狂犬病毒胶体金试纸条的制备和杂交瘤细胞株的免疫加强
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摘要
狂犬病是一种常见的人兽共患病,是由于狂犬病毒感染造成的。狂犬病具有很强的潜伏性,很多携带狂犬病毒的动物并不表现出患病症状,这对狂犬病的及早发现和预防造成了非常大的困难。本实验旨在制备方便快捷并且具有高效性特异性的狂犬病毒胶体金免疫检测试纸条,使检测狂犬病的潜在病源更加方便,从而使狂犬病从传染源上得到更好的控制。本论文的前半部分就对胶体金试纸条的研制进行了初步的研究和探索。
在制备单克隆抗体杂交瘤时,常常因为培养条件的改变、传代次数的增加以及冻存和解冻等对杂交瘤造成一定的影响,使杂交瘤分泌抗体的能力减弱甚至消失,而只能重新制备杂交瘤或者选取其他的杂交瘤,这就对实验周期和实验的一致性造成了影响。而通过体外致敏淋巴细胞可以快速获得能够分泌抗体的免疫细胞,如果能够将不能分泌抗体的杂交瘤通过体外免疫手段重新刺激,使其能够重新获得或者提高分泌抗体的能力,那么将大大缩短实验周期、提高实验的效率,本论文的后半部分就对杂交瘤进行体外致敏的方法进行了初步的研究。
单杂交瘤的选择和抗体纯化
本试验首先通过有限稀释ELISA和夹心ELISA法分别测定了7株本实验室制备的单克隆抗体杂交瘤所产生的单抗的相对亲和力和识别抗原表位,并发现杂交瘤17E3所分泌的单克隆抗体有最强的结合力,而杂交瘤17F3与17B2、17E3以及17E4识别的抗原表位最远,17E3与17B2、17E4所识别的抗原表位最近。然后选择17E3、17B2和17F3三株杂交瘤,对石蜡油处理的Balb／c小鼠进行腹腔注射,制备了具有免疫效价的腹水,并经过辛酸-硫酸铵法对腹水进行了IgG亚型抗体的纯化,分别得到了浓度为1.88mg/mL的17E3单克隆抗体和浓度为1.51mg／mL的混合抗体(17B2和17F3)；经SDS-PAGE检测发现,在50kD和25KD处有明显的条带出现,说明纯化得到的蛋白是IgG型抗体,并且具有较高的纯度,能够满足进一步试验的要求。二胶体金的制备
采用柠檬酸三钠还原法制备颗粒大小为20-30nm的胶体金。制备得到的胶体金呈现酒红色,并且在4℃和室温下存放一个月都未发生凝集沉淀现象,说明胶体金的金颗粒大小符合要求,在20-30nm之间,并且具有良好的稳定性。
三胶体金包被抗体
将制备的单克隆抗体用胶体金包被,用于胶体金试纸条结合垫的制备。首先需要确定稳定胶体金的最小蛋白浓度。通过实验得出每200μL的胶体金加入4μL的4倍稀释纯化17E3单克隆抗体IgG,能使其在添加40gL 10%的NaCl作用下保持稳定,不发生凝集,所以在包被胶体金的时候应选择最小稳定量的120%,即每毫升24gL的4倍稀释17E3单克隆抗体。
胶体金标记抗体的稳定性检测发现,在4℃保存1周以上的金标抗体未发生凝聚,说明制备的金标抗体稳定性良好。
四金标抗体试纸条的制作
金标抗体试纸条包括滴加样品的样品垫、样品与金标抗体发生抗原抗体反应的结合垫、用于检测样品是否带有抗原的检测垫以及吸水垫四部分构成。实验中首先对制作样品垫、结合垫以及检测垫的材料进行了筛选,Ahlstrom 8964玻璃纤维板对金标抗体的干燥最适合,能够结合大量的胶体金,而且凝固后颜色较为均匀,可以保证在层析时金颗粒在硝酸纤维素膜上均匀扩散。对于检测垫,选择层析速度适中的Millipore 135、Immunopore FP或者Prima 40。
其次,经过筛选发现,以pH9.0含0.2% Tween-20 Tris-HCl缓冲液包被样品垫是最理想的,将胶体金以1：2比例稀释后包被结合垫能够产生可见的沉淀线和较轻的背景色。混合抗体包被检测线可以在阳性测试中显示出沉淀线,而在阴性测试中不产生沉淀线,但是质控线无论在阳性测试还是阴性测试中都不会产生沉淀,这可能是由于包被质控线所使用的羊抗鼠IgG的浓度不足或者效价不高造成的。
五杂交瘤的体外致敏
试验选取分泌单克隆抗体能力减弱的杂交瘤作为实验对象,通过添加胸腺细胞条件培养液,使杂交瘤、杂交瘤+脾细胞、脾细胞分别与不同梯度的狂犬病疫苗抗原作用4天,用ELISA方法对产生细胞培养上清液进行检测,观察是否出现抗体分泌的增加。结果显示无论是杂交瘤单独与抗原作用,还是杂交瘤+脾细胞与抗原作用,还是脾细胞与抗原相作用,都不能获得有效的抗体增加。这可能是因为：1杂交瘤不具有被再次致敏的潜质；2试验中的体外致敏体系不具备使杂交瘤获得免疫的条件。这些还需要进一步的实验来确定。
The rabies is a common amphixenosis caused by rabies virus. It has strong latent ability, many animals carried these virus would not express the symptoms, and this causes an obstacle at the detection and precaution. This experiment aimed at preparing a convenient and specific colloidal gold test paper for rabies virus, in order to making the detection more comfortable and taking the controls at the sources of infection. Some preliminary work on the preparation of colloidal gold test paper has been done at the first half of this paper.
