牛初乳中免疫球蛋白G双抗夹心ELISA检测方法的建立

doi:10.7506/spkx1002-6630-201614013

摘要/Abstract

摘要：

将牛免疫球蛋白G（immunoglobulin G，IgG）作为免疫原免疫BALB/c小鼠，通过细胞融合、筛选获得分泌抗牛IgG单克隆抗体的细胞株。制备小鼠腹水抗体，进一步纯化获得抗牛IgG单克隆抗体。建立双抗夹心酶联免疫吸附法检测牛初乳中IgG质量浓度，该方法在7.8～1 000 ng/mL范围内有良好的线性关系，最低检出限为7.06 ng/mL，批内变异系数为4.52%，批间变异系数为4.94%，回收率为91.85%～102.45%。此法操作简便、准确度高、稳定性好，可用于实际牛初乳样品的快速检测。

关键词: 牛初乳, 免疫球蛋白G, 单克隆抗体, 双抗夹心酶联免疫吸附法

Abstract:

The hybridoma cells secreting monoclonal antibody (McAb) against bovine colostrum immunoglobulin G (IgG)
were developed by cell fusion technology after immunization of BALB/c mice with bovine IgG. McAbs were obtained by
purification of mouse ascites. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) technique was
established for the detection of bovine colostrum IgG. The linear range was 7.8–1 000 ng/mL and the limit of detection (LOD)
was 7.06 ng/mL. The intra-assay and inter-assay coefficient of variations were 4.52% and 4.94%, respectively. The recovery
rate was 91.85%–102.45%. The ELISA method can provide a simple, accurate and stabile approach for the rapid detection
of bovine colostrum IgG.

Key words: bovine colostrum, immunoglobulin G (IgG), monoclonal antibody, sandwich ELISA

中图分类号:

TS252.7

刘金，王丽威，岳喜庆. 牛初乳中免疫球蛋白G双抗夹心ELISA检测方法的建立[J]. 食品科学, doi: 10.7506/spkx1002-6630-201614013.

LIU Jin, WANG Liwei, YUE Xiqing. Development of Double Antibody Sandwich ELISA for Detection of Bovine Colostrum Immunoglobulin G[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201614013.

参考文献

[1] 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.

[2] 徐丽, 生庆海, 王玉良, 等. 牛初乳制品功能性成分的分

析[ J ] . 中国乳品工业, 2004, 32(4): 18-20. DOI:10.3969/

j.issn.1001-2230.2004.04.005.

[3] GOMES V, MADUREIRA K M, SORINANO S, et al. Factors

affecting immunoglobulin concentration in colostrum of healthy

Holstein cows immediately after delivery[J]. Pesquisa Veterinária

Brasileira, 2011, 31(Suppl 1): 53-56. DOI:10.1590/S0100-

736X2011001300009.

[4] MORETTI D B, NORDI W M, LIMA A L, et al. Lyophilized bovine

colostrum as a source of immunoglobulins and insulin-like growth

factor for newborn goat kids[J]. Livestock Science, 2012, 145(1/2/3):

223-229. DOI:10.1016/j.livsci.2012.02.007.

[5] NEAVE H W, VICKERS L, VEIRA D M. Preparation of a highimmunoglobulin

product from bovine colostrum[J]. Animal Feed

Science and Technology, 2013, 179(1/2/3/4): 133-137. DOI:10.1016/

j.anifeedsci.2012.11.007.

[6] 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.

[7] 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.

[8] 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 G

concentrations[J]. Journal of Dairy Science, 2012, 95(5): 2697-2702.

DOI:10.3168/jds.2011-5220.

[9] ABERNETHY G, OTTER D. Determination of immunoglobulin G

in bovine colostrum and milk powders, and in dietary supplements

of bovine origin by protein g affinity liquid chromatography:

collaborative study[J]. Journal of AOAC International, 2010, 93(2):

622-627.

[10] HOLLAND P T, CARGILL A, SELWOOD A L, et al. Determination

of soluble immunoglobulin g in bovine colostrum products by protein g

affinity chromatography-turbidity correction and method validation[J].

Journal of Agricultural and Food Chemistry, 2001, 59(10): 5248-5256.

DOI:10.1021/jf200839b.

[11] 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.

[12] TEIXEIRA A G V, BICALHO M L S, MACHADO V S, et al. Heat

and ultraviolet light treatment of colostrum and hospital milk: effects

on colostrum and hospital milk characteristics and calf health and

growth parameters[J]. The Veterinary Journal, 2013, 197(2): 175-181.

DOI:10.1016/j.tvjl.2013.03.032.

[13] SOUSA S G, SANTOS M D, FIDALGO L G, et al. Effect of thermal

pasteurisation and high-pressure processing on immunoglobulin

content and lysozyme and lactoperoxidase activity in human

colostrum[J]. Food Chemistry, 2014, 151: 79-85. DOI:10.1016/

j.foodchem.2013.11.024.

[14] VERWEIJ J J, KOETS A P, EISENBERG S W F. Effect of continuous

milking on immunoglobulin concentrations in bovine colostrum[J].

Veterinary Immunology and Immunopathology, 2014, 160(3/4): 225-

229. DOI:10.1016/j.vetimm.2014.05.008.

[15] GOSCH T, APPRICH S, KNEIFEL W, et al. A combination of

microfiltration and high pressure treatment for the elimination of

bacteria in bovine colostrum[J]. International Dairy Journal, 2014,

34(1): 41-46. DOI:10.1016/j.idairyj.2013.06.014.

[16] MILA H, FEUGIER A, GRELLETA, et al. Immunoglobulin G

concentration in canine colostrum evaluation and variability[J].

Journal of Reproductive Immunology, 2015, 112: 24-28. DOI:10.1016/

j.jri.2015.06.001.

[17] 任立松, 卢士英, 周玉, 等. 柠檬黄色素免疫学检测方法的

研究[ J ] . 食品科学, 2009, 30(12): 258-261. DOI:10.3321/

j.issn:1002-6630.2009.12.059.

[18] 张莹, 周玉, 李岩松, 等. 抗κ-酪蛋白单克隆抗体的制备及其特性鉴

定[J]. 中国畜牧兽医, 2014, 41(3): 24-27.

[19] 卫生部. GB/T 5009.194—2003 保健品中免疫球蛋白IgG的测定[S].

北京: 中国标准出版社, 2003: 537-542.

[20] 国家质量监督检验检疫总局. SN/T 3132—2012 出口牛乳制品中牛

免疫球蛋白G的测定: 酶联免疫法[S]. 北京: 中国标准出版社,

2012.

[21] CONESA C, LAVILLA M, SANCHEZ L, et al. Determination of IgG

levels in bovine bulk milk samples from different regions of Spain[J].

European Food Research and Technology, 2005, 220(2): 222-225.

DOI:10.1007/s00217-004-1016-0.

[22] ABDEL-SALAMA Z, GHANY S H, HARITH M A. Evaluation

of immunoglobulins in bovine colostrum using laser induced

fluorescence[J]. Talanta, 2014, 129: 15-19. DOI:10.1016/

j.talanta.2014.04.033.

[23] 马燕芬, 陈志伟, 魏晓兵, 等. 高效液相色谱法测定牛初乳中IgG

含量的方法研究[J]. 饲料工业, 2007, 28(5): 33-38. DOI:10.3969/

j.issn.1001-991X.2007.05.010.