牛γ干扰素单克隆抗体的制备与抗原捕获ELISA检测方法的建立
|
摘要
干扰素(Interferon,IFN)是在特定的诱生剂作用下,由细胞产生的一种具有高度生物学活性的糖蛋白,在同种动物细胞上具有广谱的抗病毒活性,其活性的发挥又受细胞基因组的调节和控制。IFN-γ的免疫学检测(IFN-γ实验)是在特异性抗IFN-γMcAb基础上建立的用于样本中IFN-γ定性定量分析的实验方法。国外学者已经将牛γ干扰素(Bovine Interferonγ,BoIFN-γ)的单克隆抗体用于多种牛疫病的研究。最近几年来,国内这方面的报道也逐渐增多,但检测方法建立的报道比较少。本研究克隆并表达了BoIFN-γ,制备了两株针对不同表位的高亲和力BoIFN-γ单克隆抗体,并在此基础上建立了BoIFN-γ的抗原捕获ELISA检测方法。
本研究可分为两个部分:
第一部分:本研究以原核表达并纯化的BoIFN-γ免疫6周龄雌性BALB/c小鼠,加强免疫后取脾细胞与SP2/0细胞融合,用重组杆状病毒表达的BoIFN-γ作为包被抗原采用ELISA方法筛选阳性克隆,经3-5次亚克隆,最后获得2株抗体效价较高的抗BoIFN-γ特异性单克隆抗体,分别命名为2G6和5F9,其重链分别为IgG1、IgG2b,轻链均为Kappa链。经间接ELISA、Western blot和抗病毒活性阻断实验表明两株单抗具有高度特异性。对2G6和5F9单克隆抗体进行相加ELISA实验,证明两株单抗针对BoIFN-γ不同的抗原表位。
第二部分:抗原捕获ELISA检测方法的建立。2G6和5F9腹水经Protein G纯化后,将其中效价高的5F9进行辣根过氧化物酶标记作为检测抗体,并利用2G6作为包被抗体建立了抗原捕获ELISA方法,用于检测BoIFN-γ。对ELISA各个反应条件进行优化后,得到的最佳工作条件为:包被抗体浓度0.127μg/孔,标准蛋白1:20稀释,酶标抗体浓度0.130μg/孔,包被抗体4℃过夜,标准蛋白反应时间1.0h,酶标抗体作用时间1.0h,包被液为20mM pH8.5的Tris-Cl,封闭液为3%鱼皮胶。重复性实验表明,同一样品的板内和板间的变异系数均小于10%。特异性试验表明本试验制备的单抗与牛α、β干扰素不反应。
本试验所建立的ELISA方法可检测到pET-BoIFN-γ表达的BoIFN-γ的敏感性为98.43ng/mL,可分别检测到2IU/100μL的rBac-BoIFN-γ表达的BoIFN-γ、10IU/100μL的r-NDV-lasota-BoIFN-γ表达的BoIFN-γ。
包被抗体的酶标板经老化试验和未做此试验的板同时保存于30℃、4℃和-20℃,定期进行ELISA检测,结果表明:未做老化试验的板在30℃、4℃保存时极不稳定,而做老化试验的板在30℃、4℃、-20℃可稳定保存4个月,未作老化试验的板在-20℃可稳定保存3个月。
该方法的建立为抗原捕获ELISA定量检测BoIFN-γ试剂盒的研制奠定了基础。该研究建立了高效的免疫学检测BoIFN-γ方法,在牛疫病防治和检测上具有十分重要的意义。
Interferon (IFN) is a kind of inducing glycoprotein which possesses high bioactivity and broad-spectrum antiviral activity on the homogeneity animal cells, its function is controlled and regulated by cell genome. IFN-γtest is established on the base of IFN-γMcAb which can detect the qualitation and quantity of interferon gamma. IFN-γand its monoclonal antibody also have a broad application in veterinary medicine. Some foreign scientists have reported that monoclonal antibody has been used for the research of bovine diseases. Some researches have been reported in China in recent years, but a few researches for detection. This study cloned and expressed recombinant BoIFN-γ(bovine interferon gamma), produced monoclonal antibodies against BoIFN-γand established a sandwich direct ELISA to detect BoIFN-γ.
Our study includes two parts:
Part one: The BoIFN-γgene fragment was cloned into prokaryotic expression plasmid. The recombinant protein was purified by affinity chromatography and used to immunize BALB/c mice. After three to five sub-clone,two hybridomas 2G6 and 5F9 secreting monoclonal antibodies to BoIFN-γwere established by ELISA . The two McAbs recognized BoIFN-γspecifically by western blot and indirect ELISA. The antiviral activity of BoIFN-γexpressed by recombinant baculovirus could be blocked by the two McAb ascites.The additivity ELISA demonstrated that the two McAbs recognized different antigenic regions.
Part two: A sandwich ELISA for detection of BoIFN-γwas developed utilizing 2G6 McAb and HRP-labled 5F9 McAb which could detect BoIFN-γspecifically.
The optimal working condition was listed as below. The dilution of capture McAb was 0.127μg/well, the standard protein was diluted to 1:20 and the dilution of HRP-labeled 5F9 was 0.130μg/well , the reaction time for coating antibody and HRP-labled 5F9 were all 1.0h in the assay,the coating buffer was 20mM Tris-cl pH8.5,the blocking solution was 3% gelatin from cold water fish skin, CV of intra-plate and inter-plate was lower than 10%. All the specific test in this paper demonstrated the two McAbs had good specificity .
The result showed that the ELISA had a detection limit of 98.43ng/mL BoIFN-γexpressed by pET-30a(+)-BoIFN-γin BL21, 2IU/100μL BoIFN-γexpressed by rBac-BoIFN-γ, 10IU/100μL BoIFN-γexpressed by r-NDV-lasota-BoIFN-γ.
After accelerated temperature studies for protein activity of 2G6 coating microplate the result showed that it could endure 30℃for 4 months.