Because of the culture tradition, increase of passages and freezing and defrosting, the antibody secretion of some hybridoma would be weaken, even lost. The methods to making it up would be replacing with new hybridoma. This would cause the extending of experiments duration and discontinuation. However, immune cells with antibody secretion ability could be obtained through in vitro immune. If it is possible that making the hybridoma reacquiring the secretion ability that restimulating the one lost through in vitro immune, the duration will be shorten greatly. Some basic research was placed at the last half of this paper.
1 The selection of hybridoma and antibody purification
The affinity and epitopes of seven hybridomas were tested through ELISA, the results shows that the hybridoma 17E3 has the strongest affinity and longest distances are placed between 17F3 and 17B2,17E3,17E4, the shortest distances are placed between 17E3 and 17B2,17E4. Ascites with immunopotency has been prepared throughout injection of hybridoma 17E3,17B2 and 17F3 to matured Balb/c mice treated by wax, and purifying the IgG antibodies with octanoic acid-ammonium sulfate method. The concentrations of 17E3 monoclonal antibody and mixed antibody (17B2 and 17F3) are 1.88mg/mL and 1.51mg/mL respectively. There are two lines appeared at the places of 50kD and 25kD respectively. This indicates that the protein purified is antibodies, and they are high-purity, meeting the requirements of experiments subsequently.
2 Preparation of colloidal gold
Sodium citrate reduction method has been taken to preparing the colloidal gold with 20-30nm particles. The colloidal gold obtained is wine red, and has not precipitated within a month at 4℃. This indicates that the diameters of gold particle are between 20nm and 30nm, the colloidal gold are stable.
3 Coating antibodies with colloidal gold
Coat the antibodies with colloidal gold to preparing the combining pad of test paper. The minimum concentration of monoclonal antibody to stabilizing the colloid gold must be found. 200μL colloidal gold with 4μL 17E3 antibodies diluted 4 folds would not precipitated after 40μL 10%NaCl was added. And it could be conclude that 24μL 17E3 antibodies diluted 4 folds should be added to 1 mL colloidal gold.
4 The preparation of colloidal gold test paper
There are 4 parts of colloidal gold test paper:the sample pad, combining pad, test pad and absorbent pad. The materials for sample pad, combining pad and test pad were selected, the Ahlstrom 8964 glass fiber is most suitable to coat the colloidal gold, and Millipore 135, Immunopore FP and Prima 40 are suitable to be the test pad.
The Tris-HCl buffer which contained 0.2% Tween-20 at pH9.0 is the ideal buffer to coat the sample pad, the combining pad will be most perfect with 1:2 diluted colloidal gold coated. There are still some defects on these test paper that the test line show light color in the positive tests and the control line show no color either in the positive or negative tests. What makes this happen may be the goat-anti-mouse IgG which is used to coated the control line has low concentration or pure titer.
5 The in vitro immune of hybridoma
Three different systems were taken at this experiment:hybridomas, hybridomas+spleen cells and spleen cells were cultured with gradient antigens for 4 days respectively. Thymus cells-conditioned medium was added to the system of in vitro immune. And ELISA was taken to test whether it happened that antibodies increasing or not. The results indicated that these systems could not make the antibodies secretion improving, and this would cause by:1 the hybridomas cannot be resensitized; 2 the in vitro immune systems cannot meet the requirements. After all, the reasons must be proofed with additional work.
