In vitro detection of adrenocorticotropic hormone levels by fluorescence correlation spectroscopy immunoassay for mathematical modeling of glucocorticoid-mediated feedback mechanisms

Adrenocorticotropic hormone (ACTH) is a 39-amino acid long straight-chain peptide hormone (4.5 kDa) that is derived from a 266-amino acid precursor pro-opiomelanocortin. It is secreted by the anterior pituitary gland and is considered one of the major stress hormones within the hypothalamic–pituitary–adrenal (HPA)-axis system: The hypothalamus secrets corticotrophin-releasing hormone (CRH), which stimulates the release of ACTH in the corticotrophic anterior pituitary gland [1]. Consequently, ACTH causes the production of cortisol in the adrenal glands. However, beside corticotrophic feedback actions several other feedback controls on the metabolomic or genomic level provide a complex and multifaceted system. One of the most prominent and well-studied feedback controls is the down-regulation of ACTH production by cortisol. The down-regulation is mediated via two feedback mechanisms working on a genomic and non-genomic levels (see Figure 1). Hence, we observe fast (within seconds to minutes) and slow (after several hours) negative feedback actions in response to the exposure with cortisol [2]. These feedback mechanisms are still subject of research and particularly their interplay is not fully understood. Hence, as ACTH represents the main response in regard to this glucocorticoid feedback, an accurate detection of in vitro extracellular ACTH concentration is of high relevance.The fluorescence correlation spectroscopy (FCS) has proven to be a powerful tool for studying supramolecular associations [3,4], DNA hybridization reactions [5], and detecting single molecule concentrations [6,7]. Due to its high sensitivity, short analysis time and small sample volume requirements FCS have become a valuable tool in molecular biology.In this article, we present an improved FCS setup to detect nanomolar changes of peptides in vitro by combining the fast FCS technique [8] with the highly specific routines of an immunoassay. We exemplify this procedure by means of the in vitro meas