抗HPT单克隆抗体的制备与生物学特性鉴定

目的：制备抗潮霉素B磷酸转移酶(HPT)的单克隆抗体(McAb)，建立一种快速检测转基因作物中该选择标记基因HPT编码蛋白的方法。方法：用基因重组潮霉素B磷酸转移酶(HPT)抗原免疫BALB/C小鼠，采用杂交瘤技术制备McAb。选择不同的抗原决定簇与兔抗HPT多抗配对，建立双抗夹心ELISA检测HPT抗原。结果：筛选出四株稳定分泌抗HPT单抗的杂交瘤细胞株，IgG亚类鉴定均为IgG1，ELISA检测证实单抗可特异性识别细胞培养上清、重组子菌体裂解产物中及纯化出的HPT蛋白。结合位点测定实验表明4株单抗是针对不同的抗原决定簇，与多克隆抗体组成双抗夹心ELISA法，均可较好地检测到HPT抗原。检测的灵敏度为30ng／ml，且与别的无关抗原无交叉反应性。结论：4株杂交瘤细胞株特异性好，亲和力强，组成双抗夹心ELISA法可用于快速、灵敏检测HPT抗原。

Preparation and identification of the anti-HPT monoclonal antibodies

Objective:To produce monoclonal antibodies(McAb)against hygromycin B phosphotransferase(HPT) and to establish a rapid method for the measurement of HPT antigen in the genetically modified crops(GMC).Methods:Recombinant hygromycin B phosphotransferase (HPT) was used to immune BALB/C mice.Monoclonal antibodies against HPT were produced from hybridoma.A double-Ab(McAb and polyclonal antibodies of HPT) sandwich ELISA to detect HPT was established.Results:Four hybridomasproducing antibodies against HPT were obtained.IgG isotypes of four McAb were IgG1.The antibodies were proved to be specific for HPT and recognized different epitopes on HPT by ELISA.The sandwich ELISA for HPT showed a sensitivity of 30 ng/ml and had no crossing-reaction with other proteins.Conclusion:These results show four hybridomas producing high specificity and affinity monoclonal antibodies angaist HPT and can provide for rapid assay for the measurement of HPT antigen.