抗牛免疫球蛋白G单克隆抗体的制备及鉴定
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摘要
采用杂交瘤技术从90株融合株中筛选得到分泌抗牛免疫球蛋白G(immunoglobulin G,IgG)单克隆抗体的2F6、5E3、9C6、9H8四个细胞株。其抗体亚型均为IgG1型,腹水单克隆抗体的效价可达5.12×10~6;5E3、9H8识别牛IgG重链Fc片段,2F6识别牛IgG轻链Fab片段,9C6识别牛IgG重链Fab片段;9C6与牛IgG1和牛IgG2反应,与山羊IgG、绵羊IgG、兔IgG、牛IgM、牛乳酪蛋白、牛乳β-乳球蛋白、牛乳铁蛋白、牛血清白蛋白、鸡卵清白蛋白、鱼明胶的交叉反应率均小于1%;9C6亲合力常数达牛乳8.92×10~8 L/mol。
Four hybridomas secreting monoclonal antibody (McAb) against bovine immunoglobulin G (IgG) were developed by fusion of spleen cells from immunized BALB/C mice with SP2/0 myeloma cells, and they were named as 2F6, 5E3, 9C6 and 9H8. The McAbs secreted by all four hybridomas were identified as IgG1 subtype with a titer of 5.12 × 10~6. Both 5E3 and 9H8 recognized the heavy chain Fc fragment of bovine IgG. 2F6 recognized the light chain Fab fragment of bovine IgG, and 9C6 recognized the heavy chain Fab fragment of bovine IgG. The McAb from 9C6 could react with both IgG1 and IgG2. The cross-reactivity between 9C6 and goat IgG, sheep IgG, rabbit IgG, bovine IgM, casein, β-lacto globulin, lactoferrin, bovine serum albumin, ovalbumin or fish gel was less than 1%. The affinity constant of 9C6 was 8.92 × 10~8 L/mol.
Four hybridomas secreting monoclonal antibody (McAb) against bovine immunoglobulin G (IgG) were developed by fusion of spleen cells from immunized BALB/C mice with SP2/0 myeloma cells, and they were named as 2F6, 5E3, 9C6 and 9H8. The McAbs secreted by all four hybridomas were identified as IgG1 subtype with a titer of 5.12 × 10~6. Both 5E3 and 9H8 recognized the heavy chain Fc fragment of bovine IgG. 2F6 recognized the light chain Fab fragment of bovine IgG, and 9C6 recognized the heavy chain Fab fragment of bovine IgG. The McAb from 9C6 could react with both IgG1 and IgG2. The cross-reactivity between 9C6 and goat IgG, sheep IgG, rabbit IgG, bovine IgM, casein, β-lacto globulin, lactoferrin, bovine serum albumin, ovalbumin or fish gel was less than 1%. The affinity constant of 9C6 was 8.92 × 10~8 L/mol.
引文
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[20]PENHA T R,KRüGER E R,SOCCOL V,et al.Production and characterization of monoclonal antibodies to bovine IgG-Fc[J].Veterinary Immunology and Immunopathology,2009,128(1/2/3):310.DOI:10.1590/S1516-89132010000100014.
[21]GALL-DEBRECENI A,LAZAR J,KADAS J,et al.Specific detection and quantitation of bovine IgG in bioreactor derived mouse mAb preparations[J].Journal of Immunological Methods,2016,438:26-34.DOI:10.1016/j.jim.2016.08.005.
[22]高东微,马孝斌,孙远明,等.乳制品中牛IgG含量间接竞争性ELISA检测方法的研究[J].食品工业科技,2010,31(1):395-398.DOI:10.13386/j.issn1002-0306.2010.01.074.
[23]王平,周志军,施金荣,等.抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定[J].微生物学免疫学进展,2011,39(3):18-23.DOI:10.13309/j.cnki.pmi.2011.03.007.
[24]韩秀娥,李萌,张萍,等.牛初乳免疫球蛋白Ig G单克隆抗体的制备[J].黑龙江畜牧兽医,2011,13(7):140-142.DOI:10.13881/j.cnki.hljxmsy.2011.13.041.
[25]李岩松.玉米中伏马菌素免疫学快速筛检方法研究[D].长春:吉林大学,2011:42-44.
[26]GODING J W.Monoclonal antibodies:principles and practice[M].Academic Press,Inc Ltd,1983.
[27]FARRELL H M,JIMENEZ-FLORES R,BLECK G T,et al.Nomenclature of the proteins of cows’milk:sixth revision[J].Journal of Dairy Science,2004,87(6):1641-1674.DOI:10.3168/jds.S0022-0302(04)73319-6.
