CN102580119A - Reagent for monitoring immune state of rabbit after skin grafting, and preparation method thereof - Google Patents
Reagent for monitoring immune state of rabbit after skin grafting, and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a reagent for monitoring the immune state of a rabbit after skin grafting, and a preparation method thereof. The reagent is phosphate buffer solution (PBS) suspension liquid of two rabbit spleen cells treated by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining, wherein the total concentration of the two rabbit spleen cells is (5-7)*10<7>/ml, and the proportion between the two rabbit spleen cells is 1: 1; the two rabbit spleen cells respectively come from a donor and a receptor which are treated by allogenous skin grafting; and after staining, the proportion of positive cells is more than or equal to 95%, and the proportion of living cells is more than or equal to 95%. The reagent can be used for rapidly and accurately monitoring the immune state of the rabbit after skin grafting in a visual way. The reagent is directly injected into the body of the rabbit after skin grafting, the in-vivo complex environment is not needed to be simulated, and the immune state can be reflected by testing the specificity loss of the cells, so that the monitoring results are more comprehensive and accurate.
Description
Technical field
The present invention relates to medical configuration article, be specifically related to be used for reagent of immune state monitoring after the rabbit skin transplantation and preparation method thereof.
Background technology
Must use the generation that immunosuppressant prevents acute rejection after the organ transplantation, immunosuppressant is through synthetic the working of cell proliferation, cytokine of target cells such as suppressor T cell and B cell.But still can't distinguish at present the immunological rejection immunoreation that infection causes with virus and bacteria that transplantation antigen causes.Ideal immunosuppressant therapy should and keep in immunosuppressant averaging out between immunity, and therefore transplant patient's management is a very thing of difficulty for the clinician.Preserve receptor immunity, reduce the main target that immunosuppressant consumption or inducing immune tolerance are the transplantation immunity scholars always as far as possible, it is most important therefore to transplant the back immunologic surveillance.
The mode of the monitoring of clinical receptor immune state commonly used remains the biopsy of clinical symptoms assessment combination graft at present.But the living tissue pathologic finding all is a kind of damaging inspections to graft and host, can not repeat; Immune state that can not the complete reaction transplant organ; And when pathologic finding rejection occurred, transplant rejection took place, so pathologic finding has its inherent limitation.
Except pathologic finding, immunologic surveillance method commonly used has: the mensuration of periphery blood T cell counting, killer cell CTL and NK cytoactive mensuration, MHC Molecular Detection, cytokine assay, Perforin, granzyme B, CD40L and FasL, circulating antibody detection etc.
Because immunoreactive complexity; Above-mentioned immunological detection method its limitation all arranged; It is unrealistic to rely on single immune indexes to assess modulation on immune status; Mainly a situation arises (pathological biopsy) and the graft function situation through rejection to weigh the usefulness of rejection and immunosuppressant therapy after the organ transplantation clinically, and toxic a situation arises for Liver and kidney during like renal transplantation, and liver function situation during liver transplantation (blood Cr, ALT etc.) is assessed.Pathological diagnosis remains the goldstandard of diagnosis rejection at present; But graft living tissue pathologic finding also has its limitation: one, the biopsy detection is a kind of damaging inspection method; Normal survival to graft has certain influence; Can not take sample repeatedly, in addition, take that sample is very few can to influence the correctness that pathological section is read sheet again; Two, the biopsy pathological examination is a non-quantitation property index, influences the accuracy of its clinical diagnosis, and pathological examination is read the sheet easily person's the subjective factors and the influence of technical merit produce bias, deficiency in objective property; Three, check pathological section can't be done functional judge to the wellability lymphocyte; The early diagnosis that is unfavorable for rejection. lose the receptor of function at the early stage or graft of rejection generation; Even show as the receptor of stable type clinically; The pathological biopsy section all can be seen a large amount of T lymphocytic infiltrations, or only shows as speckle property T cellular infiltration.
Summary of the invention
The object of the present invention is to provide reagent that is used for immune state monitoring after the rabbit skin transplantation and preparation method thereof.
