杂交-杂交瘤技术制备BsMAb的研究进展及其在食品安全检测中的应用

doi:10.7506/spkx1002-6630-201613045

摘要/Abstract

摘要：

双特异性单克隆抗体（bispecific monoclonal antibody，BsMAb）是指具有两个不同特异性抗原结合位点的单克隆抗体分子。杂交-杂交瘤技术因其简便易行、制备周期短，在制备BsMAb方面有较大优势。基于杂交-杂交瘤技术能制备出可以特异性识别两种或者两类小分子药物的BsMAb，在食品安全多残留免疫分析检测中具有较好的应用价值。本文综述了杂交-杂交瘤技术制备BsMAb的进展，探讨了基于该技术的BsMAb生成机制，讨论分析了杂交-杂交瘤及BsMAb筛选制备的关键技术要点，并归纳了BsMAb在检测食品中小分子污染物方面的应用进展，旨在为食品安全多残留免疫分析技术的发展应用提供参考。

关键词: 杂交-杂交瘤技术, 双特异性单克隆抗体, 多残留, 免疫分析

Abstract:

Bispecific monoclonal antibodies (BsMAb) are monoclonal antibody molecules with two different specific
antigen-binding sites. Hybrid-hybridoma technology has a great advantage in the preparation of BsMAb because of its easy
operation and less time consumption. BsMAbs with 2 distinct antigen-binding sites produced by the hybrid-hybridoma
technology could specifically recognize antigens of 2 drugs or 2 classes of drugs, and are applicable in the multi-residue
determination of foods. In this paper, we elaborate recent progress in the development of hybrid-hybridoma technology and
explore the mechanism of BsMAb generation by the hybrid-hybridoma method. Several important factors influencing the
hybrid-hybridoma technology are discussed, and the application of BsMAb in food safety detection is also reviewed. This
paper will provide some references for the development and application of BsMAb in multi-residue immunoassay of foods.

Key words: hybrid-hybridoma technology, bispecific monoclonal antibody (BsMAb), multi-residue, immunoassay

中图分类号:

TS207.3

王锋，王宇，王弘，肖治理. 杂交-杂交瘤技术制备BsMAb的研究进展及其在食品安全检测中的应用[J]. 食品科学, doi: 10.7506/spkx1002-6630-201613045.

WANG Feng, WANG Yu, WANG Hong, XIAO Zhili. Progress in Hybrid-Hybridoma Technology to Produce Bispecific Monoclonal Antibody and Its Application in Food Safety Detection[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201613045.

参考文献

[1] MILSTEIN C, CUELLO A C. Hybrid hybridomas and their

use in immunohistochemistry[J]. Nature, 1983, 305: 537-540.

DOI:10.1038/305537a0.

[2] RADER C. Chemically programmed antibodies[J]. Trends

i n B i o t e c h n o l o g y , 2 0 1 4 , 3 2 ( 4 ) : 1 8 6 - 1 9 7 . D O I : 1 0 . 1 0 1 6 /

j.tibtech.2014.02.003.

[3] SHEKHAR C. Double Whammy: bispecific antibodies help immune

cells attack tumors[J]. Chemistry Biology, 2008, 15(9): 877-878.

DOI:10.1016/j.chembiol.2008.09.002.

[4] BYRNE H, CONROY P J, WHISSTOCK J C, et al. A tale of two

specificities: bispecific antibodies for therapeutic and diagnostic

applications[J]. Trends in Biotechnology, 2013, 31(11): 621-632.

DOI:10.1016/j.tibtech.2013.08.007.

[5] YAN X, LI H X, YAN Y, et al. Developments in pesticide analysis by

multi-analyte immunoassays: a review[J]. Analtical Methods, 2014,

6(11): 3543-3554. DOI:10.1039/C3AY41946K.

[6] 李菁, 林彤, 宋帅, 等. 基因工程抗体研究进展[J]. 生物技术通报,

2009(10): 40-44.

[7] KONTERMANN R E. Bispecific antibodies[M]. Germany: Springer

Press, 2011: 29-46. DOI:10.1007/978-3-642-20910-9.

[8] KOOLWIJK P, ROZEMULLER E, STAD R K, et al. Enrichment

and selection of hybrid hybridomas by percoll density gradient

centrifugation and fluoreseent-aetivated[J]. Hybridoma, 1988, 7(2):

217-224. DOI:10.1089/hyb.1988.7.217.