[28]ZAANE D V,IJZERMAN J.Monoclonal antibodies against bovine immunoglobulins and their use in isotype-specific ELISAs for rotavirus antibody[J].Journal of Immunological Methods,1984,72(2):427-441.DOI:10.1016/0022-1759(84)90011-5.
[29]ESTES D M,TEMPLETON J W,HUNTER D M,et al.Production and use of murine monoclonal antibodies reactive with bovine IgMisotype and IgG subisotypes (IgG1,IgG2a and IgG2b) in assessing immunoglobulin levels in serum of cattle[J].Veterinary Immunology and Immunopathology,1990,25(1):61-72.DOI:10.1016/0165-2427(90)90110-E.
[30]ANTALíKOVáJ,SIMON M,JANKOVI?OVáJ,et al.Monoclonal antibody to the light chain of bovine immunoglobulin[J].Hybridoma,2006,25(5):309-312.DOI:10.1089/hyb.2006.25.309.
[2]MEHRA R,MARNILA P,KORHONEN H.Milk immunoglobulins for health promotion[J].International Dairy Journal,2006,16(11):1262-1271.DOI:10.1016/j.idairyj.2006.06.003.
[3]BOGAHAWATHTHA D,CHANDRAPALA J,VASILJEVIC T.Modulation of milk immunogenicity by thermal processing[J].International Dairy Journal,2017,69:23-32.DOI:10.1016/j.idairyj.2017.01.010.
[4]EL-LOLY M M.Bovine milk immunoglobulins in relation to human health[J].International Dairy Journal,2007,2(3):183-195.DOI:10.3923/ijds.2007.183.195.
[5]GAPPER L W,COPESTAKE D J,OTTER D E,et al.Analysis of bovine immunoglobulin G in milk,colostrums and dietary supplements:a review[J].Analytical and Bioanalytical Chemistry,2007,389(1):93-109.DOI:10.1007/s00216-007-1391-z.
[6]CHAE A,AITCHISON A,DAY A S,et al.Bovine colostrum demonstrates anti-inflammatory and antibacterial activity in in vitro models of intestinal inflammation and infection[J].Journal of Functional Foods,2017,28:293-298.DOI:10.1016/j.jff.2016.11.016.
[7]ALTOMAREA A,FASOLI E,COLZANI M.An in depth proteomic analysis based on ProteoMiner,affinity chromatography and nanoHPLC-MS/MS to explain the potential health benefits of bovine colostrum[J].Journal of Pharmaceutical and Biomedical Analysis,2016,121:297-306.DOI:10.1016/j.jpba.2016.01.013.
[8]DAVIS D G,SCHAEFER D M W,HINCHCLIFF K W,et al.Measurement of serum IgG in foals by radial immunodiffusion and automated turbidimetricimmunoassay[J].Journal of Veterinary Internal Medicine,2005,19(1):93-96.DOI:10.1111/j.1939-1676.2005.tb02664.x.
[9]LEE S H,JAEKAL J,BAE C S,et al.Enzyme-linked immunosorbent assay,single radial immunodiffusion,and indirect methods for the detection of failure of transfer of passive immunity in dairy calves[J].Journal of Veterinary Internal Medicine,2008,22(1):212-218.DOI:10.1111/j.1939-1676.2007.0013.x.
[10]GELSINGER S L,SMITH A M,JONES C M,et al.Comparison of radial immunodiffusion and ELISA for quantification of bovine immunoglobulin G in colostrum and plasma[J].Journal of Dairy Science,2015,98(6):4084-4089.DOI:10.3168/jds.2014-8491.
[11]GODDEN S,MCMARTIN S,FEIRTAG J,et al.Heat-treatment of bovine colostrum Ⅱ:effects of heating duration on pathogen viability and immunoglobulin G[J].Journal of Dairy Science,2006,89(9):3476-3483.DOI:10.3168/jds.S0022-0302(06)72386-4.
[12]DONAHUE M,GODDEN S M,BEY R,et al.Heat treatment of colostrum on commercial dairy farms decreases colostrum microbial counts while maintaining colostrum immunoglobulin Gconcentrations[J].Journal of Dairy Science,2012,95(5):2697-2702.DOI:10.3168/jds.2011-5220.
[13]MCCUE P M.Evaluation of a turbidimetric immunoassay for measurement of plasma IgG concentration in foals[J].American Journal of Veterinary Research,2007,68(9):1005-1009.DOI:10.2460/ajvr.68.9.1005.
