Hasegawa Koki, Kudoh Shinji, Ito Takaaki
Department of Pathology and Experimental Medicine, Kumamoto University, Graduate School of Medical Sciences, Kumamoto, Japan.
Center for Instrumental Analysis, Kyoto Pharmaceutical University, Kyoto, Japan.
PLoS One. 2017 Feb 9;12(2):e0172030. doi: 10.1371/journal.pone.0172030. eCollection 2017.
The confirmation of target expression in tissues is a prerequisite for molecular-targeted therapy. However, difficulties are sometimes associated with the production of appropriate antibodies against receptors. We herein developed a ligand derivative dye for the staining of receptors. The somatostatin receptor (sstr) was selected as the target and FITC-octreotate as the detective agent. We performed a blot analysis to detect sstr in the transfer membrane. The sstr2 recombinant protein or cell lysate from a small cell lung carcinoma cell line (H69) was boiled and loaded onto SDS-PAGE, and the proteins were transferred to a membrane. Even after denaturing processes, FITC-octreotate still bound sstr on the membrane. Furthermore, FITC-octreotate depicted the expression of sstr in formalin-fixed and paraffin-embedded (FFPE) sections, a method that we named ligand derivative staining (LDS). The accuracies of immunostaining and LDS were compared at the points of the detection of sstr using FFPE sections of 30 neuroendocrine tumor specimens. The sensitivity of LDS was 81.8%, while those of immunostaining using anti-sstr2 and sstr5 antibodies were 72.7% and 63.6%, respectively. Thus, LDS appears to be superior to immunostaining. A ligand derivative may be used as a substitute for antibodies, and has the potential to support economical, simple, and accurate detection methods.
在组织中确认靶点表达是分子靶向治疗的前提条件。然而,有时在制备针对受体的合适抗体方面会遇到困难。我们在此开发了一种用于受体染色的配体衍生物染料。选择生长抑素受体(sstr)作为靶点,异硫氰酸荧光素 - 奥曲肽作为检测剂。我们进行了印迹分析以检测转移膜上的sstr。将来自小细胞肺癌细胞系(H69)的sstr2重组蛋白或细胞裂解物煮沸后上样到SDS - PAGE中,然后将蛋白质转移到膜上。即使经过变性处理,异硫氰酸荧光素 - 奥曲肽仍能与膜上的sstr结合。此外,异硫氰酸荧光素 - 奥曲肽描绘了福尔马林固定石蜡包埋(FFPE)切片中sstr的表达,我们将这种方法命名为配体衍生物染色(LDS)。在使用30个神经内分泌肿瘤标本的FFPE切片检测sstr时,比较了免疫染色和LDS的准确性。LDS的敏感性为81.8%,而使用抗sstr2和sstr5抗体的免疫染色敏感性分别为72.7%和63.6%。因此,LDS似乎优于免疫染色。配体衍生物可作为抗体的替代品,并且有可能支持经济、简单且准确的检测方法。