抗河豚毒素单克隆抗体的制备及其抗毒效应

Preparation of monoclonal antibodies against tetrodotoxin and its anti-tetrodotoxin poisoning effect

目的 :为制备高毒性的小分子生物毒素河豚毒素 (TTX)的单克隆抗体 (mAb) ,探讨TTX中毒免疫抗毒的可能性。方法 :以TTX为半抗原 ,与中国鲎血蓝蛋白 (TTH)化学偶联制备人工抗原 (TTX_TTH) ;并以其免疫BALB c小鼠。用杂交瘤技术制备抗TTX的mAb ,用竞争抑制酶免疫分析法测定mAb对TTX结合活性 ,以mAb与检测抗原的结合被抑制 5 0 %时的TTX浓度 (IC50 )表示mAb的结合反应性。对小鼠预防注射抗体 ,测试抗体对抗TTX中毒的效果。结果 :建立了 2株对TTX_BSA阳性反应的mAb(1E4和 3C11) ,均属IgG1 亚类 ;具有TTX结合活性 ,其IC50 值分别为 3.2× 10 _6 mol L和 9.9× 10 _7mol L ;亲和力常数 (Kaff)分别为 3.0× 10 6 L mol和 2 .8× 10 6 L mol。小鼠预防给予mAbs ,可对抗 1×LD的TTX攻毒 ,动物存活率 71% ;抗 1.5×LD时 ,1 3存活 ;抗毒效果好于小鼠抗血清对照。结论 :用新的TTX人工抗原成功地制备了抗TTX的mAb ,具有一定的体内预防抗毒效价。

Objective:To develop monoclonal antibodies(mAbs)against neurotoxic tetrodotoxin (TTX), and to explore the possibility of immune prophylaxis and treatment of TTX intoxication.Methods:TTX was coupled to Tachypleus tridentatus hemocyanin(TTH) in presence of formaldehyde to form an artificial antigen(TTX-TTH),with which the BALB/c mice were immunized. MAbs were prepared by hybridoma technique. The quality of mAbs was examined with enzyme linked immunosorbent assay(ELISA) and competition-inhibited enzyme immunoassay(CIEIA). The binding activity of mAbs with TTX was defined by the TTX concentration (mol/L) inhibiting 50% of mAbs-detection antigen binding activity (IC 50 ). Mice were administered intraperitoneally with mAbs 8?h prior to challenge with lethal doses of TTX(1LD=13.5?μg/kg) in order to test the antitoxic effect.Results:Two TTX-specific mAbs with a titer as high as 10 5 to 10 6 in dilution were developed and named as 1E4 and 3C11. Both of them could react to TTX with the IC 50 values of 3.2×10 -6 mol/L and 9.9×10 -7 mol/L,respectively. Seventy one percent of mice administered with both mAbs survived 1×LD, and one third survived 1.5×LD of TTX challenge. MAbs appeared more effective both in antibody quality and anti-TTX poisoning efficacy than antisera of mice did.Conclusion:Two tetrodotoxin-specific mAbs were developed successfully, and could neutralize TTX in vivo. The antitoxic effect of mAbs in vivo suggested that immunoprophylaxis be the hopeful means for detoxification of TTX.