CN103589690B - ChIL-18 monoclonal antibody and its preparation method and application - Google Patents
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Abstract
The invention discloses ChIL-18 monoclonal antibody and its preparation method and application. The present invention is with the recombinant protein c hIL-18 of prokaryotic expression for immunogen, and the ChIL-18 albumen that eukaryotic cell lines is expressed is former for screening, screens hybridoma with IFA after cell fusion, and screening obtains the cell strain of 8 strains secretion ChIL-18 monoclonal antibodies; Wherein, is hybridoma cell strain 4E11 stability best, and secreted monoclonal antibody titer is the highest, specificity is the strongest, and its microbial preservation number is CGMCC? NO.7658. Invention further provides the application in detection or purification ChIL-18 albumen of hybridoma cell strain 4E11 and secreted monoclonal antibody.
Description
Technical field
The present invention relates to chicken interleukin-2 monoclonal antibody, particularly relate to ChIL-18 monoclonal antibody and secrete the hybridoma cell strain of this monoclonal antibody, the invention further relates to the application in detection or purification ChIL-18 albumen of this monoclonal antibody, belong to preparation and the application of ChIL-18 monoclonal antibody.
Background technology
Interleukin is called for short interleukin, leukocyte produce, the cytokine of mediated cell interphase interaction, plays regulatory role in the activation of cell, propagation and differentiation. Along with molecular biological development, research about chicken interleukin-2 gene and function has been increasingly becoming focus, up to now, from chicken, isolated multiple interleukin, such as: IL-1, IL-2, IL-6, IL-10, IL-12, IL-16, IL-18 etc., these interleukins can be applied to the aspects such as immunotherapeutic agent, immunostimulant, structure novel gene engineered vaccine, utilize the cytokines such as interleukin to strengthen or regulate the nonspecific immunity of body, not only can effective prevention and control livestock and poultry, moreover it is possible in Animal husbandry production, produce huge economic benefit.
There is the defects such as low, the poor stability of titer in the hybridoma cell strain being presently used for secretion ChIL-18 monoclonal antibody, secreted monoclonal antibody specificity is poor, seriously constrains production application, it would be highly desirable to improves.
Summary of the invention
An object of the present invention is to provide that a strain is stable, the hybridoma cell strain of efficient secretion ChIL-18 monoclonal antibody;
The two of the purpose of the present invention are to provide the ChIL-18 monoclonal antibody secreted by this hybridoma cell strain;
Described ChIL-18 monoclonal antibody is applied to detection or purification ChIL-18 albumen by the three of the purpose of the present invention.
The above-mentioned purpose of the present invention is achieved through the following technical solutions:
One strain is stable, the hybridoma cell strain CHIL-184E11 of efficient secretion ChIL-18 monoclonal antibody, and its deposit number is: CGMCCNO.7658; Classification And Nomenclature is: ChIL-18 monoclonal antibody hybridoma cell strain; Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; The preservation time is on May 29th, 2013: preservation address: North Star West Road, Chaoyang District, BeiJing, China 1 institute 3, Institute of Microorganism, Academia Sinica.
The present invention is with the recombinant protein c hIL-18 of prokaryotic expression for immunogen, the ChIL-18F albumen that eukaryotic cell lines is expressed is former for screening, hybridoma supernatant is screened with IFA after cell fusion, it it is that positive hybridoma is through 2-3 time cloning by preliminary judgement, it is thus achieved that the cell strain of 8 strains secretion ChIL-18 monoclonal antibodies: 2G7,2G8,3E5,4E11,5D3,1E8,2C4 and 2D11. Present invention IFA detects the cell strain culture supernatant of 8 strain secretion ChIL-18 monoclonal antibodies, measures the titer of this 8 strain positive hybridoma cell strain; 4E11 titer is 1:3200,2G7,2G8,3E5,5D3,1E8, the titer respectively 1:1800 of 2C4 and 2D11,1:1600,1:1900,1:1700,1:2000,1:1400 and 1:1300. From testing result, 4E11 odd contradictive hydroperitoneum titer to exceed far away other 7 strain of hybridoma strain monoclonal antibody titer.
