抗破伤风毒素中和单克隆抗体筛选及重组表达

Screening and recombinant expression of neutralizing monoclonal antibodies against tetanus toxin

目的制备结合不同破伤风类毒素区域的中和性鼠源单抗,并对其进行人源化。方法首先,破伤风类毒素免疫BALB/c小鼠、细胞融合及筛选后,ELISA法和小鼠中和保护实验检测阳性杂交瘤细胞株表达上清,获得4个具有中和活性的单抗。其次,采用ELISA法确定它们结合位点的竞争关系,并用重组表达的Trx-HCC为抗原对其结合区域定位。最后,将其中2个单抗进行人源化、在CHO细胞中重组表达、纯化和测定中和活性。结果筛选出4株无相互竞争关系的中和活性单克隆抗体,分别命名为S3A4、9-7A4、9-8C7和S1D2;它们的中和效价分别为52IU/ml、53IU/ml、58IU/ml和60IU/ml,9-7A4和9-8C7特异性结合HCC段,而S3A4和S1D2特异性结合非HCC段,9-7A4和S3A4其人源化后的表达产物效价分别为38IU/mg和35IU/mg,两者混合使用时,其效价可达70IU/mg。结论获得2株人源化具有中和活性的抗破伤风毒素单克隆抗体,为破伤风药物治疗或疫苗效价检测等奠定了基础。

Objective To prepare murine neutralizing monoclonal antibodies(MAbs)binding with different tetanus toxoid regions and obtain their humanized MAbs.Methods Firstly,after BALB/c mice were immunized with tetanus toxoid and their B cells were fused and screened,the expression supernatant of positive hybridoma cells was detected by ELISA and protective test,and four MAbs with protective function were obtained.Secondly,the competitive relationships of their binding sites were determined by ELISA,and the recombinant Trx-HCC was also used as antigen to locate their binding region of tetanus toxoid.Lastly,Two of Mabs were humanized,expressed in CHO cells,purified and determined for neutralization activity.Results Four non-competitive MAbs named S3A4,9-7A4,9-8C7 and S1D2 were obtained,whose neutralizing activity were 52 IU/ml,53 IU/ml,58 IU/ml and 60 IU/ml,respectively.9-7A4 and 9-8C7 can bind specifically to HCC region of tetanus toxoid while S3A4 and S1D2 can bind specifically to non-HCC region.The neutralization activity of the humanized and expressed products of 9-7A4 and S3A4 were 38 IU/mg and 35 IU/mg,respectively,and the neutralization activity could reach 70 IU/mg when they were mixed.Conclusion Two humanized MAbs against tetanus toxin with neutralizing activity were obtained,which can lay the foundation for the treatment of tetanus drug or the titer detection of the vaccine.