Ultrasensitive ELISA detection of proteins in separated lumen and membrane fractions of cancer cell exosomes
Ultrasensitive ELISA detection of proteins in separated lumen and membrane fractions of cancer cell exosomes
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DOI：
10.1016/j.ab.2022.114831
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发表时间：
2022-08-03
影响因子：
2.9
通讯作者：
Ito, Etsuro
中科院分区：
文献类型：
--
作者：
Iha, Kanako;Tsurusawa, Naoko;Ito, Etsuro
外泌体将分子水平转移到周围细胞，因此在癌症进展中起关键作用。为了阐明外泌体在癌症进展中的作用，应分别分析其管腔和膜组分中的微量蛋白质。为此，必须开发一种适当且易于使用的分离外泌体管腔和膜组分的方法。此外，由于外泌体只含有微量的蛋白质，因此需要一种超灵敏的蛋白质检测方法。为了开发一种适当且易于使用的管腔和膜组分分离方法，我们将最初为细胞开发的市上可用试剂盒应用于外泌体，并将结果与使用传统的复杂Na2CO3方法获得的结果进行了比较。为了建立一种超灵敏的蛋白检测方法，我们将癌细胞中上调并参与癌症进展的GRP78作为靶蛋白，采用超灵敏ELISA结合硫代nad循环在亚原子摩尔水平检测其。通过这些方法，我们成功地在培养癌细胞的外泌体的管腔和膜部分中定量了GRP78。本研究结果将有助于拓宽我们对肿瘤微环境的认识。
Exosomes transfer molecules horizontally to surrounding cells and therefore have a key role in cancer progression. To clarify the role of exosomes in cancer progression, trace amounts of proteins in their lumen and membrane fractions should be analyzed separately. For this purpose, an adequate and easy-to-use method of separating the lumen and membrane fractions of exosomes must be developed. Further, because exosomes contain only trace amounts of proteins, an ultrasensitive protein detection method is necessary. To develop an adequate and easy-to-use lumen and membrane fraction separation method, we applied a commercially available kit originally developed for cells to exosomes and examined the validity of the results compared with those obtained using a conventional, complicated Na2CO3 method. To develop an ultrasensitive protein detection method, we designated GRP78, which is upregulated in cancer cells and contributes to cancer progression, as the target protein and detected it at the subattomolar level using an ultrasensitive ELISA combined with thio-NAD cycling. By applying these methods together, GRP78 was successfully quantified in both the lumen and membrane fractions of exosomes obtained from cultured cancer cells. The present results will facilitate studies to broaden our understanding of the tumor microenvironment.