The technical scheme that the present invention taked is:
Be used for the reagent of immune state monitoring after the rabbit skin transplantation, this reagent is that the total concentration of two kinds of rabbit splenocytes is (5～7) * 10 through the painted two kinds of PBS suspensions with the rabbit splenocyte of strain of CFSE
7Individual/mL, the ratio of the two is 1:1, and wherein, two kinds of rabbit splenocytes are respectively from donor and receptor after transplanting through heterogenous skin, positive cell ratio>=95% after the dyeing, living cells ratio>=95%.
Preferably, donor is female rabbit.
Preferably, receptor is a male rabbit.
The above-mentioned method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation comprises the steps:
1) female rabbit and the male rabbit got with strain are respectively donor and receptor, carry out heterogenous skin and transplant;
2) heterogenous skin is transplanted the spleen of back excision in the 10th～13 day donor and receptor, processes the PBS suspension of donor spleen cells and the PBS suspension of receptor splenocyte respectively;
3) in the PBS suspension of donor spleen cells, adding 6 μ M CFSE, in the PBS suspension of receptor splenocyte, add 0.24 μ M CFSE, is to hatch dyeing under 37 ℃ in temperature then;
4) after the dyeing, with donor spleen cells and receptor splenocyte with the PBS washing after, by 1: 1 mixed of cell number, using PBS to adjust the cell total concentration is (5～7) * 10
7Individual/mL, must be used for the reagent that the rabbit skin skin is transplanted the back immune state monitoring.
Preferably, female rabbit is female White Rabbit.
Preferably, male rabbit is male White Rabbit.
Preferably, hatching dyeing time is 15～20 min.
CFSE described in the present invention is a CF 5(6)-Carboxyfluorescein diacetate succinimide ester.
Cell of the present invention derives from through the donor after the heterogenous skin transplanting and the splenocyte of receptor; Donorcells and graft are homogenic; Antigenicity and graft are identical; Can be reflected the immune attack that graft is suffered comprehensively, exactly by the immunoreation specific killing that produces because of transplanting; Recipient cell is with to carry out dermatoplastic rabbit homogenic, as the confidential reference items of immune state monitoring, is used to eliminate because cell breakage, death, a series of cells non-special error that loss brings of giving birth to such as natural death in external dyeing processing and body such as go back to the nest.
Cell of the present invention is taken from the rabbit heterogenous skin and is transplanted back the 10th～13 day donor and recipient cell; Complicated variation can take place in intravital immune environment after the skin transplantation; And heterogenous skin transplanting back rejection can occur on the 10th～13 day; Get this stages of cell and be used to monitor immune state after the rabbit skin transplantation, truer, more prepare.
The present invention is used for the monitoring method of the reagent of immune state monitoring after the rabbit skin transplantation, comprises the steps:
1) reagent that is used for immune state monitoring after the rabbit skin transplantation of the present invention is expelled in the rabbit body after the skin transplantation through auricular vein, is expelled to the intravital reagent of dermatoplastic rabbit and contains TCS and be (1～1.4) * 10
9Individual;
2) injection back 8h carries out auricular vein with rabbit and gets blood, and blood carries out fluidic cell and detects, and draws two kinds of positive cell ratios of peripheral blood;
3) calculate kill rate, according to the immune state after the skin transplantation of kill rate judgement rabbit, if injection back 8h, kill rate >=80% just can be judged as recipient rabbits graft is in the immunologic rejection state.
The immune state of rabbit according to the invention refers to the repulsion degree of receptor to graft, and promptly the allosome in the reagent (donor) spleen cell is by the degree of receptor repulsion.Monitoring method of the present invention; Kill rate is high more, and then transplant rejection is strong more, and the judgment principle of monitoring method of the present invention is: the reagent injection back 8h that is used for immune state monitoring after the rabbit skin transplantation; Kill rate >=80% just can be judged as recipient rabbits graft is in the immunologic rejection state.
The invention has the beneficial effects as follows:
The present invention is used for the reagent of immune state monitoring after the rabbit skin transplantation, can quick, intuitive and accurate monitoring transplant operation after the immune state of rabbit.In the rabbit body of reagent direct injection of the present invention after skin transplantation; Do not need complex environment in the analogue body; Specificity loss through test cell is reflected immune state; Monitoring result more comprehensively, has accurately proved in large animal that first reagent of the present invention can detect immune state after the rabbit skin transplantation effectively.
