与海洛因有完全交叉反应的抗吗啡单抗的制备

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (07): 833-836.

与海洛因有完全交叉反应的抗吗啡单抗的制备

李琳¹, 刘洁², 朱平², 徐江平¹, 富宁²

1. 南方医科大学药理学教研室, 广东, 广州, 510515;
2. 南方医科大学免疫学教研室, 广东, 广州, 510515

出版日期:2005-07-20 发布日期:2005-07-20
基金资助:
收稿日期:2005-2-26。
基金项目:广州市科技攻关项目(2002Z3-E4102)
作者简介:李琳(1975- ),女,讲师,电话020-61648235,E-mail:li751in@fimmu.com
通讯作者:富宁,第一军医大学免疫学教研室,教授,电话:020-61648221, E-mail:zfzn@fimmu.com

Preparation of anti-morphine monoclonal antibodies with complete cross reactivity with heroin

LI Lin¹, LIU Jie², ZHU Ping², XU Jiang-ping¹, FU Ning²

1. 南方医科大学药理学教研室, 广东, 广州, 510515;
2. 南方医科大学免疫学教研室, 广东, 广州, 510515

Online:2005-07-20 Published:2005-07-20

摘要/Abstract

摘要： 目的制备吗啡的单克隆抗体(mAb)并鉴定抗体的特性。方法 6-琥珀酰化吗啡与Bluecarrier(BC)载体蛋白交联制备6-琥珀酰化吗啡酯(M-6-S-BC),用其免疫BALB/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合制备杂交瘤,经筛选和3次克隆化,从制备的腹水中纯化单克隆抗体。结果获得4株杂交瘤细胞G1、G3、H4和B1,其分泌单抗的亚型均为IgG1型。经ELISA鉴定,该单克隆抗体可结合游离的吗啡、海洛因及可待因,而且对海洛因的结合优于吗啡,该单抗不与非阿片类药物结合。结论所制备的吗啡单克隆抗体可特异性结合吗啡等阿片类药,将为建立体外吗啡及海洛因的非同位素检测方法奠定基础。

Abstract: Objective To prepare and characterize anti-morphine monoclonal antibodies (mAbs) with complete cross reactivity with heroin. Methods Morphine-6-succinyl was conjugated with Blue carrier (BC) in the presence of carbodi imide.BALB/c mice were immunized with the holoantigens (M-6-S-BC) and Freund adjuvant,and hybridoma was generated with mouse spleen cells and Sp2/0 myeloma cells.After 3 fusions and cloning,4 hybridoma cell lines secreting monoclonal antibodies to morphine were obtained and the mABs purified from the mouse ascites followed by characterization with enzyme-linked immunosorbent assay (ELISA). Results Four hybridoma cell lines secreting anti-morphine mAbs were established and designated as G1,G3,H4 and B1,respectively.All the mAbs belonged to IgG1 isotype and could bind to heroin better than to morphine and codeine,which were identified by competitive inhibition ELISA. Conclusion The anti-morphine mAbs can bind with the opioids,especially heroin,and can be useful for sensitive detection of opioids.

中图分类号:

R965

李琳¹, 刘洁², 朱平², 徐江平¹, 富宁². 与海洛因有完全交叉反应的抗吗啡单抗的制备[J]. 南方医科大学学报, 2005, 25(07): 833-836.

LI Lin¹, LIU Jie², ZHU Ping², XU Jiang-ping¹, FU Ning². Preparation of anti-morphine monoclonal antibodies with complete cross reactivity with heroin[J]. Journal of Southern Medical University, 2005, 25(07): 833-836.

参考文献

[1] Plotkowiak Z, Popielarz-Brzezinska M, Luczak J, et al. Quality assessment of morphine hydrochloride solutions [J]. Acta Poi Pharm, 2004, 61(2): 103-6.
[2] Hida M, Mitsui T, Tsuge S, et al. Rapid and sensitive determination of morphine in street opium samples by thermal desorption gas chromatography using a microfurnace pyrolyzer [J]. J Sep Sci,2004, 27(12): 1030-2.
[3] Fernandez P, Vazquez C, Morales L, et al. Analysis of opiates, cocaine and metabolites in urine by high-performance liquid chromatography with diode array detection (HPLC-DAD)[J]. J Appl Toxicol, 2005, 25(3): 200-4.
[4] Sabzevari O, Abdi Kh, Amini M, et al. Application of a simple and sensitive GC-MS method for determination of morphine in the hair of opium abusers[J]. Anal Bioanal Chem, 2004, 379(1): 120-4.
[5] Narongchai P, Sribanditmonkol P, Thampithug S, et al. The duration time of urine morphine detection in heroin addicts by radioimmunoassay[J]. J Med Assoc Thai, 2002, 85(1): 82-6.
[6] Akbarzadeh A, Mehraby M, Zarbakhsh M, et al. Design and synthesis of a morphine-6- succinyl-bovine serum albumin hapten for vaccine development [J]. Biotechnol Appl Biochem, 1999, 30(2):139-45.
[7] Cirimele V, Etienne S, Villain M, et al. Evaluation of the One-Step ELISA kit for the detection ofbuprenorphine in urine, blood, and hair specimens[J]. Forensic Sci Int, 2004, 143(2-3): 153-6.
[8] 李志勇.细胞工程[M].北京:科学出版社,2005.160-76.
[9] 李琳,赵文忠,杨淑琴,等.抗吗啡抗体的制备及其特性的评价[J].第一军医大学学报,2004,24(6):673-6.Li L, ZhaoWZ, Yang SQ, et al. Prepatation and characterization of anti-morphine antiserum [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2004, 24(6): 673-6.
[10] Rahbarizadeh F, Rasaee MJ, Madani R, et al. Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine[J]. Hybridoma, 2000, 19(5): 413-7.
[11] Aoki K, Shikama YS, Kokado A, et al. Enzyme linked immunosorbant assay and latex agglutination inhibition reaction test for morphine in urine[J]. Forensic Sci Int, 1996, 81: 125-32.
[12] Wainer BH, Fitch FW, Rothberg RM, et al. The structure of morphine momohe misuccinate[J]. Science, 1972, 178: 647-8.