In vitro detection of adrenocorticotropic hormone levels by fluorescence correlation spectroscopy immunoassay for mathematical modeling of glucocorticoid-mediated feedback mechanisms
© Puchinger et al.; licensee Springer. 2012
Received: 21 December 2011
Accepted: 2 October 2012
Published: 26 October 2012
Performing quantitative, highly sensitive measurements at a single molecule level is often necessary to address specific issues related to complex molecular and biochemical systems. For that purpose, we present a technique exploiting both the flexibility of immunoassays as well as the low operating costs and high throughput rates of the fluorescence correlation spectroscopy (FCS) method. That way we have established a quantitative measurement technique providing accurate and flexibly time resolved data of single molecules. Nanomolar changes in adrenocorticotropic hormone (ACTH) levels have been detected in a short time-frame that are caused by fast feedback actions in AtT-20 anterior pituitary glands in vitro. Especially with respect to clinical diagnostic or mathematical modeling this improved FCS setup may be of high relevance in order to accurately quantify the amounts of peptide hormones—such as ACTH—as well as signaling molecules, transcription factors, etc., being involved in intra- and extracellular reaction networks.
KeywordsACTH FCS AtT-20 Cortisol CRH Glucocorticoid membrane receptor ODE model Parameter identification
The fluorescence correlation spectroscopy (FCS) has proven to be a powerful tool for studying supramolecular associations [3, 4], DNA hybridization reactions , 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  with the highly specific routines of an immunoassay. We exemplify this procedure by means of the in vitro measurement of the ACTH peptide secretion from AtT-20 mouse pituitary cells. Particularly, we use a labeled monoclonal antiACTH antibody (specific for the N-terminal epitope on the ACTH peptide) to capture the ACTH molecule, making it visible for the FCS. However, in order to detect low molecular weight peptides such as ACTH, the binding of a second unlabeled monoclonal antiACTH antibody to the C-terminal site of the ACTH peptide is necessary in order to cause a significant change in the diffusion time between the free labeled antibody and the mAb(N)-ACTH-mAb(C) immunocomplex. By measuring this discrepancy in the FCS, the concentration of the target peptide can accurately be determined.
Materials and methods
The used AtT-20 cells (ATCC no. CCL-89) were purchased from the American Type Culture Collection (ATCC, Manassas, USA) and passaged at a subcultivation ratio of 1:4 every 5 days. Cells were seeded onto polystyrene 24-well tissue culture plates (Nalge Nunc International, Japan) at a density of 1.0 × 104 cells/ml, grown in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich Inc., St. Louis, USA) supplemented with 10% fetal bovine serum, 1.5 g/l sodium bicarbonate, 10 Units/ml penicillin, and 10 μg/ml streptomycin, and maintained in an incubator (HERAcell®, Thermo Scientific, USA) at 37°C, 6% CO2 and 95% relative humidity. After 92 and 114 h of cell growing, AtT-20 cells were exposed to doses of 10 nM CRH and up to 100 nM cortisol (both from Sigma-Aldrich Inc.) for 1 min to 1 h. The supernatant was carefully removed from the cell layer and centrifuged (800 × g, 37°C, 10 min).
Focus control and pinhole adjustment
A drop of the organic fluorescent dye Rhodamine 6 G (diluted 1:200) was used to automatically position the chambered coverglass (Nalge Nunc International, Japan) in focus of the confocal optics of the spectrofluorimeter by a scanning procedure as well as to automatically adjust the pinhole to its correct position. The focus in z-direction was set 150 μm over the coverglass to record diffusions of fluorescent particles through the focal element in the drop of sample. The pinhole diameter was set to 35 μm.
Calibration of confocal volume
The confocal detection volume was determined by measuring the correlation time of a 10-nM solution of rhodamine 6 G in water with the known diffusion coefficient D of 2.8 × 10–6 cm2/s, employing the relationship D = r2/(4τ D ) and resulting in a confocal volume element of 0.17 μm in radian and 0.88 μm in axial dimension. In addition, DyLight488-labeled monoclonal antibody dilution series with known concentrations (ranging from 1 to 80 nM) were measured and showed similar molarities in FCS. Confocal volume calibrations using rhodamine 6 G were carried out on each experimental day or after 3 h measurement time.