Description of drawings
Fig. 1 is used for zooperal techniqueflow chart for reagent of the present invention;
Fig. 2 is each kill rate curve chart that divides into groups in the zoopery.
The specific embodiment
Embodiment below in conjunction with concrete is further described the present invention, but does not limit to so.
The used donor of embodiment is elected female new zealand white rabbit as, and receptor is elected male new zealand white rabbit as, and the rabbit that is used for immune state monitoring is male new zealand white rabbit, and body weight is 1.9～2.5kg, credit number: SCXK (Guangdong)-0015.
CFSE reagent is available from U.S. Molecularprobes company.
Be used for the reagent of immune state monitoring after the rabbit skin transplantation, this reagent is the PBS suspension through the painted two kinds of rabbit splenocytes of CFSE, and the total concentration of two kinds of rabbit splenocytes is 6 * 10
7Individual/mL, the ratio of the two is 1:1, wherein; Two kinds of rabbit splenocytes are respectively from donor and receptor after transplanting through heterogenous skin, and the dyeing back is observed under fluorescence microscope, and the positive cell ratio is 95%; And detecting cytoactive with trypan blue solution, the living cells ratio is 96%.
The above-mentioned method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation comprises the steps:
1) according to the principle of random pair, get female White Rabbit and male White Rabbit is respectively donor and receptor, carry out heterogenous skin and transplant;
2) heterogenous skin was transplanted the back the 12nd day, the spleen of excision donor (female White Rabbit) and receptor (male White Rabbit), and layer-by-layer suture is closed the abdominal cavity after the splenectomy, gives gauze and covers, and postoperative gives transfusion and penicillin 80,000 units/kg treatment;
3) prepare splenocyte suspension with 200 order stainless (steel) wire polishings immediately after the splenectomy, 7.5 g/L ammonium chloride splitting erythrocyte, 0.01mol/L PBS dilution prepares donor and the single splenocyte suspension of receptor respectively;
4) the receptor splenocyte suspension dyes with 0.24 μ M CFSE, and donor spleen cells dyes with 6 μ M high concentration CFSE, and the dyeing incubation temperature is 37 ℃, times 15 min;
5) after the dyeing, donor spleen cells and receptor splenocyte are washed once with PBS, then by two kinds of cells of 1: 1 mixed of cell number, and use PBS adjustment cell total concentration is 6 * 10
7Individual/mL, must mix splenocyte suspension, i.e. the reagent of immune state monitoring of the present invention.
Be used for the reagent of immune state monitoring after the rabbit skin transplantation, this reagent is the PBS suspension through the painted two kinds of rabbit splenocytes of CFSE, and the total concentration of two kinds of rabbit splenocytes is 5 * 10
7Individual/mL, the ratio of the two is 1:1, wherein; Two kinds of rabbit splenocytes are respectively from donor and receptor after transplanting through heterogenous skin, and the dyeing back is observed under fluorescence microscope, and the positive cell ratio is 97%; And detecting cytoactive with trypan blue solution, the living cells ratio is 95%.
The above-mentioned method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation comprises the steps:
1) according to the principle of random pair, get female White Rabbit and male White Rabbit is respectively donor and receptor, carry out heterogenous skin and transplant;
2) heterogenous skin was transplanted the back the 10th day, the spleen of excision donor (female White Rabbit) and receptor (male White Rabbit), and layer-by-layer suture is closed the abdominal cavity after the splenectomy, gives gauze and covers, and postoperative gives transfusion and penicillin 80,000 units/kg treatment;
3) prepare splenocyte suspension with 200 order stainless (steel) wire polishings immediately after the splenectomy, 7.5 g/L ammonium chloride splitting erythrocyte, 0.01mol/L PBS dilution prepares donor and the single splenocyte suspension of receptor respectively;
4) the receptor splenocyte suspension dyes with 0.24 μ M CFSE, and donor spleen cells dyes with 6 μ M high concentration CFSE, and the dyeing incubation temperature is 37 ℃, times 18 min;
5) after the dyeing, donor spleen cells and receptor splenocyte are washed once with PBS, then by the cell after two kinds of dyeing of 1: 1 mixed of cell number, and use PBS adjustment cell total concentration is 5 * 10
7Individual/mL, must mix splenocyte suspension, i.e. the reagent of immune state monitoring of the present invention.
