Development of a specific IgY-based ELISA for prothymosin alpha, a bioactive polypeptide with diagnostic and therapeutic potential
Chrysoula-Evangelia Karachaliou,
Ioannis V. Kostopoulos,
Vyronia Vassilakopoulou,
Persefoni Klimentzou,
Maria Paravatou-Petsotas,
Wolfgang Voelter,
Hubert Kalbacher,
Christos Zikos,
Ourania Tsitsilonis,
Evangelia Livaniou
Affiliations
Chrysoula-Evangelia Karachaliou
Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety (INRASTES), National Centre for Scientific Research “Demokritos”, Athens, Greece
Ioannis V. Kostopoulos
Department of Biology, National and Kapodistrian University of Athens, Athens, Greece
Vyronia Vassilakopoulou
Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety (INRASTES), National Centre for Scientific Research “Demokritos”, Athens, Greece
Persefoni Klimentzou
Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety (INRASTES), National Centre for Scientific Research “Demokritos”, Athens, Greece
Maria Paravatou-Petsotas
Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety (INRASTES), National Centre for Scientific Research “Demokritos”, Athens, Greece
Wolfgang Voelter
Interfaculty Institute of Biochemistry, University of Tuebingen, Tuebingen, Germany
Hubert Kalbacher
Interfaculty Institute of Biochemistry, University of Tuebingen, Tuebingen, Germany
Christos Zikos
Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety (INRASTES), National Centre for Scientific Research “Demokritos”, Athens, Greece
Ourania Tsitsilonis
Department of Biology, National and Kapodistrian University of Athens, Athens, Greece
Evangelia Livaniou
Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety (INRASTES), National Centre for Scientific Research “Demokritos”, Athens, Greece; Corresponding author.
Prothymosin alpha (ProTα) is a highly conserved polypeptide (109 amino acids in humans) with diagnostic and therapeutic potential; ProTα exerts intra- and extra-cellular biological functions associated with cell proliferation, apoptosis and immune regulation, while it has been suggested to act as a damage-associated molecular pattern (DAMP) or alarmin. In this work, chicken polyclonal anti-ProTα antibodies that had been developed several years ago were immunochemically evaluated and proven to retain immunoreactivity for ProTα, with remarkable thermal and pH stability. Moreover, the antibodies showed practically no cross-reactivity with a series of ProTα-fragments, eventually intracellularly produced -such as ProTα[1-28] (also known as Tα1) and ProTα[100-109], which exert per se biological activity and might be present in biological samples along with the intact molecule, being therefore highly specific for whole-length ProTα. Based on the above antibodies (IgYs-3e), a highly specific competitive ProTα-ELISA with well-studied analytical characteristics (intra- and inter-assay CVs: ≤5% and ≤12%, respectively, limit of detection: 2.1 ng/mL, recovery: 88–104%) was developed. The new ProTα-ELISA was applied to the analysis of supernatants of HeLa cells driven to necrosis; intact ProTα was measured in cell culture supernatants, at levels that seemed to depend on % cell necrosis.