在制备单克隆抗体杂交瘤时,常常因为培养条件的改变、传代次数的增加以及冻存和解冻等对杂交瘤造成一定的影响,使杂交瘤分泌抗体的能力减弱甚至消失,而只能重新制备杂交瘤或者选取其他的杂交瘤,这就对实验周期和实验的一致性造成了影响。而通过体外致敏淋巴细胞可以快速获得能够分泌抗体的免疫细胞,如果能够将不能分泌抗体的杂交瘤通过体外免疫手段重新刺激,使其能够重新获得或者提高分泌抗体的能力,那么将大大缩短实验周期、提高实验的效率,本论文的后半部分就对杂交瘤进行体外致敏的方法进行了初步的研究。
单杂交瘤的选择和抗体纯化
本试验首先通过有限稀释ELISA和夹心ELISA法分别测定了7株本实验室制备的单克隆抗体杂交瘤所产生的单抗的相对亲和力和识别抗原表位,并发现杂交瘤17E3所分泌的单克隆抗体有最强的结合力,而杂交瘤17F3与17B2、17E3以及17E4识别的抗原表位最远,17E3与17B2、17E4所识别的抗原表位最近。然后选择17E3、17B2和17F3三株杂交瘤,对石蜡油处理的Balb／c小鼠进行腹腔注射,制备了具有免疫效价的腹水,并经过辛酸-硫酸铵法对腹水进行了IgG亚型抗体的纯化,分别得到了浓度为1.88mg/mL的17E3单克隆抗体和浓度为1.51mg／mL的混合抗体(17B2和17F3)；经SDS-PAGE检测发现,在50kD和25KD处有明显的条带出现,说明纯化得到的蛋白是IgG型抗体,并且具有较高的纯度,能够满足进一步试验的要求。二胶体金的制备
采用柠檬酸三钠还原法制备颗粒大小为20-30nm的胶体金。制备得到的胶体金呈现酒红色,并且在4℃和室温下存放一个月都未发生凝集沉淀现象,说明胶体金的金颗粒大小符合要求,在20-30nm之间,并且具有良好的稳定性。
三胶体金包被抗体
将制备的单克隆抗体用胶体金包被,用于胶体金试纸条结合垫的制备。首先需要确定稳定胶体金的最小蛋白浓度。通过实验得出每200μL的胶体金加入4μL的4倍稀释纯化17E3单克隆抗体IgG,能使其在添加40gL 10%的NaCl作用下保持稳定,不发生凝集,所以在包被胶体金的时候应选择最小稳定量的120%,即每毫升24gL的4倍稀释17E3单克隆抗体。
胶体金标记抗体的稳定性检测发现,在4℃保存1周以上的金标抗体未发生凝聚,说明制备的金标抗体稳定性良好。
四金标抗体试纸条的制作
金标抗体试纸条包括滴加样品的样品垫、样品与金标抗体发生抗原抗体反应的结合垫、用于检测样品是否带有抗原的检测垫以及吸水垫四部分构成。实验中首先对制作样品垫、结合垫以及检测垫的材料进行了筛选,Ahlstrom 8964玻璃纤维板对金标抗体的干燥最适合,能够结合大量的胶体金,而且凝固后颜色较为均匀,可以保证在层析时金颗粒在硝酸纤维素膜上均匀扩散。对于检测垫,选择层析速度适中的Millipore 135、Immunopore FP或者Prima 40。
其次,经过筛选发现,以pH9.0含0.2% Tween-20 Tris-HCl缓冲液包被样品垫是最理想的,将胶体金以1：2比例稀释后包被结合垫能够产生可见的沉淀线和较轻的背景色。混合抗体包被检测线可以在阳性测试中显示出沉淀线,而在阴性测试中不产生沉淀线,但是质控线无论在阳性测试还是阴性测试中都不会产生沉淀,这可能是由于包被质控线所使用的羊抗鼠IgG的浓度不足或者效价不高造成的。
五杂交瘤的体外致敏
试验选取分泌单克隆抗体能力减弱的杂交瘤作为实验对象,通过添加胸腺细胞条件培养液,使杂交瘤、杂交瘤+脾细胞、脾细胞分别与不同梯度的狂犬病疫苗抗原作用4天,用ELISA方法对产生细胞培养上清液进行检测,观察是否出现抗体分泌的增加。结果显示无论是杂交瘤单独与抗原作用,还是杂交瘤+脾细胞与抗原作用,还是脾细胞与抗原相作用,都不能获得有效的抗体增加。这可能是因为：1杂交瘤不具有被再次致敏的潜质；2试验中的体外致敏体系不具备使杂交瘤获得免疫的条件。这些还需要进一步的实验来确定。
The rabies is a common amphixenosis caused by rabies virus. It has strong latent ability, many animals carried these virus would not express the symptoms, and this causes an obstacle at the detection and precaution. This experiment aimed at preparing a convenient and specific colloidal gold test paper for rabies virus, in order to making the detection more comfortable and taking the controls at the sources of infection. Some preliminary work on the preparation of colloidal gold test paper has been done at the first half of this paper.
Because of the culture tradition, increase of passages and freezing and defrosting, the antibody secretion of some hybridoma would be weaken, even lost. The methods to making it up would be replacing with new hybridoma. This would cause the extending of experiments duration and discontinuation. However, immune cells with antibody secretion ability could be obtained through in vitro immune. If it is possible that making the hybridoma reacquiring the secretion ability that restimulating the one lost through in vitro immune, the duration will be shorten greatly. Some basic research was placed at the last half of this paper.
1 The selection of hybridoma and antibody purification
The affinity and epitopes of seven hybridomas were tested through ELISA, the results shows that the hybridoma 17E3 has the strongest affinity and longest distances are placed between 17F3 and 17B2,17E3,17E4, the shortest distances are placed between 17E3 and 17B2,17E4. Ascites with immunopotency has been prepared throughout injection of hybridoma 17E3,17B2 and 17F3 to matured Balb/c mice treated by wax, and purifying the IgG antibodies with octanoic acid-ammonium sulfate method. The concentrations of 17E3 monoclonal antibody and mixed antibody (17B2 and 17F3) are 1.88mg/mL and 1.51mg/mL respectively. There are two lines appeared at the places of 50kD and 25kD respectively. This indicates that the protein purified is antibodies, and they are high-purity, meeting the requirements of experiments subsequently.