Embodiment 3
Be used for the reagent of immune state monitoring after the rabbit skin transplantation, this reagent is the PBS suspension through the painted two kinds of rabbit splenocytes of CFSE, and the total concentration of two kinds of rabbit splenocytes is 7 * 10
7Individual/mL, the ratio of the two is 1:1, wherein; Two kinds of rabbit splenocytes are respectively from donor and receptor after transplanting through heterogenous skin, and the dyeing back is observed under fluorescence microscope, and the positive cell ratio is 96%; And detecting cytoactive with trypan blue solution, the living cells ratio is 97%.
The above-mentioned method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation comprises the steps:
1) according to the principle of random pair, get female White Rabbit and male White Rabbit is respectively donor and receptor, carry out heterogenous skin and transplant;
2) heterogenous skin was transplanted the back the 13rd day, the spleen of excision donor (female White Rabbit) and receptor, and layer-by-layer suture is closed the abdominal cavity after the splenectomy, gives gauze and covers, and postoperative gives transfusion and penicillin 80,000 units/kg treatment;
3) prepare splenocyte suspension with 200 order stainless (steel) wire polishings immediately after the splenectomy, 7.5 g/L ammonium chloride splitting erythrocyte, 0.01mol/L PBS dilution prepares donor and the single splenocyte suspension of receptor respectively;
4) the receptor splenocyte suspension dyes with 0.24 μ M CFSE, and donor spleen cells dyes with 6 μ M high concentration CFSE, and the dyeing incubation temperature is 37 ℃, times 20 min;
5) after the dyeing, donor spleen cells and receptor splenocyte are washed once with PBS, then by the cell after two kinds of dyeing of 1: 1 mixed of cell number, and use PBS adjustment cell total concentration is 7 * 10
7Individual/mL, must mix splenocyte suspension, i.e. the reagent of immune state monitoring of the present invention.
The method that above-mentioned heterogenous skin is transplanted is following: get about 3 * 3cm size donor new zealand white rabbit skin of back, with eye scissors subcutaneous tissue is pruned totally, and an onesize skin is excised with scalpel in receptor White Rabbit back; Keep subcutaneous tissue; Then the donor skin graft is fixed on the receptor mouse back with 5-0 noinvasive stitching thread, binds up a wound immobilization with adhesive tape with gauze; The degree of tightness appropriateness, and with donor wound disinfection and wrapping.
Prove that through zoopery reagent of the present invention can detect immune state after the rabbit skin transplantation effectively below, the techniqueflow chart of this experiment is referring to Fig. 1.
1. laboratory animal
Select female new zealand white rabbit, 1.9～2.5kg is as donor (credit number: SCXK (Guangdong)-0015); Select male new zealand white rabbit, 1.9～2.5kg is as receptor (credit number: SCXK (Guangdong)-0015); The animal that above-mentioned animal and preparation reagent place of the present invention use is all available from Nanfang Medical Univ's Experimental Animal Center, and all animals have all received the due treatment of laboratory animal, and experimental arrangement has passed through the approval of local animal protection and research institution.
2. experiment is divided into groups
It is 5 groups (n=5) that experiment is divided into, A group: heteroplastic transplantation repulsion group; B group: auto-skin grafting group; C group: transplantation group not; D group: heteroplastic transplantation immunosuppressant low dose group; E group: heteroplastic transplantation immunosuppressant high dose group.
3. the foundation of skin transplantation model
Setting up two groups of skin transplantation models according to the grouping needs, is donor with female new zealand white rabbit, and male new zealand white rabbit is that receptor is set up the heterogenous skin transplantation model; Set up the auto-skin grafting model with male new zealand white rabbit.A, B, C are the heterogenous skin transplantation model for three groups, and the E group is the auto-skin grafting model, and the D group is equivalent to the blank group, and this group is male new zealand white rabbit.
The structure of heterogenous skin transplantation model is referring to above-mentioned heterogenous skin implantation method; The structure of auto-skin grafting model and above-mentioned heterogenous skin implantation method are basic identical, and difference is that donor and receptor all are same male White Rabbit.
