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

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

Fabienne Rayne  1 , Solène Debaisieux  1 , Annie Tu  2 , Christophe Chopard  1 , Petra Tryoen-Toth  2 , Bruno Beaumelle  3
Affiliations
Free article

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

Fabienne Rayne et al. Methods Mol Biol. 2016.
Free article

Erratum in

  • ERRATUM.
    Rayne F, Debaisieux S, Tu A, Chopard C, Tryoen-Toth P, Beaumelle B. Rayne F, et al. Methods Mol Biol. 2016;1354:E1. doi: 10.1007/978-1-4939-3046-3_26. Methods Mol Biol. 2016. PMID: 27066967 No abstract available.

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.

Keywords: ELISA; HIV-1; Immunoblots; Secretion; Tat.

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