抗CEA单抗-天花粉蛋白偶联物的研制及活性鉴定

PREPARATION OF CEA MONOCLONAL ANTIBODY-TRICHOSANTHIN CONJUGATE AND STUDY OFITS BIOLOGICAL ACTIVITY

应用SPDP化学偶联法,将天花粉蛋白通过二硫键交联与抗CEA单抗制备成免疫毒素,经Sepbadex G-150凝胶过滤和Blue Sepharose-4B离子梯度交换层析纯化,电泳鉴定显示这一免疫毒素纯度高,偶联蛋白克分子比合理。应用间接免疫荧光染色技术和ELISA法测定结果证明,其与CEA人结肠癌细胞(LoVo)具有特异结合反应,仍保留大部份原抗体活性。具有较好的导向性。体外毒性试验结果表明,免疫毒素对靶细胞(LoVo)有选择性的杀伤作用,50％细胞毒剂量(I.C50)为10^(-9)范围,比单用天花粉蛋白和天花粉蛋白与正常小鼠IgG-偶联物对靶细胞杀伤能力分别增强约150倍和2000倍,而对实验对照细胞(Hela)杀伤无增强作用。

Trichosanthin(TCS) was conjugated via a disulf ide bond with CEA MAb (B3IgG) which had been first crosslinked with N-succinimidyl-3 (2pyridyl)-dithiopropionate (SPDP)to form the immunotoxin(IT).B3IgG-TCS. On SDS-PAGE,neither free TCS nor B3IgG was dete ted to be present in the IT that had been purified through Sephadex G-150 gel filtration and Blue Sepharose-4B ion gradient exchange chromatography. The IT was verified to be well constructed (TCS/B3IgG mole ratio: 1-2/1).Our results showed that the B3IgG moiety of IT molecules retained the ability of specific binding to the CEA possitively or CEA expressed human colonic cacinoma cells (LoVo)as determined by indirect immunoflurescence technique and ELISA method.The IT showed selective killing activity to target cell lines (LoVo) in vitro. The concentration of fifty percent cytotoxicity(I. C.50)was about 10-9M for LoVo cells, thus the IT was approximately 150 times stronger than free TCS or 2000-times strongerthan the conjugate of TCS with normal mouse IgG (TCS-N-MIgG). Moreover, IT showed no enhanced killing capability for the CEA-negative carcinoma cells (HeLa) in control study.