[9] 刘静, 殷竹君, 张耀娟, 等. 次黄嘌呤-鸟嘌呤磷酸核糖转移酶缺陷型

杂交瘤细胞的快速筛选方法[J]. 南京医科大学学报(自然科学版),

2000, 20(3): 179-180. DOI:10.3969/j.issn.1007-4368.2000.03.006.

[10] WANG W W, DAS D, TANG X L, et al. Antigen targeting to

dendritic cells with bispecific antibodies[J]. Journal of Immunological

Methods, 2005, 306(1): 80-92. DOI:10.1016/j.jim.2005.07.023.

[11] LIU F, GUTTIKONDA S, SURESH M R. Bispecific monoclonal

antibodies against a viral and an enzyme: utilities in ultrasensitive

virus ELISA and phage display technology[J]. Journal of

Immunological Methods, 2003, 274(1): 115-127. DOI:10.1016/S0022-

1759(02)00511-2.

[12] SHINMOTO H, KOBORI M, TSUSHIDA T, et al. Competitive

ELISA of bovine lactoferrin with bispecific monoclonal antibodies[J].

Bioscience Biotechnology Biochemistry, 1997, 61(6): 1044-1046.

DOI:10.1271/bbb.61.1044.

[13] CAO Y, CHRISTIAN S, SURESH M R. Development of a bispecific

monoclonal antibody as a universal immunoprobe for detecting

biotinylated macromolecules[J]. Journal of Immunological Methods,

1998, 220(1): 85-91. DOI:10.1016/S0022-1759(98)00154-9.

[14] AURIOL J, GUESDON J, MAZIE J C, et al. Development of a

bispecific monoclonal antibody for use in molecular hybridisation[J].

Journal of Immunological Methods, 1994, 169(1): 123-133.

DOI:10.1016/0022-1759(94)90131-7.

[15] GANGULY A , MALA B A D I R B , LO B EN B E R G R , e t a l .

Development of an ultrasensitive hetero-sandwich ELISA assay

based on bispecific monoclonal antibody for the detection of dengue

NS1 protein[J]. Journal of Pharmacy Research, 2013, 7(5): 374-380.

DOI:10.1016/j.jopr.2013.05.013.

[16] MORECKI S, LINDHOFER H, YACOVLEV E, et al. Induction of

long-lasting antitumor immunity by concomitant cell therapy with

allogeneic lymphocytes and trifunctional bispecific antibody[J].

Experimental Hematology, 2008, 36(8): 997-1003. DOI:10.1016/

j.exphem.2008.03.005.

[17] 李荔, 陈伯权. 抗肾综合征出血热病毒和抗辣根过氧化物酶的双特

异性单克隆抗体的建立[J]. 病毒学报, 1989, 5(4): 334-339.

[18] 袁洪卫, 林汉, 朱锡霞, 等. 抗纤维蛋白-抗尿激酶双特异性单克隆抗

体的体内外溶栓效果的研究[J]. 中国医学科学院学报, 1998, 20(5):

357-360.

[19] SUNWOO H H, PALANIYAPPAN A, GANGULY A, et al.

Quantitative and sensitive detection of the SARS-CoV spike

protein using bispecific monoclonal antibody-based enzyme-linked

immunoassay[J]. Journal of Virological Methods, 2013, 187(1): 72-78.

DOI:10.1016/j.jviromet.2012.09.006.

[20] FERRINI S, PRIGIONE I, MIOTTI S, et al. Bispecific monoclonal

antibodies directed to CD16 and to a tumor-associated antigen induce

target-cell lysis by resting NK cells and by a subset of NK clones[J].

International Journal of Cancer, 1991, 48(2): 227-233. DOI:10.1002/

ijc.2910480213.

[21] LINDHOFER H, MOCIKAT R, STEIPE B, et al. Preferential

species-restricted heavy/light chain pairing in rat/mouse quadromas:

implications for a single-step purification of bispecific antibodies[J].

Journal of Immunology, 1995, 155(1): 219-225.

[22] 苏瑾, 张亚莉, 郭志兵, 等. 细胞融合法制备抗人CD3-抗人IgM μ链

双特异性抗体[J]. 第一军医大学学报, 2001, 21(12): 902-905.

[23] 王廷华, 李官成. 抗体理论与技术[M]. 北京: 科学出版社, 2013: 31-33.

[24] M O L D E N H A U E R G . B i s p e c i f i c a n t i b o d i e s f r o m h y b r i d

hybridoma[M]. Berlin: Springer, 2011: 29-46.