Monoclonal antibody secreted by positive hybridoma cell strain 2G7,2G8,3E5,4E11,5D3,1E8,2C4 and 2D11 is carried out Westernblot with prokaryotic recombinant protein ChIL-18 by the present invention respectively, result of the test finds, the monoclonal antibody of positive hybridoma cell strain 4E11 only reacts with recombinant protein c hIL-18, and do not react with empty carrier thalline, illustrate that the positive hybridoma cell strain 4E11 monoclonal antibody specificity secreted is best; There is reaction with empty carrier thalline in the monoclonal antibody secreted by positive hybridoma cell strain 2G7,2G8,3E5,5D3,1E8,2C4 and 2D11, specificity is poor.
ChIL-18 monoclonal antibody of the present invention has good specificity, the diagnostic reagent of IL-18 albumen in detection or diagnosis chicken body can be prepared into, for example, it is possible to ChIL-18 monoclonal antibody preparation of the present invention to be become the ELISA detection kit of diagnosis or detection IL-18 albumen.
Additionally, ChIL-18 monoclonal antibody of the present invention applies also for purification ChIL-18 albumen; Such as, monoclonal antibody of the present invention is adsorbed on the solid-phase matrix of inertia and prepares into chromatographic column; When sample flows through chromatographic column, antigen to be separated occurs specific binding with the monoclonal antibody of solid phase, and all the other compositions can not be in combination. After abundant for chromatographic column eluting, change ionic strength or the pH of eluent, be intended to the antigen and the antibody dissociation that separate, collect eluent and just can obtain the ChIL-18 albumen of purification.
Accompanying drawing explanation
The qualification result of Fig. 1 monoclonal antibody subclass; 1-8: the respectively supernatant of the cell culture fluid of 2G7,2G8,3E5,4E11,5D3,1E8,2C4 and 2D11.
The IFA reaction result (100 ��) of Figure 24 E11 ascites; A: ascites dilutes IFA result when 50 times;B: ascites dilutes IFA result when 3000 times; C: negative control.
The result of the westernblot of Fig. 3 monoclonal antibody and prokaryotic recombinant protein ChIL-18; 1: prokaryotic recombinant protein ChIL-18; 2: do not induce bacterium; M: protein standard.
IL-18 Protein Detection result in Fig. 4 chicken spleen; 1-5: Different Individual chicken spleen; M: protein molecular weight standard.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and apparent. But these embodiments are only exemplary, the scope of the present invention is not constituted any restriction. It will be understood by those skilled in the art that and the details of technical solution of the present invention and form can be modified or replace lower without departing from the spirit and scope of the present invention, but these amendments and replacement each fall within protection scope of the present invention.
1. biomaterial and reagent
1.1.1 laboratory animal and cell
BALB/c female mice is provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center; Mouse myeloma cell line SP2/0 is preserved by the present inventor's laboratory; The spleen infecting MDV chicken is preserved by the present inventor's laboratory.
1.1.2 main agents
50 �� HAT, 100 �� HT additive are all purchased from GIBCO; Hyclone: BIOCHROM, Berlin; DyLight594 labelling sheep anti-mouse igg is purchased from JacksonImmunoResearch company; IRDyeTM680 labelling mountain sheep anti-mouse iggs are purchased from LI-COR Life Sciences; IgG antibody subclass test kit is purchased from SouthernBiotech company;Tissue lysates is purchased from Thermo scientific & technical corporation; The monoclonal antibody of mouse-anti ��-Actin is purchased from company of Zhong Shan Golden Bridge.
The preparation of embodiment 1 ChIL-18 monoclonal antibody and qualification
1. experimental technique
The immunity of 1.1 mices
(1) preparation SDS-PAGE adhesive tape containing ChIL-18 recombiant protein;
(2) add a small amount of PBS grinding rod before immunity to grind, add appropriate dual anti-solution, can be advisable by 1mL syringe needle with micelle, i.e. immunogen;
(3) 5 6-8 week old cleaning grade BALB/c mouse are chosen, by lumbar injection, it is immune by the immunogen of preparation, every each 300 �� L, every 2w immunity once;
(4) hole blood sampling under third time immunity one week after socket of the eye, then according to conventional method separation serum;
(5) antiserum of immunized mice and CHO-ChIL-18F cell doing IFA checking test, can both detections react, and measures the titer of serum;
(6) choose the highest mice of serum titer and carry out booster immunization, do after 72h and merge.
