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.

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.