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Development of an ELISA for the quantification of the C-terminal decapeptide prothymosin α(100-109) in sera of mice infected with bacteria.

TitleDevelopment of an ELISA for the quantification of the C-terminal decapeptide prothymosin α(100-109) in sera of mice infected with bacteria.
Publication TypeJournal Article
Year of Publication2013
AuthorsSamara, Pinelopi, Hubert Kalbacher, Kyriaki Ioannou, Dorel L. Radu, Evangelia Livaniou, V. J. Promponas, Wolfgang Voelter, and Ourania Tsitsilonis
JournalJournal of immunological methods
Volume395
Issue1-2
Pagination54-62
Date Published2013 Sep 30
ISSN1872-7905
KeywordsAmino Acid Sequence, Animals, Antibody Specificity, Apoptosis, Computer Simulation, Enzyme-Linked Immunosorbent Assay, Epitopes, Humans, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Peptide Fragments, Protein Precursors, Rabbits, Streptococcal Infections, Streptococcus pyogenes, Thymosin
Abstract

Apoptosis is characterized by a series of discrete biochemical events, among which is the truncation of the nuclear polypeptide prothymosin alpha (proTα) by activated caspase-3. This early apoptotic event results in the generation of a carboxy-terminal fragment of proTα, the immunoactive decapeptide proTα(100-109). We hypothesized that the detection of increased levels of proTα(100-109) in serum can be directly correlated with the induction of massive cell apoptosis, resulting from a severe bacterial infection. Thus, using high-affinity-purified polyclonal antibodies (Abs), raised in rabbits and a prototype antibody-capture system, we developed a highly sensitive and specific competitive ELISA for proTα(100-109). The sensitivity of the ELISA (0.1ng/mL to 10μg/mL) is acceptable for the quantification of the decapeptide in serum samples. To assess our initial hypothesis, we determined the concentration of proTα(100-109) in the sera of mice infected with the bacterium Streptococcus pyogenes over the course of the infection. We show that serum concentration of proTα(100-109) was marginal to undetectable before infection, increased over time and peaked at 72h postinfection. In silico analysis suggests that the Abs generated are unlikely to cross-react with any other unrelated mouse or bacterial protein. Further validation of our ELISA using serum samples from humans, infected with bacteria, may provide a useful tool to differentiate the causative agent of a potentially lethal septic infection.

DOI10.1016/j.jim.2013.06.011
Alternate JournalJ. Immunol. Methods


by Dr. Radut.