60 nM antiACTH(N-term)-monoclonal IgG1 antibodies (Phoenix Pharmaceuticals Inc., Belmont, USA) and 60 nM-labeled antiACTH(C-term)-IgG1 antibodies (Fitzgerald Industries International, Concord, USA; labeled with DyLight488 from Pierce Biotechnology, Rockford, USA) were added in the cell-free supernatant and incubated (30°C, 15 min) to ensure a quick and absolute ACTH-capture of both monoclonal antibodies. Western blotting analysis and ELISA measurements prior to FCS showed no crossreactivity between both antibodies in absence of ACTH. A drop (25 μl) of sample was pipetted on the chambered coverglass, excited with an 488-nm Ar-laser attenuated by an optical density filter (1.0–in. diameter), and the fluctuations in fluorescence intensity of the mAb(N)-ACTH-mAb(C) immunocomplex compared to the free labeled antibody were monitored in series of 50 measurements with identical setup (measurement time: 10 s; correlator scaling: 10 s) for each sample (see Figure 2).
where the angular brackets in the function represent the ensemble average, δF(t) denotes the corresponding variance, and τ is known as the delay or correlation time interval over which the fluctuations are compared.
where N is the particle number and τ D the molecular diffusion time of the excited fluorophores moving in a three-dimensional confocal volume through an axial (z) to radial (r) dimension.
The obtained autocorrelation functions were evaluated using a two-component model by fixing the diffusion time of the unbound labeled antibody fraction (τD 1) which was achieved from one-component fitting procedure.
Dose response relation of CRH and cortisol to ACTH secretion
In vitro studies in AtT-20s within 22 h
Cell population (cells/ml)
205164 ± 12889
15.016 ± 0.201
335938 ± 20522
16.764 ± 0.593
324219 ± 23935
17.427 ± 0.422
302084 ± 28168
16.146 ± 0.564
237500 ± 15630
15.364 ± 0.575
312500 ± 28125
16.673 ± 0.467
298438 ± 20625
15.679 ± 0.337
Fast feedback regulation of ACTH release
In order to demonstrate the capabilities of the method we focused on the fast negative feedback control by cortisol. Due to the fast sampling and the low sample volume we were able to detect significant differences in ACTH response within 5–15 min after CRH and/or cortisol incubation (see Figure 4).
Validation of FCS results
Validation of FCS results
% change to FCS
+10 nM CRH
+10 nM CRH + 100 nM cortisol
Mathematical modeling of feedback mechanisms
The improved setup for the FCS method is particularly suitable for experiments which have to be conducted repeatedly and demand a high (quantitative) accuracy of the data. A central motivation to develop such a technique comes from a mathematical modeling task initiated by the research presented in . We are interested in the interplay of the genomic and non-genomic negative feedback of cortisol on the secretion of ACTH and its effect on the dynamics of the HPA-axis. This research goal demands to model both intracellular mechanisms as well as interactions of the different glands. This cannot be achieved in full detail. Consequently, we concentrated on the main feedback mechanisms related to the anterior pituitary gland and the basic controls between the hypothalamus and the adrenal glands. This approach of bridging several levels of complexity eventually needs validation by experimental data. In that regard the measurement of the ACTH secretion in response to CRH and cortisol serves two goals. First of all the secretion of ACTH is targeted by the main feedback controls we focus on in our model and thus allows us at least a basic assessment of the model. And secondly, we introduce no bias concerning the overall dynamics of the HPA-axis, as we consider only the anterior pituitary cells in our in vitro experiment and thus have no interaction with other tissues or glands.
In 2005, Maier et al.  were able to provide evidence of a glucocorticoid receptor in the anterior pituitary cell membrane which may regulate fast response of anterior pituitary cells to cortisol. With our improved FCS setup it was possible to detect lowest changes in extracellular ACTH molarities (±0.3 nM) that arise from signaling of these activated G-protein coupled membrane receptors in vitro (see Figure 1). Even 5–15 min after cortisol administration (100 nM) we were able to monitor an inhibition of CRH-induced ACTH secretion by cortisol. Extracellular ACTH levels of 14.387 ± 0.428 nM compared to 15.131 ± 0.254 nM without addition of cortisol were measured. The fast inhibitory effects on CRH-induced ACTH secretion have become evident within at most 5 min after cortisol administration (see Figure 4). However, a detailed temporal restriction of fast and slower feedback actions on extracellular ACTH secretion was not studied with this FCS setup so far, but our results suggested that immediate ACTH secretion which has occurred within minutes after cortisol treatment can only be caused by fast non-genomic feedback actions (see Figure 1) and not by genomic-slow feedback mechanisms which have been shown to occur after several hours .
FCS provides a highly flexible, easy-to-use assay format with very small sample volumes (approximately 20 μl), and increased throughput, as particle numbers can be measured directly after calibrating the confocal volume once, a major disadvantage of ELISAs which need to be calibrated quite often. In addition, FCS makes it possible to extract complex signals from high background due to the different characteristic time scales over which signal and noise occur.
FCS-immunoassay versus ELISA
μM to sub-nM
nM to sub-nM
Sample volume required
Approximately 20 μl
Approximately 25 min
On the surface
Washing steps required
Calibration curve required
Yes (confocal volume)
Size-based fluctuations in signal intensities
Changes in signal intensity
Enzyme-linked immunosorbent assay
Fluorescence correlation spectroscopy
This study was supported by the Vienna Science and Technology Fund (WWTF) Grant no. MA07-030.
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