[14]QUIGLEY J D,LAGO A,CHAPMAN C E,et al.Evaluation of the Brix refractometer to estimate immunoglobulin G concentration in bovine colostrum[J].Journal of Dairy Science,2013,96(2):1148-1155.DOI:10.3168/jds.2012-5823.
[15]KUMMER A,KITTS D,LI-CHAN E,et al.Quantification of bovine IgG in milk using enzyme-linked immunosorbent assay[J].Food and Agricultural Immunology,1992,4(2):93-102.DOI:10.3168/jds.2014-8491.
[16]PEREIRA R V,BICALHO M L,MACHADO V S,et al.Evaluation of the effects of ultraviolet light on bacterial contaminants inoculated into whole milk and colostrum,and on colostrum immunoglobulin G[J].Journal of Dairy Science,2014,97(5):2866-2875.DOI:10.3168/jds.2013-7601.
[17]FOSTER D M,POULSEN K P,SYLVESTER H J,et al.Effect of high-pressure processing of bovine colostrum on immunoglobulin Gconcentration,pathogens,viscosity,and transfer of passive immunity to calves[J].Journal of Dairy Science,2016,99(11):8575-8588.DOI:10.3168/jds.2016-11204.
[18]SRIKUMARAN S,GUIDRY A J,GOLDSBY R A.Production and characterization of monoclonal antibodies to bovine immunoglobulin G2[J].American Journal of Veterinary Research,1982,43(1):21-25.
[19]HENNING D,NIELSEN K.Cross-reactivity of monoclonal antibodies to bovine immunoglobulins with immunoglobulins of other species[J].Veterinary Immunology and Immunopathology,1992,34(3/4):235-243.DOI:10.1016/0165-2427(92)90167-O.
[20]PENHA T R,KRüGER E R,SOCCOL V,et al.Production and characterization of monoclonal antibodies to bovine IgG-Fc[J].Veterinary Immunology and Immunopathology,2009,128(1/2/3):310.DOI:10.1590/S1516-89132010000100014.
[21]GALL-DEBRECENI A,LAZAR J,KADAS J,et al.Specific detection and quantitation of bovine IgG in bioreactor derived mouse mAb preparations[J].Journal of Immunological Methods,2016,438:26-34.DOI:10.1016/j.jim.2016.08.005.
[22]高东微,马孝斌,孙远明,等.乳制品中牛IgG含量间接竞争性ELISA检测方法的研究[J].食品工业科技,2010,31(1):395-398.DOI:10.13386/j.issn1002-0306.2010.01.074.
[23]王平,周志军,施金荣,等.抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定[J].微生物学免疫学进展,2011,39(3):18-23.DOI:10.13309/j.cnki.pmi.2011.03.007.
[24]韩秀娥,李萌,张萍,等.牛初乳免疫球蛋白Ig G单克隆抗体的制备[J].黑龙江畜牧兽医,2011,13(7):140-142.DOI:10.13881/j.cnki.hljxmsy.2011.13.041.
[25]李岩松.玉米中伏马菌素免疫学快速筛检方法研究[D].长春:吉林大学,2011:42-44.
[26]GODING J W.Monoclonal antibodies:principles and practice[M].Academic Press,Inc Ltd,1983.
[27]FARRELL H M,JIMENEZ-FLORES R,BLECK G T,et al.Nomenclature of the proteins of cows’milk:sixth revision[J].Journal of Dairy Science,2004,87(6):1641-1674.DOI:10.3168/jds.S0022-0302(04)73319-6.
[28]ZAANE D V,IJZERMAN J.Monoclonal antibodies against bovine immunoglobulins and their use in isotype-specific ELISAs for rotavirus antibody[J].Journal of Immunological Methods,1984,72(2):427-441.DOI:10.1016/0022-1759(84)90011-5.
[29]ESTES D M,TEMPLETON J W,HUNTER D M,et al.Production and use of murine monoclonal antibodies reactive with bovine IgMisotype and IgG subisotypes (IgG1,IgG2a and IgG2b) in assessing immunoglobulin levels in serum of cattle[J].Veterinary Immunology and Immunopathology,1990,25(1):61-72.DOI:10.1016/0165-2427(90)90110-E.
[30]ANTALíKOVáJ,SIMON M,JANKOVI?OVáJ,et al.Monoclonal antibody to the light chain of bovine immunoglobulin[J].Hybridoma,2006,25(5):309-312.DOI:10.1089/hyb.2006.25.309.