本研究可分为两个部分:
第一部分:本研究以原核表达并纯化的BoIFN-γ免疫6周龄雌性BALB/c小鼠,加强免疫后取脾细胞与SP2/0细胞融合,用重组杆状病毒表达的BoIFN-γ作为包被抗原采用ELISA方法筛选阳性克隆,经3-5次亚克隆,最后获得2株抗体效价较高的抗BoIFN-γ特异性单克隆抗体,分别命名为2G6和5F9,其重链分别为IgG1、IgG2b,轻链均为Kappa链。经间接ELISA、Western blot和抗病毒活性阻断实验表明两株单抗具有高度特异性。对2G6和5F9单克隆抗体进行相加ELISA实验,证明两株单抗针对BoIFN-γ不同的抗原表位。
第二部分:抗原捕获ELISA检测方法的建立。2G6和5F9腹水经Protein G纯化后,将其中效价高的5F9进行辣根过氧化物酶标记作为检测抗体,并利用2G6作为包被抗体建立了抗原捕获ELISA方法,用于检测BoIFN-γ。对ELISA各个反应条件进行优化后,得到的最佳工作条件为:包被抗体浓度0.127μg/孔,标准蛋白1:20稀释,酶标抗体浓度0.130μg/孔,包被抗体4℃过夜,标准蛋白反应时间1.0h,酶标抗体作用时间1.0h,包被液为20mM pH8.5的Tris-Cl,封闭液为3%鱼皮胶。重复性实验表明,同一样品的板内和板间的变异系数均小于10%。特异性试验表明本试验制备的单抗与牛α、β干扰素不反应。
本试验所建立的ELISA方法可检测到pET-BoIFN-γ表达的BoIFN-γ的敏感性为98.43ng/mL,可分别检测到2IU/100μL的rBac-BoIFN-γ表达的BoIFN-γ、10IU/100μL的r-NDV-lasota-BoIFN-γ表达的BoIFN-γ。
包被抗体的酶标板经老化试验和未做此试验的板同时保存于30℃、4℃和-20℃,定期进行ELISA检测,结果表明:未做老化试验的板在30℃、4℃保存时极不稳定,而做老化试验的板在30℃、4℃、-20℃可稳定保存4个月,未作老化试验的板在-20℃可稳定保存3个月。
该方法的建立为抗原捕获ELISA定量检测BoIFN-γ试剂盒的研制奠定了基础。该研究建立了高效的免疫学检测BoIFN-γ方法,在牛疫病防治和检测上具有十分重要的意义。
Interferon (IFN) is a kind of inducing glycoprotein which possesses high bioactivity and broad-spectrum antiviral activity on the homogeneity animal cells, its function is controlled and regulated by cell genome. IFN-γtest is established on the base of IFN-γMcAb which can detect the qualitation and quantity of interferon gamma. IFN-γand its monoclonal antibody also have a broad application in veterinary medicine. Some foreign scientists have reported that monoclonal antibody has been used for the research of bovine diseases. Some researches have been reported in China in recent years, but a few researches for detection. This study cloned and expressed recombinant BoIFN-γ(bovine interferon gamma), produced monoclonal antibodies against BoIFN-γand established a sandwich direct ELISA to detect BoIFN-γ.
Our study includes two parts:
Part one: The BoIFN-γgene fragment was cloned into prokaryotic expression plasmid. The recombinant protein was purified by affinity chromatography and used to immunize BALB/c mice. After three to five sub-clone,two hybridomas 2G6 and 5F9 secreting monoclonal antibodies to BoIFN-γwere established by ELISA . The two McAbs recognized BoIFN-γspecifically by western blot and indirect ELISA. The antiviral activity of BoIFN-γexpressed by recombinant baculovirus could be blocked by the two McAb ascites.The additivity ELISA demonstrated that the two McAbs recognized different antigenic regions.
Part two: A sandwich ELISA for detection of BoIFN-γwas developed utilizing 2G6 McAb and HRP-labled 5F9 McAb which could detect BoIFN-γspecifically.
The optimal working condition was listed as below. The dilution of capture McAb was 0.127μg/well, the standard protein was diluted to 1:20 and the dilution of HRP-labeled 5F9 was 0.130μg/well , the reaction time for coating antibody and HRP-labled 5F9 were all 1.0h in the assay,the coating buffer was 20mM Tris-cl pH8.5,the blocking solution was 3% gelatin from cold water fish skin, CV of intra-plate and inter-plate was lower than 10%. All the specific test in this paper demonstrated the two McAbs had good specificity .
The result showed that the ELISA had a detection limit of 98.43ng/mL BoIFN-γexpressed by pET-30a(+)-BoIFN-γin BL21, 2IU/100μL BoIFN-γexpressed by rBac-BoIFN-γ, 10IU/100μL BoIFN-γexpressed by r-NDV-lasota-BoIFN-γ.
After accelerated temperature studies for protein activity of 2G6 coating microplate the result showed that it could endure 30℃for 4 months.
引文
1.杜平主编.医用干扰素学.北京:解放军出版社,1985:39～56.
2.杜贤宇,周如娇,杨玲,等.单克隆抗体亲和层析纯化Namalva细胞干扰素.药物生物技术,2002,8(6):310～312.
3.额尔敦,方建华,郭美香,等.人IFN-γ免疫PCR检测方法的建立及其敏感性的比较.内蒙古大学学报(自然科学版),1998,29(1):91～95.
4.龚非力.医学免疫学.北京:科学出版社,1998.
5.沈关心,周汝麟.现代免疫学实验技术,武汉:湖北科学技术出版社, 1998.
6.屠宗青,沈幼美,童葵塘.用国产单克隆抗体纯化重组干扰素α1b的研究.微生物学免疫学进展,2001,29(2):53～56.
7.吴美英,艾艳萍,任韧,等.重组人α干扰素单克隆抗体的研制及应用.中华实验和临床病毒学杂志,2002,16(3):261～263.