2 Preparation of colloidal gold
Sodium citrate reduction method has been taken to preparing the colloidal gold with 20-30nm particles. The colloidal gold obtained is wine red, and has not precipitated within a month at 4℃. This indicates that the diameters of gold particle are between 20nm and 30nm, the colloidal gold are stable.
3 Coating antibodies with colloidal gold
Coat the antibodies with colloidal gold to preparing the combining pad of test paper. The minimum concentration of monoclonal antibody to stabilizing the colloid gold must be found. 200μL colloidal gold with 4μL 17E3 antibodies diluted 4 folds would not precipitated after 40μL 10%NaCl was added. And it could be conclude that 24μL 17E3 antibodies diluted 4 folds should be added to 1 mL colloidal gold.
4 The preparation of colloidal gold test paper
There are 4 parts of colloidal gold test paper:the sample pad, combining pad, test pad and absorbent pad. The materials for sample pad, combining pad and test pad were selected, the Ahlstrom 8964 glass fiber is most suitable to coat the colloidal gold, and Millipore 135, Immunopore FP and Prima 40 are suitable to be the test pad.
The Tris-HCl buffer which contained 0.2% Tween-20 at pH9.0 is the ideal buffer to coat the sample pad, the combining pad will be most perfect with 1:2 diluted colloidal gold coated. There are still some defects on these test paper that the test line show light color in the positive tests and the control line show no color either in the positive or negative tests. What makes this happen may be the goat-anti-mouse IgG which is used to coated the control line has low concentration or pure titer.
5 The in vitro immune of hybridoma
Three different systems were taken at this experiment:hybridomas, hybridomas+spleen cells and spleen cells were cultured with gradient antigens for 4 days respectively. Thymus cells-conditioned medium was added to the system of in vitro immune. And ELISA was taken to test whether it happened that antibodies increasing or not. The results indicated that these systems could not make the antibodies secretion improving, and this would cause by:1 the hybridomas cannot be resensitized; 2 the in vitro immune systems cannot meet the requirements. After all, the reasons must be proofed with additional work.
引文
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[1]沈关心,周汝麟。现代免疫学实验技术[M]；武汉：湖北科学技术出版社,1998；
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[11]Yeon Suk Jung,Shin-ei Matsumoto, et al., propionibacterium acnes acts as an adjuvant in in vitro immunization of human peripheral blood mononuclear cells [J], Biosci. Biotechnol. Biochem.2007; 71(8),1963-1969
[12]Roman M, Martin-Orozco E, Goodman JS, et al. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants [J]. Nature Med.1998; 3(8):849-854.
[13]温建新,李俊,周顺等,不同序列的CpG ODN对猪体外免疫细胞免疫刺激作用的研究[J],西北农业学报,2007,16(4)：71-75
[1]Kohler G, Milsten C. Continuous cultures of fused cells secreting antibody of Predefined specificity [J]. Nature,1975; 256:495-497
[2]Olsson L. and Kaplan HS., Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity [J]. PNAS,1980; 77(9):5429-5431
[3]Samoilovich SR, Dugan CB, Macario AJ.Hybridoma technology:new developments of practical interest [J]. Immunol Methods 1987;101:153-170
[4]Knudsen KA. Proteins transferred to nitrocellulose for use as immunogens [J].Anal Biochem 1985;147(2):285-288
[5]Ho MK., Rand N. Murray J. et al. In vitro immunization of human lymphocytes. I. Production of human monoclonal antibodies against bombesin and tetanus toxoid [J]. J Immunol 1985; 135 (6):3831-3838
[6]Sheng H. Z., Hoogenraad J., Carnegie P. R., et al. Use of Protein-bearing Nitrocellulose as Immunogen for in vitro Production of Monodonal Antibodies:Application to Myelin Basic Protein Electrophoretically Separated from a Complex Brain Protein Mixture [J]. Immunology Lettes.1987; 16:75-81;
[7]Dinjis W. N. M.,Van der Linden E. P. S.,Signet C. M.,et al.,Solid-phase Adsorption of for Efficient Production of Antibodies Reactive with Native and Fixed Tissue Antigens [J]. J Immunol Methods.1990; 126:175-182;
