抗赭曲霉毒素B单克隆抗体的制备与鉴定

Production and characterization of monoclonal antibodies against ochratoxin B.

作者信息

Heussner Alexandra H, Moeller Ines, Day Billy W, Dietrich Daniel R, O'Brien Evelyn

机构信息

Environmental Toxicology, University of Konstanz, P.O. Box X-918, 78457 Konstanz, Germany.

出版信息

Food Chem Toxicol. 2007 May;45(5):827-33. doi: 10.1016/j.fct.2006.10.027. Epub 2006 Nov 7.

DOI:10.1016/j.fct.2006.10.027

PMID:17161512

Abstract

Monoclonal antibodies against ochratoxin B (OTB) were generated by immunizing Balb/c mice with OTB conjugated to keyhole limpet hemocyanin (KLH) via carbodiimide reactions with CHMC and EDAC. A stable hybridoma cell line 2F1.E10 was produced by fusion of murine splenocytes and myeloma cells. The obtained antibodies were characterized using an indirect competitive ELISA. The detection limit was calculated (27+/-2 nM OTB) and 50% binding inhibition was reached at 500 nM free OTB. A low cross-reactivity to ochratoxin A (OTA) of 3.3% and no cross-reactivities to either coumarin or DL-phenylalanine were observed, suggesting a highly specific OTB antibody. The antibody type was identified as IgG class 1 with the light chain being of the kappa configuration. These antibodies can be used in an indirect competitive ELISA to detect OTB in the nanomolar to micromolar concentration range and may be useful for the analysis of contaminated food items.

摘要

通过用经碳二亚胺反应与氯甲基碳酸酯（CHMC）和1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDAC）偶联到钥孔血蓝蛋白（KLH）上的赭曲霉毒素B（OTB）免疫Balb/c小鼠，产生了抗赭曲霉毒素B的单克隆抗体。通过鼠脾细胞与骨髓瘤细胞融合产生了稳定的杂交瘤细胞系2F1.E10。使用间接竞争ELISA对获得的抗体进行表征。计算出检测限（27±2 nM OTB），在500 nM游离OTB时达到50%结合抑制。观察到对赭曲霉毒素A（OTA）的交叉反应率低至3.3%，对香豆素或DL-苯丙氨酸均无交叉反应，表明该抗体具有高度特异性。抗体类型被鉴定为IgG1类，轻链为κ构型。这些抗体可用于间接竞争ELISA，以检测纳摩尔至微摩尔浓度范围内的OTB，可能有助于分析受污染的食品。