8. Anthony D, Lehmann P V. T-cell epitope mapping using the ELISPOT approach. Methods, 2003, 29(3): 260～269.
9. Bach EA, Aguet M, Schreiber RD. The interferon-γreceptor: a paradigm for cytokine receptor signaling. Annu Rev Immunol ,1997, 15: 563.
10. Barbara AO.Transcriptional control of T cell development.Curr Opoi Immunol, 2000,12: 301～306.
11. Barouch D H, Letvin N L. CD8+ cytotoxic T lymphocyte responses to lentiviruses and herpes- viruses. Curr Opin Immunol, 2001, 13(4): 479～482.
12. Boehm U,Klamp T,Groot,M. Cellular response to interferon-gamma. Annal Rev Immunol, 1997, 15: 749～795.
13. Bogdan C. The function of type I interferons in antimicrobial immunity. Curr Opin Immunol, 2000 , 12(4): 419～424.
14. Bonanomi A, Kojic D, Giger B, Rickenbach Z, Jean-Richard-Dit-Bressel L, Berger C, Niggli F K, Nadal D. Quantitative cytokine gene expression in human tonsils at excision and during histoculture assessed by standardized and calibrated real-time PCR and novel data processing. J Immunol Methods, 2003, 283(1-2): 27～43.
15. Borden E C, Hogan T F, Voelkel J G. Comparative antiproliferative activity in vitro of natural interferons alpha and beta for diploid and transformed human cells. Cancer Res, 1982, 42(12): 4948～4953.
16. Boss J M, Strominger J L. Regulation of a transfected human class II major histocompatibility complex gene in human fibroblasts. Proc Natl Acad Sci U S A, 1986, 83(23):9139～9143.
17. Canosi U, Mascia M, Gazza L, Serlupi-Crescenzi O, Donini S, Antonetti F, Galli G. A highly precise reporter gene bioassay for type I interferon. J Immunol Methods, 1996 , 199(1): 69～76.
18. Capellier M, Levy-Benezra R, Poindron P. Critical study of the titration of human leukocytic and lymphoblastoid interferon on MDBK cells, in microplaque by the so-called dye-uptake method. II. Statistical analysis. Ann Pharm Fr, 1986, 44(4):285～291
19. Cassatella MA, Hartman L, Perussia B, et al. Tumor necrosis factor and immune interferon gamma and upopolysaccharide enhancement of phagocyte of phagocyte respiratory burst capability, studies on gene expression of several NAPDH oxidase in human leukeemic myeloid cells. J Chin Invest, 1989, 83: 1570～1579.
20. Cerretti DP, Mckoreghan K, Larsen A, et al. Cloning and expression of bovine interferon gamma.J imuunol, 1986, 136(12): 4561～4564.
21. Chilakamarti V,Ramana,Pilar Gil,et al.Statl-dependent and independent pathways in IFN-γdependent signaling.Trends in immunology,2002,23(2):96～101.
22. Chin YE, Kitagawam, Su WC, et al. Cell growth arrest and induction of cyclin- dependent kinase inhibitor P21 mediated by STAT1. Science, 1996, 272: 719～722.
23. Bromberg IF, Horvath CM, Wen Z, et al. Transcriptionally active stat1 is required for the anti-protiferative effects of both interferon-αand interferon-γ. Proc Natl Acad Sci, 1996, 93: 7673～7678.
24. Clay T M, Hobeika A C, Mosca P J, Lyerly H K, Morse M A. Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin Cancer Res, 2001 , 7(5): 1127～1135.
25. Darnell JR, Kerr IM, Stark GR. Jak-stat pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science, 1994, 264:1415～1421.
26. Daubener W, Wanagat N, Pilz K, Seghrouchni S, Fischer H G, Hadding U. A new, simple, bioassay for human IFN-gamma. J Immunol Methods, 1994, 168(1):39～47.
27. Dayton MA, Knobloch TJ, Benjamin D. Human B cell lines express the interferon gamma gene. Cytokine, 1992, 4(6): 454～460.
28. Desem N , Jones SL. Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis infection. Clin Diagn Lab Immunol , 1998 , 5 : 531～536.
29. Douglas P C,Kate M,Alf L. Cloning structure and expression of bovine interferon-γ.The Journal of Immunology,1986,136(12):4561～4564.
30. Douglas PC, Kata M, Alf L. Cloning, sequence, and expression of bovine interferon-γ. The Journal of Immunology, 1986, 12 :4561～4564.
31. Duhe R J,Farrar W L. Struetural and mechanistic aspects of Janus kinases:how-faced god wields a double-edged sword. Interferon Cytokine Res,1998,18:l～5
32. Dupuis S,Impairment of mycobacterial but not viral immunity by a germline hunman STAT1 mutation.Science,2001,293:300～303.
33. Eberl G, Macdonald HR. Selective induction of NK cell proliferation and cytoxicity by active- ted NK T cells. Eur J Immunol, 2000, 30(4):985～992.
34. Elewaut D, Kronenberg M. Molecular biology of NK T cell specificity and development. Semin Immunol, 2000, 12(6): 561～568.
35. Farrar MA, Schreiber RD .The molecular biology of interferon-gamma and its receptor. Annual Rev Immunal, 1993, 11: 571～611.
36. Farrar WL. Structural and mechanistic aspects of Janus kinases: how the two-faced god wields a double-edged sword. Interferon Cytokine Res, 1998, 18: 1～5.
37. Ferreira P C, Peixoto M L, Silva M A, et al. Assay of human interferon in Vero cells by seve- ral methods . J Clin Microbiol, 1979, 9(4):471～475.
38. Finkelman F D, Holmes J, Katona I M, Urban J F Jr, Beckmann M P, Park L S, Schooley K A, Coffman R L, Mosmann T R, Paul W E. Lymphokine control of in vivo immunoglobulin isotype selection. Annu Rev Immunol, 1990, 8:303～333.