[8]Larsson A. Nilsso BO., Immunization with Nanogram Quantities of Nitrocellose-bound Antigen, Electroblotted from Sodium Dedecyl Sulphate-Polyacrylamide Gels [J]. Scand J Imrnunol.1988;27:305-309
[9]Guzman J., Schoedon G., Blau N., In Vitro Immunization with Antigen Directly Blotted from SDS-polyacrylamide Gels to Polyvinylidene Difluoride Membranes [J]. J Immunol Methods. 1993; 158:37-47
[10]Geeta NS., Ramanadham M., In Vitro Immunization of Murine Lymphocytes Using Immobilized Immunogens [J]. Biotech. Appl. Biochem.1996;24:61-64
[11]Sheng HZ, Hoogenraad J, Carngie PR,et al. Use of Protein-bearing Nitrocellulose as Immunogen for in vitro Production of Monoclonal Antibodies:Applifacion to Myelin Basic Protein Electrophoretically Separated from a Complex Brain Protein Mixture [J]. Immunology Letters.1987;16:75-81
[12]Gathuru J. K., Miyoshi I., Naiki M., In Vitro Immunization of Rat Spleen Lymphocytes with Forssman Glycosphingolipid Antigen and the Generation of a Monoclonal Antibody [J]. J Immunol Methods.1991;137:95-102
[13]Yarchoan R, Murphy BR, Strober W, et al.; Specific anti-influenza virus antibody production in vitro by human peripheral blood mononuclear cells [J]. J Immunol,1981;127(6):2588-2594;
[14]Borrbaeck CAK., In Vitro Immunization for Production of Murine and Human Monoclonal Antibodies:Present Status [J]. Trends Biotechnol.1986;4:147～153
[15]Steinman RM, Colin ZA. Identification of novel cell type in peripheral lymphoid organs of mice:Morphology, quantification, tissue distribution. [J]. J Exp. Med.1973; 137:1142-1162
[16]Liu L., Rich BE., Inobe J., et al. Induction of Th2 cell differentiation in the primary immune response:dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4F T cells to secrete IL-4 [J]. Int. Immunol.1998;10(8):1017-1026
[17]Gramaglia I.,Weinberg AD.,Lemon M. et al. Ox-40 ligand:a potent costimulatory molecule for sustaining primary CD40 T cell response [J].J Immunol,1998; 161(12):6510-6517.
[18]Tanaka H, Demeure C E, Rubio M. et al. Human monocyte-derived dendritic cells induce naive T cell differentiation into T helper cell type 2(Th2) or Th1/Th2 effectors, role of stimulator/responder ratio [J]. J Exp Med.2000;192 (3):405-412.
[19]Hochrein, Keeffe HMO, Luft T. et al. Interleukin(IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells [J]. J Exp Med.2000; 192:823-834.
[20]Tesciuba AG, Subudhi S, Rother RP. et al. Induciable costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease [J]. J Immunol. 2001; 167(4):97-101.
[21]Wang S D, Zhu GF, Chapoval A A. et al. Costimulation of T cells by B7H2, a B7-like molecule that binds ICOS [J]. Blood.2000; 96:2808-2813.
[22]Liang L, Porter EM., Sha WC. Constitutive expression of B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling [J]. J Exp Med. 2002; 196 (1):97-108.
[23]Saman K., Andreas H.,Kerstin B. et a. ICOS2-ligand, expressed on human endothelial cells, costimulates Th1 and Th2 cytokine secretion by memory CD4+T cells [J]. Molecular Immunology,2002; 99(9):6198-6203
[24]Paul, WE. Pleiotrophy and redundancy:T cell-derived lymphokines in the immune response [J]. Cell.1989.57:521.
[25]Howard, M., J. Farrar, M. Hilfiker, B. et al. Identification of a T cell derived B cell growth factor distinct from interleukin 2 [J].J, Exp. Med.1982; 155:914.
[26]Vitetta, ES., Ohara J., Myers C., et al., Serologic, biochemical and functional identity of B cell stimulatory factor-1 and B cell differentiation factor for IgG [J]. J. Exp Med.1985; 162:1726.
[27]Coffman RL. and Carty JA. T cell activity that enhances polyclonal IgE production and its inhibition by interferony [J]. J. Immunol.1986; 136:949.
[28]Coffman RL., Ohara J., Bond MW.,et al. B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells [J].J. Immunol.1986;136(10):4538.
[29]Mosmann TR. and Coffman RL.. TH1 and TH2 cells:different patterns of lymphokine secretion lead to different functional properties [J]. Annu. Rev. Immunol.1989; 7:145.
[30]Swain SL., McKenzie DT., Weinberg AD., et al. Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells [J]. J. Immunol.1988;141:3445.
[31]Powers GD., Abbas AK. and Miller RA. Frequencies of IL-2 and IL-4-secreting T cells in naive and antigen stimulated lymphocyte populations [J]. J. Immunol.1988:140:3352.
