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背景与目的:前哨淋巴结活检是临床腋窝淋巴结阴性早期乳腺癌患者治疗的标准。准确定位前哨淋巴结对分期、预后及治疗至关重要。该研究将利妥昔单抗与荧光示踪剂吲哚菁绿偶联,制备新型前哨淋巴结示踪剂,确定最佳偶联比例,并对其生物学特性、安全限度及定位性能进行研究。方法:直接偶联法制备新型前哨淋巴结吲哚菁绿-利妥昔单抗,双层析快速薄层层析-硅胶层析纸法测定标记率,非还原型SDS聚丙烯酰胺凝胶电泳法和双抗体夹心间接酶联免疫测定法检测新型示踪剂中单抗分子完整性和免疫活性,按中华药典要求检测新型示踪剂的安全限度及在小鼠体内前哨淋巴结的定位性能。结果:新型示踪剂中利妥昔单抗分子完整且保持了单抗的免疫活性,利妥昔单抗大分子上吲哚菁绿的标记率为100%,新型示踪剂为无菌、无致热原的溶液且局部注射不会产生危害。利妥昔单抗与吲哚菁绿质量比例为4∶1、6∶1偶联形成的新型示踪剂,前哨淋巴结显像效果最佳。前哨淋巴结定位与核素法一致。结论:吲哚菁绿-利妥昔单抗偶联的新型前哨淋巴结示踪剂的制备工艺简单且无放射性危害,其中单抗的分子完整性和免疫活性无破坏,为无菌、无致热原、无急性毒性的示踪剂,能够用于前哨淋巴结显像。
BACKGROUND & AIM: Sentinel lymph node biopsy is the standard of treatment for patients with clinical stage axillary lymph node negative early breast cancer. Accurate localization of sentinel lymph nodes is crucial for staging, prognosis and treatment. In this study, rituximab was conjugated with indocyanine green fluorescent probe to prepare a new SLN tracer, to determine the optimal coupling ratio, and to study its biological characteristics, safety margin and localization. Methods: A new type of sentinel lymph node was prepared by direct coupling method. Indol greenine-rituximab was separated by dual-layer flash chromatography-silica gel chromatography. The non-reducing SDS polyacrylamide gel electrophoresis And indirect antibody sandwich enzyme-linked immunosorbent assay (ELISA) were used to detect the molecular integrity and immunological activity of the new tracers. The safety limits of the new tracers as required by the Chinese Pharmacopoeia and the localization of sentinel lymph nodes in mice were determined. Results: The new tracer rituximab molecular integrity and maintain the immunological activity of monoclonal antibody rituximab macrolide indocyanine green labeling rate of 100%, the new tracer is sterile, Pyrogen-free solution and topical injection will not cause harm. Rituximab and indocyanine green mass ratio of 4: 1, 6: 1 coupled to form a new tracers, sentinel lymph node imaging the best. Sentinel lymph node location and nuclide method. Conclusion: Indocyanine green-rituximab-conjugated new SLNs are simple and non-radioactive. The molecular integrity and immunocompetence of the monoclonal antibodies are non-destructive, and are sterile and non-pyrogenic The original, non-acute toxic tracer, can be used for sentinel lymph node imaging.