39. Foster G R, Rodrigues O, Ghouze F, Schulte-Frohlinde E, Testa D, Liao M J, Stark G R, Leadbeater L, Thomas H C. Different relative activities of human cell-derived interferon-alpha subtypes: IFN-alpha has very high antiviral potency. J Interferon Cytokine Res, 1996, 16(12): 1027～1033.
40. Friguet B, Djavadi-Ohaniance L, Pages J. A convenient enzyme-Linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the subunit of Escherichia coli tryptophan synthase. J Immunol Methods,1983, 60: 351～358.
41. Fukao T, Frdcht DM, Yap G, et al. Inducible expression of STAT4 in dendritic cell and macrophages and its critical role in innate and adaptive immune respones. J Immunol,2001, 166(7): 4446～4455.
42. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 2001, 25(4): 386～401.
43. Glimcher LH, Singh H. Transcription factors in lymphocyte development-T and B cell getogether.Cell, 1999, 96(1):13～23.
44. Goorha R M. Preparation and assay of vesicular stomatitis virus . Methods Enzymol, 1981, 78(Pt A):309～312.
45. Grrenland AC, Morales MO, Vivano BL, et al. STAT recruifment by tyrosine phosphorylated cytokine receptor:an ordered reversible affinity-driven process. Immunity, 1995, 2: 677～687.
46. Hao XS , Le JM, Vilcek J . Determination of human T cell activity in response to allogeneic cells and mitogens. An immunochemical assay for gamma-interferon is more sensitive and specific than a proliferation assay. J Immunol Methods , 1986 , 92 : 59～63.
47. Harton JA, Ting JP. ClassⅡtransativator: mastering the art of major histocom- patibility complex expression. Mol Cell Biol, 2000, 20(17): 6185～6194.
48. Heldin C H,Signal transduction via platelet-derived growth-factor receptors.Biochim Biophy Acta,1998,1378:79～113.
49. Higgns DP,Hemsley S,Canfield PJ.Assessment of anti-bovine IL4 and IFN-gamma antibodies to label IL-4 and IFN gamma in lymphocytes of the koala and brussum.Vet Immunol Immunopathol,2004,101(3-4):153～160.
50. Hiroshi S,Kenji M,Yasuo I,et al.Anti-viral effect of recombinant bovine interferonγon bovine Leukaemia virus.Cytokine,2001,6:227～231.
51. Horavth CM, Darnell JE. The state of the STSTs: recent developments in the study of signal transduction to the nucleus. Curr Opin Cell boil, 1997, 9: 233～240.
52. Imanishi J, Hoshino S, Hoshino A, et al. New simple dye-uptake assay for interferon . Biken J, 1981, 24(3):103～108.
53. Inmaculada E,Juan C,Eladio V.Effect of interferon-γand tumour Necrosis Factor on African Swine Fever Virus Replication in Porcine Monocytes.J Virol,1998,69:2973～2980.
54. Isaacs A,Lindanmann J.Virus interferon I.The interferon.Proc R Soc Ser B,1957,147:258～267.
55. Jameson P, Grossberg S E. Virus yield-reduction assays for interferon: picornavirus hemagg- lutination measurements . Methods Enzymol, 1981, 78(Pt A):357～368`.
56. Joseph C, Beyer,Roger W, et al. Cloning and expression of caprine interferon gamma.Gene, 1998, 210: 103～108.
57. Kaplan D H.Demonstration of an IFN-γdependent tumor surveillance system in immunoc- ompetent mice.Proc Natl Acad Sci USA,1998,95:7556～7561.
58. KawakamiK,Kinjo Y,Uezu K,et al;Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18.Microbes Infect,2004,6(4):339～349.
59. Kee H Ng,Franks E Aldwell,D Neil Wedlock,et al.Antigen-induced interferon-γand interleukin-2 responses of cattle inoculated with Mycobacterium bovis .Vet Immunol Immunopathol,1997,57:59～68.
60. Kendall C, Ionescu-Matiu I, Dreesman G R. Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods, 1983, 56(3): 329～339.
61. Kim JI,Ho IC,Grusby MJ,et al. The transcription factor C-maf controls the production of interleukin-4 but not other Th2 cytokine.Immunity,1998:745～751.
62. Kim TK, Maniatiis T. Regulation of interferon-gamma activated STAT by upqutin-proteasome pathway, Science, 1996, 273: 1717～1719.
63. Kim Y M, Son K. A nitric oxide production bioassay for interferon-gamma. J Immunol Methods, 1996, 198(2): 203～209.
64. Kubota T.C-teminal Cys-rich region of mumps virus structural V protein correlates with block of interferon-αandγthough decrease of STAT1α.Biochem Biophy Res Commun ,2001, 283: 255～259.
65. Kuo CT,Leiden JM. Transcriptional regulation of T lymphocyte development and function.Annu Rev Immunol,1997,17:149～187.
66. Letsch A, Scheibenbogen C. Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining. Methods, 2003, 31(2):143～149.
67. Levieux D, Venien A, Levieux A . Epitopic anaLysis and quantification of bovine myoglobin with monocLonaL antibodies. Hybridoma ,1995,14: 435～442.
68. MacMicking J D, North R J, LaCourse R, Mudgett J S, Shah S K, Nathan C F. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci U S A, 1997, 13: 94(10):5243～5248
69. Malu S, Srinivasan S, Kumar Maiti P, Rajagopal D, John B, Nandi D. IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice. J Immunol Methods, 2003, 272(1-2): 55～65.
70. Mashishi T, Gray C M. The ELISPOT assay: an easily transferable method for measuring cellular responses and identifying T cell epitopes. Clin Chem Lab Med, 2002, 40(9): 903～910.
71. Meager A. Bioimmunoassays for proinflammatory cytokines involving cytokine-induced cellular adhesion molecule expression in human glioblastoma cell lines. J Immunol Methods, 1996, 190(2): 235～244.