[32]Ben-Sasson SZ., Le Gros G., Conrad DH., et al. IL-4 production by T cells from naive donors. IL-2 is required for IL-4 production [J].J. Immunol.1990;145(4):1127-1136,
[33]Graham Le Gros, Shlomo Z., Ben-Sasson, et al. Generation of Interleukin 4 (IL4)-producing Cells In Vivo and In Vitro:IL-2 and IL-4 Are Required For In Vitro Generation of IL-4 producing Cells [J]. J. Exp. Med.,1990; 172:921-929
[34]Sean Diehl, Mercedes Rincon. The two faces of IL-6 on Th1/Th2 differentiation [J]. Mol. Immuno.2002; 19:531-536
[35]Choe J and Choi YS., IL-10 interrupts memory B cell expansion in the germinal center by inducing differentiation into plasma cells [J]. Eur.J.Immunol.1998; 28:508-515;
[36]Kindler V. and Zubler RH., Memory, but not naive, peripheral blood B lymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimulation with IL-4 and the differentiation factors IL-2, IL-10, and IL-3 [J]. J. Immunol.1997; 159:2085-2090
[37]Kobayashi N., Nagumo H. and Agematsu K., IL-10 enhances B-cell IgE synthesis by promoting differentiation into plasma cells, a process that is inhibited by CD27/CD70 interaction [J]. Clin.Exp.Immunol.2002;129:446-452
[38]Qianghua Xu,Yoshinori K. et al., IL-10 Augments Antibody Production in in Vitro Immunized Lymphocytes by Inducing a Th2-Type Response and B Cell maturation [J], Biosci. Biotechnol. Biochem.,68 (11),2279-2284,2004
[39]Leyden JJ., McGinley KJ., and Vowels B., Propionibacterium acnes colonization in acne and nonacne [J]. Dermatology,1998;196:55-58
[40]Green S., Dobrjansky A., Chiasson MA.,et al., Corynebacterium parvum as the priming agent in the production of tumor necrosis factor in the mouse [J]. J. Natl. Cancer Inst. 1977;59:1519-1522
[41]Halpern BN., Biozzi G., Stiffel C., et al., Inhibition of tumour growth by administration of killed Corynebacterium parvum [J]. Nature,1966;212:853-854
[42]Woodruff M.F., McBride WH., and Dunbar N., Tumour growth, phagocytic activity and antibody response in Corynebacterium parvum-treated mice[J]. Clin. Exp. Immunol.1974; 17: 509-518
[43]Warr GW., and James K., Effect of Corynebacterium parvum on the class and subclass of antibody produced in the response of different strains of mice to sheep erythrocytes [J]. Immunology,1975;28:431-442
[44]Abath FG., Coutinho EM., Montenegro SM., et al., The use of nonspecific immunopotentiators in experimental Trypanosoma cruzi infection [J]. Trans. R. Soc. Trop. Med. Hyg., 1988;82:73-76
[45]Hill JO., Modulation of the pattern of development of experimental disseminated leishmaniasis by Corynebacterium parvum[J]. J. Leukoc. Biol.,1987;41:165-169
[46]Smith SR., Calzetta AB.et al., Lipopolysaccharide-induced cytokine production and mortality in mice treated with Corynebacterium parvum[J]. J. Leukoc. Biol.,1993;54:23-29
[47]Tsuji H., Mukaida N., Harada A., Kaneko S.,et al., Alleviation of lipopolysaccharide-induced
acute liver injury in Propionibacterium acnes-primed IFN-gamma-deficient mice by a concomitant reduction of TNF-alpha, IL-12, and IL-18 production [J]. J. Immunol.1999; 162: 1049-1055
[48]Yeon Suk Jung, Shin-ei Matsumoto, et al.,propionibacterium acnes acts as an adjuvant in in vitro immunization of human peripheral blood mononuclear cells [J], Biosci. Biotechnol. Biochem.,2007;71(8),1963-1969
[49]Roman M, Martin-Orozco E, Goodman JS, Nguyen M-D, Sato Y, Ronaghy A, Kornbluth RS, Richman DD, Carson DA, Raz E. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nature Med 1998;3(8):849-54.
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[1]Kohler G and Milsten C. Continuous cultures of fused cells secreting antibody of Predefined specificity [J]. Nature.1975; 256:495-497
[2]沈关心,周汝麟。现代免疫学实验技术[M],武汉：湖北科学技术出版社,1998.10
[3]Thiele DL, Kurosaka M and Lipsky PE. Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood:delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester [J]. The Journal of Immunology. 1983; 131 (5):2282-2290
[4]Paul, WE. Pleiotrophy and redundancy:T cell-derived lymphokines in the immune response [J]. Cell.1989.57:521.
[5]Howard, M., J. Farrar, M. Hilfiker, B. et al. Identification of a T cell derived B cell growth
factor distinct from interleukin 2 [J].J. Exp. Med.1982; 155:914.
[6]Vitetta, ES., Ohara J., Myers C., et al., Serologic, biochemical and functional identity of B cell stimulatory factor-1 and B cell differentiation factor for IgG [J]. J. Exp Med.1985; 162:1726.
[7]Coffman RL. and Carty JA. T cell activity that enhances polyclonal IgE production and its inhibition by interferony [J]. J. Immunol.1986; 136:949.
[8]Coffman RL., Ohara J., Bond MW.,et al. B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells [J].J. Immunol.1986;136(10):4538.
[9]Mosmann TR. and Coffman RL.. TH1 and TH2 cells:different patterns of lymphokine secretion lead to different functional properties [J]. Annu. Rev. Immunol.1989; 7:145.