[25] KUFER P, LUTTERBÜSE R, BAEUERLE P A. A revival of

bispecific antibodies[J]. Trends in Biotechnology, 2004, 22(5):

238-244. DOI:10.1016/j.tibtech.2004.03.006.

[26] 居漪, 徐肇华, 胡畅中. 亲和层析法提纯双特异性单克隆抗体[J]. 上

海免疫学杂志, 1998, 18(5): 302-305.

[27] M A S S I N O Y S , D E R G U N O V A N N , K I Z I M E A , e t a l .

Quantitative analysis of the products of IgG chain recombination

in hybrid hybridomas based on affinity chromatography and

radioimmunoassay[J]. Journal of Immunological Methods, 1997,

201(1): 57-66. DOI:10.1016/S0022-1759(96)00212-8.

[28] TAKAHASHI M, FULL S A. Production of murine hybrid-hybridomas

secreting bispecific monoclonal antibodies for use in urease-based

immunoassays[J]. Clinical Chemistry, 1988, 34(9): 1693-1696.

[29] STAERZ U D, BEVAN M J. Hybrid hybridoma producing a bispecific

monoclonal antibody that can focus effector T-cell activity[J].

Proceedings of the National Academy of Sciences of the United Stases

of America, 1986, 83(5): 1453-1457. DOI:10.1073/pnas.83.5.1453.

[30] de PREVAL C, FOUGEREAU M. Specific interaction between VH

and VL regions of human monoclonal immunoglobulins[J]. Journal

of Molecular Biology, 1976, 102(3): 657-678. DOI:10.1016/0022-

2836(76)90340-5.

[31] KRANZ D M, VOSS E W. Restricted reassociation of heavy and light

chains from hapten-specific monoclonal antibodies[J]. Proceedings of

the National Academy of Sciences of the United Stases of America,

1981, 78(9): 5807-5811. DOI:10.1073/pnas.78.9.5807.

[32] STEVENSON G T, MOLE L E. The specificity of chain interactions

among immunoglobulins. combinations of gamma chains with kappa

chains of the same subgroup as in the parent immunoglobulin G[J].

Biochemical Journal, 1974, 139(2): 369-374. DOI:10.1042/bj1390369.

[33] 裘雪梅, 刘仁荣, 徐玲, 等. 次黄嘌呤-鸟嘌呤磷酸核糖转移酶缺陷

型产抗苏丹红I单克隆抗体杂交瘤细胞的筛选[J]. 时珍国医国药,

2013, 23(11): 2669-2670.

[34] 刘瑛, 陶玉珍, 陈受霓. 分泌抗HBsAg-抗HRP双特异单克隆抗体杂

交-杂交瘤的建株[J]. 中国生物制品学杂志, 1992, 5(3): 111-113.

[35] 孟文霞, 王润田, 张玉芬, 等. 抗HIV p24和人A型红细胞双特异性单

克隆抗体的研制[J]. 细胞与分子免疫学杂志, 2004, 20(5): 582-584.

[36] 李刚. 抗吡虫啉-甲基对硫磷双特异性单克隆抗体的研究[D]. 南京:

南京农业大学, 2011: 1-85.

[37] de LAU W B, HEIJE K, NEEFJES J J, et al. Absence of preferential

homologous H/L chain association in hybrid hybridomas[J]. Journal

Immunology, 1991, 146(3): 906-914.

[38] 全昱东, 赵莉, 程明, 等. 角蛋白和过氧化物酶双特异性杂交杂交瘤

的建立[J]. 单克隆抗体通讯, 1990, 6(1): 20-23.

[39] 刘明旭, 张瑛勋, 王建安, 等. 抗TNF和抗CALLA三体瘤的研制及其

分泌抗体的初步纯化[J]. 单克隆抗体通讯, 1993, 9(2): 1-4.

[40] 徐青, 汪厚平, 巫山, 等. 抗人IgG-抗HRP双特异性单克隆抗体细胞

株的建立与鉴定[J]. 免疫学杂志, 2000, 16(3): 225-227; 238.

[41] LANZAVECCHIA A, SCHEIDEGGER D. The use of hybrid

hybridomas to target human cytotoxic T lymphocytes[J]. European

Journal of Immunology, 1987, 17(1): 105-111. DOI:10.1002/

eji.1830170118.