72. Michalski WP,Shiell BJ,O’Neil TE, et al.Recombinant chicken IFN-gamma expressed in Escherichia coli:Analysis of C-terminal truncation and effect on biologic activity.Interferon Cytokine Res,1999,19:383～392.
73. Migliorini P, Corradin G, Corradin S B. Macrophage NO2- production as a sensitive and rapid assay for the quantitation of murine IFN-gamma. J Immunol Methods, 1991, 139(1):107～114.
74. Mosmann T R, Moore K W. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol Today, 1991, 12(3):A49～53.
75. Mylrea PJ . Eradication of bovine tubercμLosis from New South Wales—a century of endeavour. Aust Vet J ,1990 , 67 : 104～107.
76. Nagorsen D, Scheibenbogen C, Thiel E, Keilholz U. Immunological monitoring of cancer vaccine therapy. Expert Opin Biol Ther, 2004, 4(10): 1677～1684.
78. Nederman T, Karlstrom E, Sjodin L. An in vitro bioassay for quantitation of human interf- erons by measurements of antiproliferative activity on a continuous human lymphoma cell line. Biologicals, 1990, 18(1): 29～34
78. Niemeyer C M, Adler M, Wacker R. Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol, 2005, 23(4): 208～216.
79. Nussler AK, Billiar TR. Inflammation, immunoregulation and inducible nitric oxide synthase. Leuk BIOl, 1993, 54: 171～178.
80. Ogiso S, Shirai J, Tuchiya Y, et al. Use of Getah virus for antiviral assay of human interferon. Uirusu, 2005, 55(2):317～326.
81. Okamoto Y, Abe T, Niwa T, Mizuhashi S, Nishida M. Development of a dual color enzyme- linked immunospot assay for simultaneous detection of murine T helper type 1- and T helper type 2-cells. Immunopharmacology, 1998, 39(2): 107～116.
82. Pala P, Hussell T, Openshaw P J. Flow cytometric measurement of intracellular cytokines. J Immunol Methods, 2000, 243(1-2): 107～124.
83. Perler L, Pfister H, Schweizer M, et al. A bioassay for interferon type I based on inhibition of Sendai virus growth . J Immunol Methods, 1999, 222(1-2):189～196.
84. Perler L,Schweizer M,Jungi T,et al.Bovine viral diarrhea virus and bovine herpesvirus-1 prime uninfected macropgages for lipoplysaccharide-triggered apoptosis by interferon dependent and independent pathways.J Gen Virol,2000,81:881～887.
85. Pestka S, Meager A. Interferon standardization and designations . J Interferon Cytokine Res, 1997, 17 Suppl 1:S9～114.
86. Rane S G,Reddy EP.Janus kinases: components of multiple signaling pathways.Oneoge, 2000,19:5662～5679.
87. Rane SG, Reddy EP. Janus kinases:components of multiple signaling path ways. Oncogene, 2000, 19: 5662～5679.
88. Rao A,OC,Hogan PG.Transcription factors of NFAT family;regulation and function.Annu Rev Immunol,1997,15:707～747.
89. Rigby W F, Ball E D, Guyre P M, Fanger M W. The effects of recombinant-DNA-derived interferons on the growth of myeloid progenitor cells. Blood, 1985, 65(4): 858～861.
90. Roy S K .CCAAT/enhancer-binding protein-βregulates interferon-induced transcription through a novel element. Biol Chem,2000,275:12626～12632.
91. Schindler H, Lutz MB, Rollinghoff M, et al. The production of IFN-γby IL-2 /IL-18-activated macrophage requires STAT4 signaling and is inhibited by IL-4. Immunol, 2001, 166(5): 3075～3082.
92. Sen G C, Lengyel P. The interferon system. A bird's eye view of its biochemistry. Biol Chem, 1992, 267(8): 5017～5020.
93. Sikand VK, Rothel JS , Martin RM. Diagnosis of Lyme borreliosis by a whole-blood gamma interferon assay for cell-mediated immune responses. Clin Diagn Lab lmmunol , 1999 , 6 : 445.
94. Snapper C M, Paul W E. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science, 1987, 236(4804): 944～947.
95. Stanton G J, Langford M P, Dianzani F. Virus yield-reduction assay for interferon by titration of Sindbis virus hemagglutinin . Methods Enzymol, 1981, 78(Pt A):351～357
96. Stark G R, Kerr I M, Williams B R, Silverman R H, Schreiber R D. How cells respond to interferons. Annu Rev Biochem, 1998, 67:227～264.
97. Stark GR, Kerr IM, Willians BR, et al.Hoe cells respond to interferon. Annu Rev Biochem ,1998, 67: 227～264.
98. Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides . science, 1995, 269(5230): 1585～1588.
99. Weigent D A, Stanton G J, Langford M P, et al. Virus yield-reduction assay for interferon by titration of infectious virus . Methods Enzymol, 1981, 78(Pt A):346～351.
100. Wiley SR, Kubin MZ, Sedger LM, et al. Monocyte-mediated tumoricidal activity via tumor mecrosis factor-related cytokine, TRAIL. Exp Med, 1999, 189: 1343～1354.
101. Yoshida H,Nishina H,Takimoto H,et al. The transcription factor NF-ATC 1 regulates proliferation and Th2 cytokine production.Immunity,1998:745～751.
102. Ziemiecki A, Harpur AG, Wilks AF. Jak protein tyrosine kinase: their role in cytokine signaling. Trends Cell Biol, 1994, 4: 204～215.
2.杜贤宇,周如娇,杨玲,等.单克隆抗体亲和层析纯化Namalva细胞干扰素.药物生物技术,2002,8(6):310～312.
3.额尔敦,方建华,郭美香,等.人IFN-γ免疫PCR检测方法的建立及其敏感性的比较.内蒙古大学学报(自然科学版),1998,29(1):91～95.
4.龚非力.医学免疫学.北京:科学出版社,1998.
