食品科学 ›› 2010, Vol. 31 ›› Issue (13): 180-183.doi: 10.7506/spkx1002-6630-201013041

• 生物工程 • 上一篇    下一篇

重金属铅多克隆抗体的制备及鉴定

翟一凡1,向军俭1 ,* ,唐 勇1,郭常伟1,刘小锋2,杨红宇1   

  1. 1.暨南大学抗体工程中心
    2.广州市千江企业有限公司
  • 收稿日期:2009-11-24 出版日期:2010-07-01 发布日期:2010-12-29
  • 通讯作者: 向军俭 E-mail:txjj@jnu.edu.cn
  • 基金资助:

    国家“863”计划项目(2008AA10Z427);广州市天河区科技计划项目(085G079)

Preparation and Identification of Anti-Lead-chelate Complex Polyclonal Antibody

ZHAI Yi-fan1,XIANG Jun-jian1,*,TANG Yong1,GUO Chang-wei1,LIU Xiao-feng2,YANG Hong-yu1   

  1. 1. Antibody Engineering Center, Jinan University, Guangzhou 510632, China ;
    2. Guangzhou Rivers Enterprise Co. Ltd., Guangzhou 510632, China
  • Received:2009-11-24 Online:2010-07-01 Published:2010-12-29
  • Contact: XIANG Jun-jian E-mail:txjj@jnu.edu.cn

摘要:

目的:合成铅离子完全抗原,并对其进行鉴定;用合成的完全抗原免疫Balb/c 小鼠制备多克隆抗体,鉴定效价和特异性。方法:用双功能螯合剂p-SCN-Bn-DTPA 将Pb2+ 与载体蛋白BSA 和OVA 偶联在一起,合成免疫抗原(Pb-DTPA-BSA)和检测抗原(Pb-DTPA-OVA),并通过SDS-PAGE、紫外扫描、电感耦合等离子体(ICP) 检测抗原中Pb2+ 含量,以及通过免疫Balb/c 小鼠,采集血清进行间接ELISA 和竞争ELISA 检测。结果:两种合成的抗原相对于各自的载体蛋白均出现了蛋白条带滞后,紫外吸收峰偏移等现象;免疫的4 号和5 号Balb/c 小鼠的抗血清效价达1:400000;与OVA 无交叉反应,ELISA 检测IC50 值为1.5ng/mL,与Fe3+ 有近5% 的交叉反应,与其他重金属离子的交叉反应均小于1%。结论:铅离子多克隆抗体制备成功。

关键词: 铅离子, 合成抗原, ELISA, 多克隆抗体

Abstract:

Objective: BAS and OVA were individually conjugated with lead-chelate complex (Pb-DTPA) to prepare 2 antigens, namely Pb-DTPA-BSA, immunogen and Pb-DTPA-OVA, detective antigen. The synthesized antigens were then used to immunize BALB/c mice for preparing polyclonal antibodies. Finally, the antisera withdrawn from immunized BALB/c mice were assessed for their titers and specificities. Methods: The artificial antigens were identified by SDS-PAGE and scanned with ultraviolet spectrophotometer. The Pb2+ concentrations of the synthesized antigens were determined using inductively coupled plasma (ICP) spectrophotometer. Indirect ELISA and competitive ELISA were used to determine antiserum titer and specificity, respectively. Results: Compared with carrier proteins, the synthesized antigens exhibited lagged protein bands in SDS-PAGE pattern and blue shift of UV absorption peaks. The antiserum titers of the fourth and fifth mice reached up to 1:400,000 without cross-reactivity with OVA. The IC50 determined by IC-ELISA was 1.5 ng/mL and the cross reactivity with the other heavy ions were less than 1% although the cross-reactivity with Fe3+ was close to 5%. Conclusion: The above results suggest that anti-leadchelate complex polyclonal antibodies are prepared successfully.

Key words: lead ion, antigen synthesis, ELISA, polyclonal antibody

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