[10]Choe,J and Choi,Y.S., IL-10 interrupts memory B cell expansion in the germinal center by inducing differentiation into plasma cells [J]. Eur. J.Immunol.1998; 28:508-515;
[11]Yeon Suk Jung,Shin-ei Matsumoto, et al., propionibacterium acnes acts as an adjuvant in in vitro immunization of human peripheral blood mononuclear cells [J], Biosci. Biotechnol. Biochem.2007; 71(8),1963-1969
[12]Roman M, Martin-Orozco E, Goodman JS, et al. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants [J]. Nature Med.1998; 3(8):849-854.
[13]温建新,李俊,周顺等,不同序列的CpG ODN对猪体外免疫细胞免疫刺激作用的研究[J],西北农业学报,2007,16(4)：71-75
[1]Kohler G, Milsten C. Continuous cultures of fused cells secreting antibody of Predefined specificity [J]. Nature,1975; 256:495-497
[2]Olsson L. and Kaplan HS., Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity [J]. PNAS,1980; 77(9):5429-5431
[3]Samoilovich SR, Dugan CB, Macario AJ.Hybridoma technology:new developments of practical interest [J]. Immunol Methods 1987;101:153-170
[4]Knudsen KA. Proteins transferred to nitrocellulose for use as immunogens [J].Anal Biochem 1985;147(2):285-288
[5]Ho MK., Rand N. Murray J. et al. In vitro immunization of human lymphocytes. I. Production of human monoclonal antibodies against bombesin and tetanus toxoid [J]. J Immunol 1985; 135 (6):3831-3838
[6]Sheng H. Z., Hoogenraad J., Carnegie P. R., et al. Use of Protein-bearing Nitrocellulose as Immunogen for in vitro Production of Monodonal Antibodies:Application to Myelin Basic Protein Electrophoretically Separated from a Complex Brain Protein Mixture [J]. Immunology Lettes.1987; 16:75-81;
[7]Dinjis W. N. M.,Van der Linden E. P. S.,Signet C. M.,et al.,Solid-phase Adsorption of for Efficient Production of Antibodies Reactive with Native and Fixed Tissue Antigens [J]. J Immunol Methods.1990; 126:175-182;
[8]Larsson A. Nilsso BO., Immunization with Nanogram Quantities of Nitrocellose-bound Antigen, Electroblotted from Sodium Dedecyl Sulphate-Polyacrylamide Gels [J]. Scand J Imrnunol.1988;27:305-309
[9]Guzman J., Schoedon G., Blau N., In Vitro Immunization with Antigen Directly Blotted from SDS-polyacrylamide Gels to Polyvinylidene Difluoride Membranes [J]. J Immunol Methods. 1993; 158:37-47
[10]Geeta NS., Ramanadham M., In Vitro Immunization of Murine Lymphocytes Using Immobilized Immunogens [J]. Biotech. Appl. Biochem.1996;24:61-64
[11]Sheng HZ, Hoogenraad J, Carngie PR,et al. Use of Protein-bearing Nitrocellulose as Immunogen for in vitro Production of Monoclonal Antibodies:Applifacion to Myelin Basic Protein Electrophoretically Separated from a Complex Brain Protein Mixture [J]. Immunology Letters.1987;16:75-81
[12]Gathuru J. K., Miyoshi I., Naiki M., In Vitro Immunization of Rat Spleen Lymphocytes with Forssman Glycosphingolipid Antigen and the Generation of a Monoclonal Antibody [J]. J Immunol Methods.1991;137:95-102
[13]Yarchoan R, Murphy BR, Strober W, et al.; Specific anti-influenza virus antibody production in vitro by human peripheral blood mononuclear cells [J]. J Immunol,1981;127(6):2588-2594;
[14]Borrbaeck CAK., In Vitro Immunization for Production of Murine and Human Monoclonal Antibodies:Present Status [J]. Trends Biotechnol.1986;4:147～153
[15]Steinman RM, Colin ZA. Identification of novel cell type in peripheral lymphoid organs of mice:Morphology, quantification, tissue distribution. [J]. J Exp. Med.1973; 137:1142-1162
[16]Liu L., Rich BE., Inobe J., et al. Induction of Th2 cell differentiation in the primary immune response:dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4F T cells to secrete IL-4 [J]. Int. Immunol.1998;10(8):1017-1026
[17]Gramaglia I.,Weinberg AD.,Lemon M. et al. Ox-40 ligand:a potent costimulatory molecule for sustaining primary CD40 T cell response [J].J Immunol,1998; 161(12):6510-6517.
[18]Tanaka H, Demeure C E, Rubio M. et al. Human monocyte-derived dendritic cells induce naive T cell differentiation into T helper cell type 2(Th2) or Th1/Th2 effectors, role of stimulator/responder ratio [J]. J Exp Med.2000;192 (3):405-412.
[19]Hochrein, Keeffe HMO, Luft T. et al. Interleukin(IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells [J]. J Exp Med.2000; 192:823-834.
[20]Tesciuba AG, Subudhi S, Rother RP. et al. Induciable costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease [J]. J Immunol. 2001; 167(4):97-101.
