通过酶联免疫吸附测定法（ELISA）或蛋白质免疫印迹法（Western Blot）检测细胞培养上清液中的HIV-1反式激活因子（Tat）

Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot.

作者信息

Rayne Fabienne, Debaisieux Solène, Tu Annie, Chopard Christophe, Tryoen-Toth Petra, Beaumelle Bruno

机构信息

CPBS, UMR 5236 CNRS, University of Montpellier, 1919 Route de Mende, 34923, Montpellier Cedex 05, France.

Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, University of Strasbourg, Strasbourg, France.

出版信息

Methods Mol Biol. 2016;1354:329-42. doi: 10.1007/978-1-4939-3046-3_22.

DOI:10.1007/978-1-4939-3046-3_22

PMID:26714722

Abstract

HIV-1 Tat is efficiently secreted by HIV-1-infected or Tat-transfected cells. Accordingly, Tat concentrations in the nanomolar range have been measured in the sera of HIV-1-infected patients, and this protein acts as a viral toxin on bystander cells. Nevertheless, assaying Tat concentration in media or sera is not that straightforward because extracellular Tat is unstable and particularly sensitive to oxidation. Moreover, most anti-Tat antibodies display limited affinity. Here, we describe methods to quantify extracellular Tat using a sandwich ELISA or Western blotting when Tat is secreted by suspension or adherent cells, respectively. In both cases it is important to capture exported Tat using antibodies before any Tat oxidation occurs; otherwise it will become denatured and unreactive toward antibodies.

摘要

HIV-1反式激活因子（Tat）可由HIV-1感染的细胞或转染了Tat的细胞有效分泌。因此，在HIV-1感染患者的血清中已检测到纳摩尔浓度范围的Tat，并且这种蛋白质对旁观者细胞起病毒毒素的作用。然而，检测培养基或血清中的Tat浓度并非那么简单，因为细胞外的Tat不稳定，尤其对氧化敏感。此外，大多数抗Tat抗体的亲和力有限。在此，我们描述了分别在悬浮细胞或贴壁细胞分泌Tat时，使用夹心酶联免疫吸附测定法（ELISA）或蛋白质印迹法来定量细胞外Tat的方法。在这两种情况下，在任何Tat氧化发生之前使用抗体捕获分泌出的Tat都很重要；否则它会变性并对抗体失去反应性。