title: Development of an ELISA for the quantification of the C-terminal decapeptide prothymosin α(100-109) in sera of mice infected with bacteria.
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.
authors.affiliation: Department of Animal and Human Physiology, Faculty of Biology, University of Athens, Greece.
authors.forename: Pinelopi
authors.initials: P
authors.lastname: Samara
authors.forename: Hubert
authors.initials: H
authors.lastname: Kalbacher
authors.forename: Kyriaki
authors.initials: K
authors.lastname: Ioannou
authors.forename: Dorel L
authors.initials: DL
authors.lastname: Radu
authors.forename: Evangelia
authors.initials: E
authors.lastname: Livaniou
authors.forename: Vasilis J
authors.initials: VJ
authors.lastname: Promponas
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Voelter
authors.forename: Ourania
authors.initials: O
authors.lastname: Tsitsilonis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D017209:Apoptosis / Q000276:immunology*; D003198:Computer Simulation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000097:blood* / Q000235:genetics / Q000276:immunology*; D011498:Protein Precursors / Q000097:blood* / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D013290:Streptococcal Infections / Q000097:blood* / Q000276:immunology / Q000473:pathology; D013297:Streptococcus pyogenes; D013947:Thymosin / Q000031:analogs & derivatives* / Q000097:blood / Q000235:genetics / Q000276:immunology
chemical_list: D000939:Epitopes; D010446:Peptide Fragments; D011498:Protein Precursors; C040782:prothymosin alpha; D013947:Thymosin
keywords: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); ABTS; BSA; Bacterial infection; CFA; CFU; ELISA; HRP; Ig; Immunoreactive peptide; In vivo model; KLH; PBS; Prothymosin alpha; RIA; S. pyogenes; Streptococcus pyogenes.; bovine serum albumin; colony-forming unit; complete Freund's adjuvant; enzyme-linked immunosorbent assay; horseradish peroxidase; immunoglobulin; keyhole limpet hemocyanin; phosphate buffered saline; proTα; prothymosin alpha; radioimmunoassay