1.2 cell fusion
1.21 prepare feeder layer cells
Carry out the previous day of cell fusion, prepare feeder layer cells in accordance with the following methods:
(1) taking 8-10 week old BALB/c female mice, extract eyeball and take blood, separate serum as negative control, then cervical dislocation is lethal, with the alcohol-pickled sterilization 5min of 75%;
(2) super-clean bench is moved into, extremity 1mL syringe needle is fixed on sterilized foam fixing plate, the outside of belly is upwards, mouse part skin is cut off an osculum by sterile working, blunt separation skin and peritoneum, draw the 5mL DMEM culture medium containing HAT with 5mL syringe and inject abdominal cavity, rub mouse web portion with cotton ball soaked in alcohol gently, again by the syringe Intraabdominal liquid of slow sucking-off, repeatable operation 2 times;
(3) add about 90mLHAT culture fluid suspension cell gently, be dispensed into 96 porocyte culture plates according to 100 �� L/ holes, put 5%CO2, 37 DEG C of incubators are cultivated.
1.2.2 the preparation of myeloma cell SP2/0
(1) SP2/0 2w recovery before merging, gets out superclean bench, adds the DMEM culture medium containing 10%FBS, 1% glutamine in a minicell bottle;
(2) take out from liquid nitrogen container containing SP2/0 cell cryopreservation tube, put into rapidly in 37 DEG C of water-baths, it is desirable to melt in 1min; With the 75% alcohol disinfecting mouth of pipe, moving in superclean bench, sucking-off cell suspension joins in ready culture bottle, is placed in 37 DEG C, 5%CO2Incubator is cultivated;
(3) after 12h, the DMEM complete medium more renewed.
1.2.3 the preparation (polishing) of splenocyte
(1) selecting the immune mouse that serum antibody titer is higher, eyeball is taken a blood sample, positive control when collecting serum for detection; Cervical dislocation is lethal, puts into 75% alcohol-pickled 5min and carries out body surface sterilization;
(2) mice moving into super-clean bench, extremity 1mL syringe needle is fixed on sterilized foam fixing plate, and the outside of belly is upwards.
(3) mouse part skin is cut off an osculum by work by aseptic behaviour, blunt separation skin and peritoneum, then peritoneum is cut off, expose spleen, spleen is moved into one and is placed with in 10mLDMEM plate, reject connective tissue around, take out spleen, then put it in another plate pouring 10mLDMEM in advance into and clean;
(4) being then transferred on the copper mesh of sterilizing, copper mesh is placed on another one and fills on the plate of sky DMEM; Grind on copper mesh lightly with the piston portion of syringe, copper mesh is immersed in culture fluid and makes splenocyte be rinsed;
(5) being transferred to by splenocyte suspension in 15mL centrifuge tube, 1500r/min is centrifuged 8min.
1.2.4 cell fusion
(1) by SP2/0 good for growth conditions and splenocyte, mix in 50mL conical centrifuge tube according to the ratio of 1:5-1:10, add sky DMEM in certain volume, mix gently;
(2) in the centrifugal 8min of horizontal centrifuge 1500r/min, clean supernatant, flicks bottom centrifuge tube with finger, makes two kinds of loose mixings of cell, is put in the beaker equipped with 37 DEG C of water and is incubated;
(3) in 1min, it is added dropwise over the PEG of 1mL50% along inside pipe wall with pipettor3350, uniform rotation centrifuge tube while adding;
(4) PEG is terminated with 30mLDMEM culture fluid3350Effect, add in 90s, stand 10min;
(5) the centrifugal 8min of 1500r/min. Supernatant is abandoned in suction, adds the DMEM containing HAT, 10%FBS of 50mL37 DEG C of insulation, mixing;
(6) on the cell plates completing feeder cells in advance, every hole adds 100 �� L fused cell suspensions, is placed in 37 DEG C, the CO of 5%2Incubator is cultivated;
(7) after merging 5 days, change HAT culture fluid, adopt the mode partly changing liquid to carry out changing liquid;
(8) it is cultured to about 9 days, observes culture fluid color, if can detect when culture fluid becomes yellow.