[42] 王宇. 孔雀石绿及其代谢物免疫分析技术研究[D]. 广州: 华南农业

大学, 2010: 112-123.

[43] BRANSCOMB E E, RUNGE M S, SAVARD C E, et al. Bispecific

monoclonal antibodies produced by somatic cell fusion increase

the potency of tissue plasminogen activator[J]. Thrombosis and

Haemostasis, 1990, 64(2): 260-266.

[44] SARKAR S, TANG X L, DAS D, et al. A bispecific antibody based

assay shows potential for detecting tuberculosis in resource constrained

laboratory settings[J]. PLoS ONE, 2012, 7(2): e32340. DOI:10.1371/

journal.pone.0032340.

[45] KARAWAJEW L, MICHEEL B, BEHRSING O, et al. Bispecific

antibody-producing hybrid hybridomas selected by a fluorescence

activated cell sorter[J]. Journal of Immunological Methods, 1987,

96(2): 265-270. DOI:10.1016/0022-1759(87)90323-1.

[46] ARAGAY G, PINO F, MERKOCI A. Nanomaterials for sensing and

destroying pesticides[J]. Chemical Reviews, 2012, 112(10): 5317-5338.

DOI:10.1021/cr300020c.

[47] 陈晓钎, 沈德诚, 孙同哲. CD3/CD19双特异性单克隆抗体的制

备、鉴定及其再导向的研究[J]. 中国医学科学院学报, 1998,

20(5): 351-356.

[48] 崔运昌. 单克隆抗体研究新进展(双特异性单克隆抗体和杂交-杂交

瘤技术)[J]. 第四军医大学学报, 1986, 7(2): 175-176.

[49] MOROZOVA V S, LEVASHOVA A I, EREMIN S A. Determination

of pesticides by pesticide by enzyme immunoassay[J]. Journal of

Analytical Chemistry, 2005, 60(3): 202-217. DOI:10.1007/s10809-

005-0075-0.

[50] ZHOU Q, PENG D P, WANG Y L, et al. A novel hapten and

monoclonal-based enzyme-linked immunosorbent assay for

sulfonamides in edible animal tissues[J]. Food Chemistry, 2014, 154:

52-62. DOI:10.1016/j.foodchem.2014.01.016.

[51] GAO B L, LIU J, DONG L X, et al. Broad specific enzyme-linked

immunosorbent assay for determination of residual phenothiazine

drugs in swine tissues[J]. Analtical Biochemistry, 2014, 454(1): 7-13.

DOI:10.1016/j.ab.2014.02.032.

[52] DONG J X, LI Z F, LEI H T, et al. Development of a single-chain

variable fragment-alkaline phosphatase fusion protein and a sensitive

direct competitive chemiluminescent enzyme immunoassay for

detection of ractopamine in pork[J]. Analtical Chimica Acta, 2012,

736: 85-91. DOI:10.1016/j.aca.2012.05.033.

[53] JIANG H, FAN M T. Multi-analyte immunoassay for pesticides: a

review[J]. Analtical Letters, 2012, 45(11): 1347-1364. DOI:10.1080/0

0032719.2012.675493.

[54] 金仁耀, 朱国念. 基于双特异性单克隆抗体的克百威和三唑磷多残

留ELISA分析方法研究及在环境样品中应用[J]. 核农学报, 2013,

27(4): 515-522; 531.

[55] OUYANG H, WANG L M, YANG S J, et al. Chemiluminescence

reaction kinetics-resolved multianalyte immunoassay strategy using a

bispecific monoclonal antibody as the unique recognition reagent[J].

Analtical Chemistry, 2015, 87(5): 2952-2958. DOI:10.1021/

ac5045093.

[56] HUA X D, WANG L M, LI G, et al. Multi-analyte enzymelinked

immunosorbent assay for organophosphorus pesticides and

neonicotinoid insecticides using a bispecific monoclonal antibody[J].

Analtical Methods, 2013, 5(6): 1556-1563. DOI:10.1039/c3ay26398c.

[57] 余厚美, 崔廷婷, 冯才伟, 等. 抗3种β-激动剂双特异性单克隆抗体的

建立与鉴定[J]. 化学试剂, 2012, 34(7): 607-611.

[58] GUO Y R, LIU S Y, GUI W J, et al. Gold immunochromatographic

assay for simultaneous detection of carbofuran and triazophos in

water samples[J]. Analtical Biochemistry, 2009, 389(1): 32-39.

DOI:10.1016/j.ab.2009.03.020.