5.沈关心,周汝麟.现代免疫学实验技术,武汉:湖北科学技术出版社, 1998.
6.屠宗青,沈幼美,童葵塘.用国产单克隆抗体纯化重组干扰素α1b的研究.微生物学免疫学进展,2001,29(2):53～56.
7.吴美英,艾艳萍,任韧,等.重组人α干扰素单克隆抗体的研制及应用.中华实验和临床病毒学杂志,2002,16(3):261～263.
8. Anthony D, Lehmann P V. T-cell epitope mapping using the ELISPOT approach. Methods, 2003, 29(3): 260～269.
9. Bach EA, Aguet M, Schreiber RD. The interferon-γreceptor: a paradigm for cytokine receptor signaling. Annu Rev Immunol ,1997, 15: 563.
10. Barbara AO.Transcriptional control of T cell development.Curr Opoi Immunol, 2000,12: 301～306.
11. Barouch D H, Letvin N L. CD8+ cytotoxic T lymphocyte responses to lentiviruses and herpes- viruses. Curr Opin Immunol, 2001, 13(4): 479～482.
12. Boehm U,Klamp T,Groot,M. Cellular response to interferon-gamma. Annal Rev Immunol, 1997, 15: 749～795.
13. Bogdan C. The function of type I interferons in antimicrobial immunity. Curr Opin Immunol, 2000 , 12(4): 419～424.
14. Bonanomi A, Kojic D, Giger B, Rickenbach Z, Jean-Richard-Dit-Bressel L, Berger C, Niggli F K, Nadal D. Quantitative cytokine gene expression in human tonsils at excision and during histoculture assessed by standardized and calibrated real-time PCR and novel data processing. J Immunol Methods, 2003, 283(1-2): 27～43.
15. Borden E C, Hogan T F, Voelkel J G. Comparative antiproliferative activity in vitro of natural interferons alpha and beta for diploid and transformed human cells. Cancer Res, 1982, 42(12): 4948～4953.
16. Boss J M, Strominger J L. Regulation of a transfected human class II major histocompatibility complex gene in human fibroblasts. Proc Natl Acad Sci U S A, 1986, 83(23):9139～9143.
17. Canosi U, Mascia M, Gazza L, Serlupi-Crescenzi O, Donini S, Antonetti F, Galli G. A highly precise reporter gene bioassay for type I interferon. J Immunol Methods, 1996 , 199(1): 69～76.
18. Capellier M, Levy-Benezra R, Poindron P. Critical study of the titration of human leukocytic and lymphoblastoid interferon on MDBK cells, in microplaque by the so-called dye-uptake method. II. Statistical analysis. Ann Pharm Fr, 1986, 44(4):285～291
19. Cassatella MA, Hartman L, Perussia B, et al. Tumor necrosis factor and immune interferon gamma and upopolysaccharide enhancement of phagocyte of phagocyte respiratory burst capability, studies on gene expression of several NAPDH oxidase in human leukeemic myeloid cells. J Chin Invest, 1989, 83: 1570～1579.
20. Cerretti DP, Mckoreghan K, Larsen A, et al. Cloning and expression of bovine interferon gamma.J imuunol, 1986, 136(12): 4561～4564.
21. Chilakamarti V,Ramana,Pilar Gil,et al.Statl-dependent and independent pathways in IFN-γdependent signaling.Trends in immunology,2002,23(2):96～101.
22. Chin YE, Kitagawam, Su WC, et al. Cell growth arrest and induction of cyclin- dependent kinase inhibitor P21 mediated by STAT1. Science, 1996, 272: 719～722.
23. Bromberg IF, Horvath CM, Wen Z, et al. Transcriptionally active stat1 is required for the anti-protiferative effects of both interferon-αand interferon-γ. Proc Natl Acad Sci, 1996, 93: 7673～7678.
24. Clay T M, Hobeika A C, Mosca P J, Lyerly H K, Morse M A. Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin Cancer Res, 2001 , 7(5): 1127～1135.
25. Darnell JR, Kerr IM, Stark GR. Jak-stat pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science, 1994, 264:1415～1421.
26. Daubener W, Wanagat N, Pilz K, Seghrouchni S, Fischer H G, Hadding U. A new, simple, bioassay for human IFN-gamma. J Immunol Methods, 1994, 168(1):39～47.
27. Dayton MA, Knobloch TJ, Benjamin D. Human B cell lines express the interferon gamma gene. Cytokine, 1992, 4(6): 454～460.
28. Desem N , Jones SL. Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis infection. Clin Diagn Lab Immunol , 1998 , 5 : 531～536.
29. Douglas P C,Kate M,Alf L. Cloning structure and expression of bovine interferon-γ.The Journal of Immunology,1986,136(12):4561～4564.
30. Douglas PC, Kata M, Alf L. Cloning, sequence, and expression of bovine interferon-γ. The Journal of Immunology, 1986, 12 :4561～4564.
31. Duhe R J,Farrar W L. Struetural and mechanistic aspects of Janus kinases:how-faced god wields a double-edged sword. Interferon Cytokine Res,1998,18:l～5
32. Dupuis S,Impairment of mycobacterial but not viral immunity by a germline hunman STAT1 mutation.Science,2001,293:300～303.
33. Eberl G, Macdonald HR. Selective induction of NK cell proliferation and cytoxicity by active- ted NK T cells. Eur J Immunol, 2000, 30(4):985～992.
34. Elewaut D, Kronenberg M. Molecular biology of NK T cell specificity and development. Semin Immunol, 2000, 12(6): 561～568.
35. Farrar MA, Schreiber RD .The molecular biology of interferon-gamma and its receptor. Annual Rev Immunal, 1993, 11: 571～611.
36. Farrar WL. Structural and mechanistic aspects of Janus kinases: how the two-faced god wields a double-edged sword. Interferon Cytokine Res, 1998, 18: 1～5.