1.3 screen the screening of positive hybridoma cell with IFA
(1) CHO-ChIL-18F cell line is gone down to posterity according to a conventional method it is laid on 96 porocyte culture plates, be placed in 37 DEG C, 5%CO2Incubator is cultivated;
(2) when cell monolayer length is to 80%-90%, outwell culture fluid, wash 3 times with PBS, each 5min, with the acetone of room temperature: methanol (1:1) (v/v) room temperature fix 15min, gets rid of fixative, stand-by after unlimited culture plate natural drying;
(3) growing state of fused cell is observed, after its culture fluid turns yellow, draw 100 �� L of supernatant liquid and be added on fixing Chinese hamster ovary celI plate, with immune mouse serum, for positive control, (1:50 dilutes, diluent is PBS), with prepare feeder layer cells mice serum for negative control, with PBS for blank, 37 DEG C of water-bath 1h;PBS washs 3 times, each 5min; Adding 50 �� LDyLight594 labelling sheep anti-mouse iggs is two anti-(l:300 dilutes, and PBS dilutes), and 37 DEG C of water-baths 40min, PBS wash 3 times, each 5min; Red fluorescence is observed under fluorescence inverted microscope.
(4) cell in the hole that selection red fluorescence is the strongest is cloned.
The monoclonal of 1.4 positive hybridoma cells
Adopt limiting dilution assay monoclonal, determine the number of times of clone according to practical situation. Carry out as follows:
(1) feeder layer cells is prepared according to 4.1.5.1 the previous day of cloning;
(2) with pipettor by hole to be cloned inner cell piping and druming mixing, culture fluid is selected to be diluted in each hole by hole inner cell 1 cell with the HT containing 20% serum;
(4) cell diluent is added to according to 100 �� L/ holes the cell plates of existing feeder cells, puts 5%CO2, the incubator of 37 DEG C is cultivated;
(5), when being cultured to the 4th day, under inverted microscope, observe and record cell monoclonal growth hole; It is cultured to about 1w, when cell culture fluid turns yellow, carries out labelling, draw 100 �� L of supernatant, with the IFA method detection cell hole previously established.
(6) treated that culture fluid turned yellow every about 3 days, again initial survey positive hole is done IFA detection;
(7) the hole inner cell that twice detection is strong positive carries out 2-3 sub-clone, until every hole can detect strong red fluorescence;
(8) the strong positive hybridoma of amplification culture frozen cloning.
The Detection of Stability of 1.5 strong positive hybridomies
(1) with elbow straw, the positive hybridoma cell being in exponential phase good for growth conditions is blown down gently from bottle wall;
(2) the centrifugal 5min of 1000r/min, abandons supernatant;
(3) by cell precipitation suspension frozen stock solution (DMEM culture fluid: FBS:DMSO is 5:4:1), by cell subpackage in aseptic cryopreservation tube, 4 DEG C of refrigerator 30min ,-20 DEG C of refrigerator 1h ,-70 DEG C of refrigerator overnight, put into the medium-term and long-term preservation of liquid nitrogen container; Carry out respective record.
(4) recover according to the method for resuscitation of Chinese hamster ovary celI. After cultivation, detect cells and supernatant with IFA, measure titer.
The preparation of 1.5 odd contradictive hydroperitoneums
(1) choosing 10-13 week old BALB/c mouse 4, lumbar injection liquid paraffin 0.5mL/ is only;
After (2) 7 days, intraperitoneal inoculation is through the PBS 4E11 positive hybridoma cell being cultured to logarithmic (log) phase diluted, every mice 5 �� 105/ 0.2mL;
Note the abdominal cavity observing mice after (3) 5 days, when mouse web portion substantially expands, extract ascites with the syringe of 5mL, collected once every 3 days, until dead mouse.