D organizes (heteroplastic transplantation immunosuppressant low dose group): receptor gives 2mg/kg ciclosporin A (CsA), intravenous injection, receptor preceding 8 hours of transplanting with once, once a day later on;
E organizes (heteroplastic transplantation immunosuppressant high dose group): receptor gives 15mg/kg ciclosporin A (CsA), intravenous injection, receptor preceding 8 hours of transplanting with once, once a day later on.
4. adopt the immune state monitoring reagent infusion of embodiment 1
1) the male White Rabbit (receptor) of each group is fixing;
2) the ear edge of White Rabbit being used volume fraction is 75% ethanol disinfection;
3) with the immune state monitoring reagent of 4 number sword-shaped needles puncture auricular vein success back injection embodiment 1, consumption 20 mL/, the mixing spleens cell number of every male White Rabbit injection is 1.2 * 10
9Individual.
5. collection of specimens and streaming
Male White Rabbit after every group of skin moves and grows, 1h, 2h, 4h, 8h behind immune state monitoring reagent infusion of the present invention get blood through auricular vein and carry out two kinds of positive cell ratios of streaming monitoring peripheral blood, that is: donor spleen cells number/receptor donor spleen cells number.Each about 200 μ L, with erythrocyte cracked liquid (BD company, the U.S.) splitting erythrocyte at room temperature, flow cytometer (BD company, the U.S.) detects after PBS washing 3 times, and selecting the lymphocyte analysis is analytic target, and every part of BIAO and BEN measures about 10
5Individual cell.
6. calculating kill rate
After reagent was expelled in the rabbit body, because the difference of donorcells MHC phenotype receives the repulsion of receptor, the degree that we are ostracised the donorcells (allogene) of injecting reagent was called kill rate, calculates by following formula:
Kill rate (R)=1-donor spleen cells number/receptor spleens cell number * 100%.
7. statistical analysis
Experimental data is carried out statistical analysis with SPSS 13.0 softwares and is handled, data result with mean and standard deviation represent (± SD), the comparative statistics method adopts unidirectional variance (F) analysis between each group of each time point.Compare with check of Dunnett t (2-sided) method and A group (heteroplastic transplantation repulsion group) between group.< 0.05 has been considered to statistical significance to P, makes the kill rate curve chart.The kill rate result of each group experiment sees table 1, and the kill rate curve chart is seen Fig. 2.Criterion: 8h behind the injection reagent, kill rate >=80%, decidable are graft-rejection.
Can know that by table 1 and Fig. 2 the recipient rabbits of A group (heteroplastic transplantation repulsion group) and D group (heteroplastic transplantation immunosuppressant low dose group) is in the immunologic rejection state to graft.
Treated after the male White Rabbit skin transplantation of 5 experimental grouies the 12nd day, and got its graft tissue and do pathologic finding, HE dyeing; Microscopically is observed; The A group has produced strong rejection with the D group, and B group and E group do not produce intensive rejection, and the experimental result of experimental result and table 1 is coincide.
Interpretation is following:
Because repelling the White Rabbit of model group, heteroplastic transplantation after skin transplantation, produces intensive rejection; Store a large amount of lymphocyte, NK cell, antibody, complement in the body to donorcells; This moment, donorcells contacted with the receptor of sensitization once more; The cellular immunization and the humoral immune reaction of mediations such as the lymphocyte that therefore prestores in the White Rabbit body of heteroplastic transplantation repulsion model, NK cell, antibody, complement produce dead cat to donor spleen cells (being the doe splenocyte); Intensive rejection takes place; So there be specific killing and wounding in the receptor White Rabbit to donor spleen cells, promptly be ostracised basically after 8 hours and remove totally in injection, kill rate reached 86.19 ± 6.95% in 8 hours.
The autotransplantation group and not the female White Rabbit splenocyte of transplantation group do not show by specific killing; But be ostracised gradually; Kill rate was respectively 31.58 ± 3.41% and 33.51 ± 3.49% in 8 hours; Because autotransplantation group and the intravital immune system of transplantation group un-activation not, there be not lymphocyte and antibody, the complement of sensitization, promptly receptor is not in the sensitization state; So receptor can not attacked female White Rabbit splenocyte at once, treat back just can the repulsion gradually such as immunocyte and cytokine, antibody, complement of female White Rabbit splenocyte activated receptor and remove.