37. Ferreira P C, Peixoto M L, Silva M A, et al. Assay of human interferon in Vero cells by seve- ral methods . J Clin Microbiol, 1979, 9(4):471～475.
38. Finkelman F D, Holmes J, Katona I M, Urban J F Jr, Beckmann M P, Park L S, Schooley K A, Coffman R L, Mosmann T R, Paul W E. Lymphokine control of in vivo immunoglobulin isotype selection. Annu Rev Immunol, 1990, 8:303～333.
39. Foster G R, Rodrigues O, Ghouze F, Schulte-Frohlinde E, Testa D, Liao M J, Stark G R, Leadbeater L, Thomas H C. Different relative activities of human cell-derived interferon-alpha subtypes: IFN-alpha has very high antiviral potency. J Interferon Cytokine Res, 1996, 16(12): 1027～1033.
40. Friguet B, Djavadi-Ohaniance L, Pages J. A convenient enzyme-Linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the subunit of Escherichia coli tryptophan synthase. J Immunol Methods,1983, 60: 351～358.
41. Fukao T, Frdcht DM, Yap G, et al. Inducible expression of STAT4 in dendritic cell and macrophages and its critical role in innate and adaptive immune respones. J Immunol,2001, 166(7): 4446～4455.
42. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 2001, 25(4): 386～401.
43. Glimcher LH, Singh H. Transcription factors in lymphocyte development-T and B cell getogether.Cell, 1999, 96(1):13～23.
44. Goorha R M. Preparation and assay of vesicular stomatitis virus . Methods Enzymol, 1981, 78(Pt A):309～312.
45. Grrenland AC, Morales MO, Vivano BL, et al. STAT recruifment by tyrosine phosphorylated cytokine receptor:an ordered reversible affinity-driven process. Immunity, 1995, 2: 677～687.
46. Hao XS , Le JM, Vilcek J . Determination of human T cell activity in response to allogeneic cells and mitogens. An immunochemical assay for gamma-interferon is more sensitive and specific than a proliferation assay. J Immunol Methods , 1986 , 92 : 59～63.
47. Harton JA, Ting JP. ClassⅡtransativator: mastering the art of major histocom- patibility complex expression. Mol Cell Biol, 2000, 20(17): 6185～6194.
48. Heldin C H,Signal transduction via platelet-derived growth-factor receptors.Biochim Biophy Acta,1998,1378:79～113.
49. Higgns DP,Hemsley S,Canfield PJ.Assessment of anti-bovine IL4 and IFN-gamma antibodies to label IL-4 and IFN gamma in lymphocytes of the koala and brussum.Vet Immunol Immunopathol,2004,101(3-4):153～160.
50. Hiroshi S,Kenji M,Yasuo I,et al.Anti-viral effect of recombinant bovine interferonγon bovine Leukaemia virus.Cytokine,2001,6:227～231.
51. Horavth CM, Darnell JE. The state of the STSTs: recent developments in the study of signal transduction to the nucleus. Curr Opin Cell boil, 1997, 9: 233～240.
52. Imanishi J, Hoshino S, Hoshino A, et al. New simple dye-uptake assay for interferon . Biken J, 1981, 24(3):103～108.
53. Inmaculada E,Juan C,Eladio V.Effect of interferon-γand tumour Necrosis Factor on African Swine Fever Virus Replication in Porcine Monocytes.J Virol,1998,69:2973～2980.
54. Isaacs A,Lindanmann J.Virus interferon I.The interferon.Proc R Soc Ser B,1957,147:258～267.
55. Jameson P, Grossberg S E. Virus yield-reduction assays for interferon: picornavirus hemagg- lutination measurements . Methods Enzymol, 1981, 78(Pt A):357～368`.
56. Joseph C, Beyer,Roger W, et al. Cloning and expression of caprine interferon gamma.Gene, 1998, 210: 103～108.
57. Kaplan D H.Demonstration of an IFN-γdependent tumor surveillance system in immunoc- ompetent mice.Proc Natl Acad Sci USA,1998,95:7556～7561.
58. KawakamiK,Kinjo Y,Uezu K,et al;Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18.Microbes Infect,2004,6(4):339～349.
59. Kee H Ng,Franks E Aldwell,D Neil Wedlock,et al.Antigen-induced interferon-γand interleukin-2 responses of cattle inoculated with Mycobacterium bovis .Vet Immunol Immunopathol,1997,57:59～68.
60. Kendall C, Ionescu-Matiu I, Dreesman G R. Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods, 1983, 56(3): 329～339.
61. Kim JI,Ho IC,Grusby MJ,et al. The transcription factor C-maf controls the production of interleukin-4 but not other Th2 cytokine.Immunity,1998:745～751.
62. Kim TK, Maniatiis T. Regulation of interferon-gamma activated STAT by upqutin-proteasome pathway, Science, 1996, 273: 1717～1719.
63. Kim Y M, Son K. A nitric oxide production bioassay for interferon-gamma. J Immunol Methods, 1996, 198(2): 203～209.
64. Kubota T.C-teminal Cys-rich region of mumps virus structural V protein correlates with block of interferon-αandγthough decrease of STAT1α.Biochem Biophy Res Commun ,2001, 283: 255～259.
65. Kuo CT,Leiden JM. Transcriptional regulation of T lymphocyte development and function.Annu Rev Immunol,1997,17:149～187.
66. Letsch A, Scheibenbogen C. Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining. Methods, 2003, 31(2):143～149.
67. Levieux D, Venien A, Levieux A . Epitopic anaLysis and quantification of bovine myoglobin with monocLonaL antibodies. Hybridoma ,1995,14: 435～442.
68. MacMicking J D, North R J, LaCourse R, Mudgett J S, Shah S K, Nathan C F. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci U S A, 1997, 13: 94(10):5243～5248
69. Malu S, Srinivasan S, Kumar Maiti P, Rajagopal D, John B, Nandi D. IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice. J Immunol Methods, 2003, 272(1-2): 55～65.