[21]Wang S D, Zhu GF, Chapoval A A. et al. Costimulation of T cells by B7H2, a B7-like molecule that binds ICOS [J]. Blood.2000; 96:2808-2813.
[22]Liang L, Porter EM., Sha WC. Constitutive expression of B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling [J]. J Exp Med. 2002; 196 (1):97-108.
[23]Saman K., Andreas H.,Kerstin B. et a. ICOS2-ligand, expressed on human endothelial cells, costimulates Th1 and Th2 cytokine secretion by memory CD4+T cells [J]. Molecular Immunology,2002; 99(9):6198-6203
[24]Paul, WE. Pleiotrophy and redundancy:T cell-derived lymphokines in the immune response [J]. Cell.1989.57:521.
[25]Howard, M., J. Farrar, M. Hilfiker, B. et al. Identification of a T cell derived B cell growth factor distinct from interleukin 2 [J].J, Exp. Med.1982; 155:914.
[26]Vitetta, ES., Ohara J., Myers C., et al., Serologic, biochemical and functional identity of B cell stimulatory factor-1 and B cell differentiation factor for IgG [J]. J. Exp Med.1985; 162:1726.
[27]Coffman RL. and Carty JA. T cell activity that enhances polyclonal IgE production and its inhibition by interferony [J]. J. Immunol.1986; 136:949.
[28]Coffman RL., Ohara J., Bond MW.,et al. B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells [J].J. Immunol.1986;136(10):4538.
[29]Mosmann TR. and Coffman RL.. TH1 and TH2 cells:different patterns of lymphokine secretion lead to different functional properties [J]. Annu. Rev. Immunol.1989; 7:145.
[30]Swain SL., McKenzie DT., Weinberg AD., et al. Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells [J]. J. Immunol.1988;141:3445.
[31]Powers GD., Abbas AK. and Miller RA. Frequencies of IL-2 and IL-4-secreting T cells in naive and antigen stimulated lymphocyte populations [J]. J. Immunol.1988:140:3352.
[32]Ben-Sasson SZ., Le Gros G., Conrad DH., et al. IL-4 production by T cells from naive donors. IL-2 is required for IL-4 production [J].J. Immunol.1990;145(4):1127-1136,
[33]Graham Le Gros, Shlomo Z., Ben-Sasson, et al. Generation of Interleukin 4 (IL4)-producing Cells In Vivo and In Vitro:IL-2 and IL-4 Are Required For In Vitro Generation of IL-4 producing Cells [J]. J. Exp. Med.,1990; 172:921-929
[34]Sean Diehl, Mercedes Rincon. The two faces of IL-6 on Th1/Th2 differentiation [J]. Mol. Immuno.2002; 19:531-536
[35]Choe J and Choi YS., IL-10 interrupts memory B cell expansion in the germinal center by inducing differentiation into plasma cells [J]. Eur.J.Immunol.1998; 28:508-515;
[36]Kindler V. and Zubler RH., Memory, but not naive, peripheral blood B lymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimulation with IL-4 and the differentiation factors IL-2, IL-10, and IL-3 [J]. J. Immunol.1997; 159:2085-2090
[37]Kobayashi N., Nagumo H. and Agematsu K., IL-10 enhances B-cell IgE synthesis by promoting differentiation into plasma cells, a process that is inhibited by CD27/CD70 interaction [J]. Clin.Exp.Immunol.2002;129:446-452
[38]Qianghua Xu,Yoshinori K. et al., IL-10 Augments Antibody Production in in Vitro Immunized Lymphocytes by Inducing a Th2-Type Response and B Cell maturation [J], Biosci. Biotechnol. Biochem.,68 (11),2279-2284,2004
[39]Leyden JJ., McGinley KJ., and Vowels B., Propionibacterium acnes colonization in acne and nonacne [J]. Dermatology,1998;196:55-58
[40]Green S., Dobrjansky A., Chiasson MA.,et al., Corynebacterium parvum as the priming agent in the production of tumor necrosis factor in the mouse [J]. J. Natl. Cancer Inst. 1977;59:1519-1522
[41]Halpern BN., Biozzi G., Stiffel C., et al., Inhibition of tumour growth by administration of killed Corynebacterium parvum [J]. Nature,1966;212:853-854
[42]Woodruff M.F., McBride WH., and Dunbar N., Tumour growth, phagocytic activity and antibody response in Corynebacterium parvum-treated mice[J]. Clin. Exp. Immunol.1974; 17: 509-518
[43]Warr GW., and James K., Effect of Corynebacterium parvum on the class and subclass of antibody produced in the response of different strains of mice to sheep erythrocytes [J]. Immunology,1975;28:431-442
[44]Abath FG., Coutinho EM., Montenegro SM., et al., The use of nonspecific immunopotentiators in experimental Trypanosoma cruzi infection [J]. Trans. R. Soc. Trop. Med. Hyg., 1988;82:73-76
[45]Hill JO., Modulation of the pattern of development of experimental disseminated leishmaniasis by Corynebacterium parvum[J]. J. Leukoc. Biol.,1987;41:165-169
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