Same; Low dosage immunosuppressant group is not enough because of immunosuppressant dosage; Fail to suppress well the rejection of receptor to donor skin; Immune mediators such as a large amount of primed lymphocytes and complement, antibody are arranged in the body, so its performance is consistent with transplant rejection group White Rabbit model, its kill rate and transplant rejection group do not have significant difference.
High dose immunosuppressant group is owing to restrained the receptor immune system well, and is consistent with capable transplantation group and the performance of autotransplantation tolerance group kill rate so there were significant differences for its kill rate and heteroplastic transplantation repulsion group White Rabbit model, do not have significant difference.
This shows; Donor specific cells in vivo toxicity test can reflect the immune state of transplanting the back receptor exactly; According to monitoring reagent injection back 8h, kill rate >=80% judges that recipient rabbits is in the immunologic rejection state to graft; This monitoring method has antigenic specificity, is a kind of method of monitoring transplanting back receptor immune state of excellent specificity.
Claims (7)
1. be used for the reagent of immune state monitoring after the rabbit skin transplantation, this reagent is that the total concentration of two kinds of rabbit splenocytes is (5～7) * 10 through the painted two kinds of PBS suspensions with the rabbit splenocyte of strain of CFSE
7Individual/mL, the ratio of the two is 1:1, and wherein, two kinds of rabbit splenocytes are respectively from donor and receptor after transplanting through heterogenous skin, positive cell ratio>=95% after the dyeing, living cells ratio>=95%.
2. the reagent that is used for immune state monitoring after the rabbit skin transplantation according to claim 1, it is characterized in that: donor is female rabbit.
3. the reagent that is used for immune state monitoring after the rabbit skin transplantation according to claim 1, it is characterized in that: receptor is a male rabbit.
4. like each described method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation of claim 1～3, comprise the steps:
1) female rabbit and the male rabbit got with strain are respectively donor and receptor, carry out heterogenous skin and transplant;
2) heterogenous skin is transplanted the spleen of back excision in the 10th～13 day donor and receptor, processes the PBS suspension of donor spleen cells and the PBS suspension of receptor splenocyte respectively;
3) in the PBS suspension of donor spleen cells, adding 6 μ M CFSE, in the PBS suspension of receptor splenocyte, add 0.24 μ M CFSE, is to hatch dyeing under 37 ℃ in temperature then;
4) after the dyeing, with donor spleen cells and receptor splenocyte with the PBS washing after, by 1: 1 mixed of cell number, using PBS to adjust the cell total concentration is (5～7) * 10
7Individual/mL, must be used for the reagent that the rabbit skin skin is transplanted the back immune state monitoring.
5. the method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation according to claim 4, it is characterized in that: female rabbit is female White Rabbit.
6. the method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation according to claim 4, it is characterized in that: male rabbit is male White Rabbit.
7. the method for preparing that is used for the reagent of immune state monitoring after the rabbit skin transplantation according to claim 4, it is characterized in that: hatching dyeing time is 15～20 min.
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Citations (3)
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| CN1427258A (en) * | 2001-12-18 | 2003-07-02 | 杜凤鸣 | Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying low density lipoprotein content in human urine and its preparation method |
| CN1598586A (en) * | 2004-09-07 | 2005-03-23 | 江苏省农业科学院 | Immune antibody for detecting pesticide organic phosphorus residus and its application |
| CN101149371A (en) * | 2007-10-26 | 2008-03-26 | 南方医科大学珠江医院 | Mouse immune state monitoring reagent and its preparing process |
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|---|---|---|---|---|
| CN1427258A (en) * | 2001-12-18 | 2003-07-02 | 杜凤鸣 | Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying low density lipoprotein content in human urine and its preparation method |
| CN1598586A (en) * | 2004-09-07 | 2005-03-23 | 江苏省农业科学院 | Immune antibody for detecting pesticide organic phosphorus residus and its application |
| CN101149371A (en) * | 2007-10-26 | 2008-03-26 | 南方医科大学珠江医院 | Mouse immune state monitoring reagent and its preparing process |
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