70. Mashishi T, Gray C M. The ELISPOT assay: an easily transferable method for measuring cellular responses and identifying T cell epitopes. Clin Chem Lab Med, 2002, 40(9): 903～910.
71. Meager A. Bioimmunoassays for proinflammatory cytokines involving cytokine-induced cellular adhesion molecule expression in human glioblastoma cell lines. J Immunol Methods, 1996, 190(2): 235～244.
72. Michalski WP,Shiell BJ,O’Neil TE, et al.Recombinant chicken IFN-gamma expressed in Escherichia coli:Analysis of C-terminal truncation and effect on biologic activity.Interferon Cytokine Res,1999,19:383～392.
73. Migliorini P, Corradin G, Corradin S B. Macrophage NO2- production as a sensitive and rapid assay for the quantitation of murine IFN-gamma. J Immunol Methods, 1991, 139(1):107～114.
74. Mosmann T R, Moore K W. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol Today, 1991, 12(3):A49～53.
75. Mylrea PJ . Eradication of bovine tubercμLosis from New South Wales—a century of endeavour. Aust Vet J ,1990 , 67 : 104～107.
76. Nagorsen D, Scheibenbogen C, Thiel E, Keilholz U. Immunological monitoring of cancer vaccine therapy. Expert Opin Biol Ther, 2004, 4(10): 1677～1684.
78. Nederman T, Karlstrom E, Sjodin L. An in vitro bioassay for quantitation of human interf- erons by measurements of antiproliferative activity on a continuous human lymphoma cell line. Biologicals, 1990, 18(1): 29～34
78. Niemeyer C M, Adler M, Wacker R. Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol, 2005, 23(4): 208～216.
79. Nussler AK, Billiar TR. Inflammation, immunoregulation and inducible nitric oxide synthase. Leuk BIOl, 1993, 54: 171～178.
80. Ogiso S, Shirai J, Tuchiya Y, et al. Use of Getah virus for antiviral assay of human interferon. Uirusu, 2005, 55(2):317～326.
81. Okamoto Y, Abe T, Niwa T, Mizuhashi S, Nishida M. Development of a dual color enzyme- linked immunospot assay for simultaneous detection of murine T helper type 1- and T helper type 2-cells. Immunopharmacology, 1998, 39(2): 107～116.
82. Pala P, Hussell T, Openshaw P J. Flow cytometric measurement of intracellular cytokines. J Immunol Methods, 2000, 243(1-2): 107～124.
83. Perler L, Pfister H, Schweizer M, et al. A bioassay for interferon type I based on inhibition of Sendai virus growth . J Immunol Methods, 1999, 222(1-2):189～196.
84. Perler L,Schweizer M,Jungi T,et al.Bovine viral diarrhea virus and bovine herpesvirus-1 prime uninfected macropgages for lipoplysaccharide-triggered apoptosis by interferon dependent and independent pathways.J Gen Virol,2000,81:881～887.
85. Pestka S, Meager A. Interferon standardization and designations . J Interferon Cytokine Res, 1997, 17 Suppl 1:S9～114.
86. Rane S G,Reddy EP.Janus kinases: components of multiple signaling pathways.Oneoge, 2000,19:5662～5679.
87. Rane SG, Reddy EP. Janus kinases:components of multiple signaling path ways. Oncogene, 2000, 19: 5662～5679.
88. Rao A,OC,Hogan PG.Transcription factors of NFAT family;regulation and function.Annu Rev Immunol,1997,15:707～747.
89. Rigby W F, Ball E D, Guyre P M, Fanger M W. The effects of recombinant-DNA-derived interferons on the growth of myeloid progenitor cells. Blood, 1985, 65(4): 858～861.
90. Roy S K .CCAAT/enhancer-binding protein-βregulates interferon-induced transcription through a novel element. Biol Chem,2000,275:12626～12632.
91. Schindler H, Lutz MB, Rollinghoff M, et al. The production of IFN-γby IL-2 /IL-18-activated macrophage requires STAT4 signaling and is inhibited by IL-4. Immunol, 2001, 166(5): 3075～3082.
92. Sen G C, Lengyel P. The interferon system. A bird's eye view of its biochemistry. Biol Chem, 1992, 267(8): 5017～5020.
93. Sikand VK, Rothel JS , Martin RM. Diagnosis of Lyme borreliosis by a whole-blood gamma interferon assay for cell-mediated immune responses. Clin Diagn Lab lmmunol , 1999 , 6 : 445.
94. Snapper C M, Paul W E. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science, 1987, 236(4804): 944～947.
95. Stanton G J, Langford M P, Dianzani F. Virus yield-reduction assay for interferon by titration of Sindbis virus hemagglutinin . Methods Enzymol, 1981, 78(Pt A):351～357
96. Stark G R, Kerr I M, Williams B R, Silverman R H, Schreiber R D. How cells respond to interferons. Annu Rev Biochem, 1998, 67:227～264.
97. Stark GR, Kerr IM, Willians BR, et al.Hoe cells respond to interferon. Annu Rev Biochem ,1998, 67: 227～264.
98. Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides . science, 1995, 269(5230): 1585～1588.
99. Weigent D A, Stanton G J, Langford M P, et al. Virus yield-reduction assay for interferon by titration of infectious virus . Methods Enzymol, 1981, 78(Pt A):346～351.
100. Wiley SR, Kubin MZ, Sedger LM, et al. Monocyte-mediated tumoricidal activity via tumor mecrosis factor-related cytokine, TRAIL. Exp Med, 1999, 189: 1343～1354.
101. Yoshida H,Nishina H,Takimoto H,et al. The transcription factor NF-ATC 1 regulates proliferation and Th2 cytokine production.Immunity,1998:745～751.
102. Ziemiecki A, Harpur AG, Wilks AF. Jak protein tyrosine kinase: their role in cytokine signaling. Trends Cell Biol, 1994, 4: 204～215.