary can detecting α GST respectively in human plasma and urine, so that help the situation of heteroplastic liver and kidney, is exactly this situation really.But, can notice that as if blood plasma matrix incompatible with half competitive immunometric assay mode, may be because IgG and immobilized a-protein interact, cause unusual reading.In addition, other all liquid show with two kinds of mensuration modes to have compatibility.What is interesting is especially and can in organizing Supporting Media, detect pig α GST, this should help heterograft before transplanting is assessed or the analysis (being present in cartridge case) of the viability of the porcine hepatocyte that separates, because these systems remain in the conventional Supporting Media usually.In addition, the compatibility of described matrix/sample should help to detect the α GST at the nutrient culture media that is used for keeping external donor organ widely, thereby donor organ (for example liver) is kept contacting with nutrient culture media outside the human body.
Embodiment 5
The assessment that people α GST disturbs in the enzyme immunoassay (EIA) of pig α GST interlayer
Interference to people α GST in the pig α GST interlayer enzyme immunoassay (EIA) of embodiment 1 is assessed.As what measure, when being lower than 25 micrograms per litre, the concentration of droplet plate hole do not prove interference (being equal to the sample concentration of 125 micrograms per litre in the sample diluting liquid with 1/5 dilution) by the absorption value that increases.The results are shown in table 3.Table 3
| (α GST) p (α GST) h (micrograms per litre) | Absorption value A 450／630nm | |
| 20 | - | 0．869 |
| 20 | 6 | 0．875 |
| 20 | 12 | 0．875 |
| 20 | 25 | 0．881 |
| 20 | 50 | 0．975 |
Main points:
α GSTp-pig α GST
α GSTh-people α GST
The result proof that is shown in table 3 is used to detect the specificity of the immunoassays of pig α GST.Can prove from these data, in addition sample concentration during up to 125 micrograms per litre (25 micrograms per litre * 5) people α GST do not disturb described enzyme immunoassay (EIA), this represents the normal higher limit of the α GST of 15 and 6 times human serum and urine respectively.(＞250 micrograms per litre when the higher level of people α GST; 50 micrograms per litre * 5), some interference obviously occurs, calculated the cross reactivity of about 5-10% between people α GST and IgG (anti--pig α GST).But, should calculate the accurate amount of existing people α GST when in quantitative immunoassays, measuring the level of people and pig α GST altogether, therefore in to people's biological fluid, should consider during the quantitative measurement pig α GST.
Should be noted that further immunoassays of the present invention also can be used for detecting pig α GST in the biological fluid that pig derives, as relate to the situation of toxicological study in the external or body of pig or pig organ.This result is significant, because known pig and life Neo-Confucianism/biological chemistry are very similar, so pig often is used as the animal model of prediction to people's drug toxicity.
Claims (14)
1. measure or detect the method for learning the analyte that donor organ that the indication donor organ that exists in the liquid damages derives at the heteroplastic transplantation artifact, the damage of heterograft organ in thus can identification receptor, described method comprises by the described analyte of antibody capture and directly or indirectly measures the analyte that donor organ is derived that described antibody is:
A) be specific to the analyte that donor organ is derived; Or
B) the analyte generation cross reaction that can derive with described donor organ.
2. method according to claim 1, wherein acceptor is the people.
3. method according to claim 1, wherein acceptor is inhuman primate.
4. according to each described method of claim 1-3, wherein donor organ derives from pig.
5. according to each described method of claim 1-4, wherein donor organ is a kidney.
6. according to each described method of claim 1-4, wherein donor organ is a liver.
7. according to the described method of each claim of front, wherein the donor organ analyte of deriving is a protein.
8. method according to claim 7, wherein the donor organ analyte of deriving is an enzyme.
9. method according to claim 8, wherein enzyme is glutathione S-transferase (GST).
10. method according to claim 9, wherein enzyme is α GST.
11. according to the described method of each claim of front, when being subordinated to claim 4, wherein antibody is anti--pig α GST.
12. according to each described method of claim 1-10, when being subordinated to claim 2, wherein antibody is Anti-Human α GST, it shows to have 100% cross reactivity with pig α GST basically.
13., wherein before heteroplastic transplantation, test the viability of donor material according to the described method of each claim of front.
14. contain the test kit or the pack of one or more compositions, be used to implement each described method of claim 1-13.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95197996 CN1205081A (en) | 1995-12-08 | 1995-12-08 | Method of determining or detecting donor organ demage following xenotransplantation based on donor organ-derived analytes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 95197996 CN1205081A (en) | 1995-12-08 | 1995-12-08 | Method of determining or detecting donor organ demage following xenotransplantation based on donor organ-derived analytes |
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| CN1205081A true CN1205081A (en) | 1999-01-13 |
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| CN 95197996 Pending CN1205081A (en) | 1995-12-08 | 1995-12-08 | Method of determining or detecting donor organ demage following xenotransplantation based on donor organ-derived analytes |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107660234A (en) * | 2015-04-14 | 2018-02-02 | 伊万基因诊断中心有限公司 | The method of prediction organ-graft refection is sequenced using two generations |
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1995
- 1995-12-08 CN CN 95197996 patent/CN1205081A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107660234A (en) * | 2015-04-14 | 2018-02-02 | 伊万基因诊断中心有限公司 | The method of prediction organ-graft refection is sequenced using two generations |
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