(4) by ascites with 2000r/min, centrifugal 5min; Stay-70 DEG C of Refrigerator stores after supernatant subpackage. 1.6 monoclonal antibody Identification of Biological Characteristics
1.6.1 the qualification of monoclonal antibody subclass
The 8 strain monoclonal antibodies filtered out are carried out subgroup identification by Subclass of antibody identification kit to specifications that utilize SouthernBiotech company:
(1) by ChIL-18 recombiant protein through SDS-PAGE electrophoresis, glue cuts purpose band, puts it in EP pipe and pulverize as much as possible, be subsequently adding appropriate PBS after reclaiming, and 4 DEG C overnight;
(2) next day, centrifugal, 5000r/min is centrifuged 5min; Take supernatant, survey protein concentration;
(3) according to 4ug/mL coated elisa plate, every hole 100 �� L, 37 DEG C are overnight;
(4) next day, getting rid of unconjugated albumen, PBST washs 3 times, each 5min; Every hole adds the 0.5%BSA of 100 �� L and closes, and places 1h for 37 DEG C;
(5) in the ELISA Plate closed, 2G7,2G8,3E5,4E11,5D3,1E8,2C4 and 2D11 cell culture supernatant it are separately added into, 100 �� L/ holes, hatch 1h for 37 DEG C; PBST washs 3 times, each 5min;
(6) the sheep anti mouse detection two that is sequentially added into HRP labelling with PBS1:250 dilution in ELISA Plate anti-(respectively against murine ��, ��, IgM, IgA, IgG1��IgG2a��IgG2bAnd IgG3, 100 �� L/ holes, place 1h, PBST for 37 DEG C and wash 3 times, each 5min;
(7) add the colour developing of ABTS substrate, 100 �� L/ holes, hatch 10-15min;
(8) by microplate reader at 405nm wavelength place reading light absorption value (OD value).
1.6.2 the reactivity of the westernblot of monoclonal antibody and prokaryotic recombinant protein ChIL-18
Respectively by prokaryotic recombinant protein ChIL-18 and the RT-PCR bacterium do not induced after SDS-PAGE electrophoresis, it is transferred to pvdf membrane, with respectively with the cells and supernatant of 8 strain monoclonal antibodies that filters out for primary antibodie, with IRDyeTM700DX labelling goat against murine IgG(1:5000 dilute) be two resist.
1.6.3 the mensuration of odd contradictive hydroperitoneum IFA titer
By ascites PBS according to following dilution proportion, l:50, l:100, l:200, l:400, l:600, l:800, l:1000, l:1200, l:1500, l:2000,1:3000,1:4000,1:5000 measure IFA titer. 1.6.4 the IL-18 albumen in monoclonal antibody detection MDV infected chicken spleen is utilized
(1) chicken spleen utilizing T-PER Tissue lysates 5 Different Individual of cracking extracts albumen, liquid nitrogen grinding spleen, weighs the 0.1g spleen powder ground, adds 0.5mLT-PER lysate;
(2) 2h, 10000g are cracked on ice, centrifugal 5min, take supernatant;
(3) add 5 �� SDS lysate and boil 10min; Albumen is carried out SDS-PAGE electrophoresis, loading volume 28 �� L;
(4) after electrophoresis terminates, conventionally carrying out transferring film, wherein primary antibodie is cell culture supernatant and the mouse-anti ��-Actin monoclonal antibody (1:1000 dilution) of 4E11 positive hybridoma cell strain, and two resist for IRDyeTM700DX labelling goat against murine IgG(1:4000 dilutes). Concrete operation step is with reference to 3.1.9.3.
2. experimental result
The screening of 2.1ChIL-18 monoclonal antibody
With the recombinant protein c hIL-18 of prokaryotic expression for immunogen, the ChIL-18F albumen that eukaryotic cell lines is expressed is former for screening, screening hybridoma supernatant is carried out with IFA after cell fusion, preliminary judgement is that the hybridoma cell clone of the positive is through 2-3 time cloning, it is thus achieved that the cell strain of 8 strains secretion ChIL-18 monoclonal antibodies. Wherein 2G7,2G8,3E5,4E11 and 5D3 have passed through 2 sub-clones, and 1E8,2C4 and 2D11 have passed through 3 sub-clones.
The subgroup identification result of 2.2 monoclonal antibodies
The immunoglobulin standard subgroup identification test kit utilizing SouthernBiotech company carries out the 2G7 obtained, 2G8,3E5,4E11,5D3, this 8 strain monoclonal antibody of 1E8,2C4 and 2D11 are carried out subgroup identification, its heavy chain respectively IgM, light chain is Kappa chain (Fig. 1).
The Detection of Stability of 2.3 strong positive hybridomies
Detection of Stability is it was found that the stability of strong positive hybridoma 4E11 secretion ChIL-18 monoclonal antibody is best; 2G7,2G8,3E5,5D3,1E8,2C4 and 2D11 less stable.
