Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects
Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects
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DOI：
10.3892/ijo_00000551
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发表时间：
2010-04-01
影响因子：
5.2
通讯作者：
Frejd, Fredrik Y.
中科院分区：
文献类型：
--
作者：
Gostring, Lovisa;Chew, Ming Tsuey;Frejd, Fredrik Y.
靶向剂的肿瘤细胞内化是令人感兴趣的，因为内化影响放射性核素的局部保留时间，从而影响PET和SPECT中的成像质量以及放射性核素治疗的效果。在核方法不适用于细胞水平的情况下，定量荧光技术是有用的，如本文所述。应用两种基于荧光的方法来研究细胞内化：CypHer和Alexa 488猝灭方法，两者都用于荧光显微镜和流式细胞术。在A431细胞中分析了两种EGFR结合亲和体分子：单体Z1907和二聚体（Z1907）（2）。EGF、西妥昔单抗和非特异性亲和体分子用作对照。做个比较。用酸洗内化测定法研究In-111标记的Z1907的内化。Cypher方法简单明了，但需要对所有化合物进行同等标记以进行准确定量。Alexa 488猝灭法是优选的，因为它不依赖于染料与蛋白质的比率。根据该方法，约45%的EGF和19-24%的结合的亲和体分子和西妥昔单抗在1小时内内化。In-111-Z1907在酸洗法中观察到类似的结果，而（Z1907）（2）不能被酸除去，因此不能以这种方式进行研究。基于荧光的Alexa 488猝灭方法非常适合于定量分析靶向剂的内化，也适用于抗酸洗的靶向剂。内化的级分显示，预期单体和二聚体亲和体分子均提供良好的摄取，从而提供良好的保留：金属放射性核素，这将使良好的肿瘤达到背景值。
Tumor cell internalization of targeting agents is of interest, since internalization influences the local retention time of a radionuclide and thereby imaging quality in PET and SPECT and effects of radionuclide therapy. In cases where nuclear methods are not applicable at the cellular level, quantitative fluorescent techniques are useful as described in this article. Two fluorescence-based methods to study cellular internalization were applied: the CypHer and the Alexa488-quenching methods, both utilized in fluorescence microscopy and flow cytometry. Two EGFR-binding Affibody molecules were analyzed in A431 cells: the monomer Z1907 and the dimer (Z1907)(2). EGF, cetuximab and non-specific Affibody molecules were used as controls. For comparison. internalization of In-111-labeled Z1907 was studied with the acid wash internalization assay. The Cypher method is straightforward, but requires equal labeling of all compounds for accurate quantification. The Alexa488-quenching method is preferable since it is independent of the dye-to-protein ratio. According to this method, about 45% of EGF and 19-24% of the bound Affibody molecules and cetuximab were internalized within one hour. Similar results were seen with In-111-Z1907 in the acid wash method, while (Z1907)(2) was not removed by acid and thus could not be studied this way. The fluorescence-based Alexa488-quenching method is well suited to quantitatively analyze internalization of targeting agents, also those that resist acid wash. The internalized fraction showed that both the monomeric and dimeric Affibody molecules are expected to give good uptake and thereby good retention of: metallic radionuclides which will render good tumor to background values.