抗癌胚抗原单抗与生物素及链霉亲和素偶联物的制备及性能鉴定

Preparation and bioactivity evaluation of the conjugates of carcinoembryonic antigen monoclonal antibody with biotin or streptavidin

目的 研究抗癌胚抗原(CEA)单抗与生物素及链霉亲和素(streptavidin, SA)偶联物的制备方法。方法 将CEA单抗按抗体与生物素活化酯物质的量之比为1:15～1:50进行生物素化。抗CEA单抗与SA的偶联采用3-（2-吡啶二巯基丙酸）-N-琥珀酰亚胺酯（SPDP）化学偶联法制备。抗CEA单抗偶联物的生物活性及免疫活性分别采用间接ELISA方法和放射免疫分析法进行测定。 结果 生物素化单抗每分子抗体中约偶联3分子生物素，具有良好的SA结合活性，经SDS-PAGE电泳证实为单一蛋白条带，仍保留95%的免疫活性。SA每分子中含有1～2个巯基，而抗CEA单抗每分子中含有2～3个3-（2-吡啶二巯基丙酸）-N-琥珀酰亚胺酯间接ELISA方法证实偶联物具有良好的生物素结合活性， SDS-PAGE电泳显示单抗-SA偶联物相对分子质量为210 000左右，仍保留着原单抗活性的70%左右。结论 所制备的两类抗CEA单抗偶联物基本保持完整的特异性结合肿瘤CEA的性质，可为进一步的预定位显像和治疗应用提供可靠的靶向载体。

Objective To explore the approach to conjugate streptavidin or biotin with carcinoembryonic antigen (CEA) monoclonal antibody (CEA mAb) for future studies of pretargeting technique with the avidin-biotin system. Methods Biotin was conjugated to CEA mAb using biotin reagent N-hydroxysuccinimido-biotin and streptavidin was conjugated via a disulfide bond with CEA mAb that was previously crosslinked with N-succinimidyl-3(2-pyridyl)-dithiopropionate (SPDP) to form the immunoconjugate (CEA mAb-SA). The immunoreactivity of the immunoconjugates was evaluated by means of radioimmunoassay, and the biotin or streptavidin-binding capacity tested by indirect enzyme-linked immunoassay. Results On average, each CEA mAb molecule conjugates with 3 biotin molecules, and the resulted the biotinylated CEA mAb retained 95% immunoreactivity of its original antibody and the homogeneity of a pure protein (with relative molecular weight of 150 000) as shown by SDS-PAGE analysis. Each streptavidin molecule possesses 1 or 2 sulfhydryl and a CEA mAb molecule may conjugate approximately 2 or 3 PDP, with the final CEA mAb-SA having a relative molecular weight of 210 000 and the CEA-binding activity about 70% of the original. Indirect enzyme-linked immunoassay showed that immunoconjugate of CEA mAb with biotin or streptavidin had good binding capacity with streptavidin or biotin. Conclusion The immunoconjugates of CEA mAb with streptavidin and biotin retain specific binding capacity to CEA, which provides reliable carrier for pretargeting radioimmunoimaging and therapy.