The measurement result of 2.4 odd contradictive hydroperitoneum IFA titers
With PBS, as primary antibodie, ascites dilution being carried out IFA reaction, 4E11 titer is 1:3200(Fig. 2). 2G7,2G8,3E5,5D3,1E8, the titer respectively 1:1800 of 2C4 and 2D11,1:1600,1:1900,1:1700,1:2000,1:1400 and 1:1300.From bioactivity result, 4E11 odd contradictive hydroperitoneum titer to exceed far away other 7 strain of hybridoma strain monoclonal antibody titer.
The result of the westernblot of 2.5 monoclonal antibodies and prokaryotic recombinant protein ChIL-18
Respectively by prokaryotic recombinant protein ChIL-18 and the RT-PCR bacterium do not induced after SDS-PAGE electrophoresis, it is transferred to pvdf membrane, with respectively with the cells and supernatant of 8 strain monoclonal antibodies that filters out for primary antibodie, with IRDyeTM700DX labelling mountain sheep anti-mouse igg be two resist.
Fig. 3 is the westernblot result that cells and supernatant is primary antibodie of 4E11 positive hybridoma cell strain, it was shown that monoclonal antibody only reacts with recombinant protein c hIL-18, and does not react with the recombinant bacterium do not induced, and illustrates that the specificity of monoclonal antibody is fine. 2G7,2G8,3E5,5D3,1E8,2C4 and 2D11 monoclonal antibody except reacting with recombinant protein c hIL-18, also react with empty carrier thalline, illustrate that specificity is poor.
2.6 utilize monoclonal antibody detection to infect the IL-18 albumen result in MDV chicken spleen
Infecting MDV chicken spleen with the cracking of T-PER Tissue lysates and extract albumen, utilizing 4E11 strain cell culture supernatant and mouse-anti ��-Actin monoclonal antibody (1:1000 dilution) is primary antibodie, with IRDyeTM700DX labelling goat against murine IgG(1:4000 dilutes) it is two anti-carry out westernblot reaction, 5 Different Individual chicken spleens all detect the ��-Actin albumen of the 43kDa as internal reference, 4 Different Individual chicken spleens all can detect the albumen of a 28kDa size wherein, wherein in the spleen of a chicken, band is more weak, but the albumen detected is all compared with natural ChIL-18 protein 24 kDa slightly larger (Fig. 4).
Claims (8)
1. the hybridoma cell strain 4E11 of a strain stably excreting ChIL-18 monoclonal antibody, it is characterised in that its microbial preservation number is: CGMCCNO.7658.
2. the monoclonal antibody of hybridoma cell strain 4E11 secretion described in claim 1.
3. the purposes in the IL-18 protein reagent in preparation detection or diagnosis chicken body of the hybridoma cell strain 4E11 described in claim 1.
4. the purposes in preparing purification ChIL-18 protein reagent of the hybridoma cell strain 4E11 described in claim 1.
5. the purposes in the IL-18 protein reagent in preparation detection or diagnosis chicken body of the monoclonal antibody described in claim 2.
6. the purposes in preparing purification ChIL-18 protein reagent of the monoclonal antibody described in claim 2.
7. the purposes described in claim 4 or 6, it is characterised in that including: the monoclonal antibody that claim 1 hybridoma cell strain 4E11 secretes is adsorbed on the solid-phase matrix of inertia and prepares into chromatographic column; When sample flows through chromatographic column, antigen to be separated occurs specific binding with the monoclonal antibody of solid phase, and all the other compositions can not be in combination; After abundant for chromatographic column eluting, change ionic strength or the pH of eluent, be intended to the antigen and the antibody dissociation that separate, collect eluent and just obtain the ChIL-18 albumen of purification.
8. the ELISA detection kit of a diagnosis or detection ChIL-18 albumen, it is characterised in that: containing the monoclonal antibody of hybridoma cell strain 4E11 secretion described in claim 1.
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| 检测马白细胞介素-18双抗体夹心ELISA方法的建立;童铁钢等;《细胞与分子免疫学杂志》;20081231;第24卷(第4期);364-365,368 * |
| 稳定表达鸡IL-18蛋白细胞系的建立及鸡IL-18单克隆抗体的制备;李行;《中国优秀硕士学位论文全文数据库农业科技辑》;20121015(第10期);D050-58 * |
| 重组马白细胞介素-18生物学活性的测定及单克隆抗体中和活性鉴定;童铁钢等;《农业生物技术学报》;20091231;第17卷(第3期);370-374 * |
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