Measurement of human growth hormone using a chemiluminescence assay and a "glow" luminometer.

The gene that codes for human growth hormone (hGH) is used as a reporter gene to assess changes an effector may have on gene expression of a specific DNA sequence (6). hGH is secreted into the medium of transfected cells following the addition of an appropriate effector; measurement of hGH levels allows for quantification of the degree of activation (or suppression) of gene expression relative to untreated transfected cells. Many laboratories use the gene that codes for firefly luciferase as a reporter (1,5) because it affords extreme sensitivity in monitoring changes in gene expression. While luciferase activity can be monitored using a “flash” luminometer (one that quantifies a pulse of light), other types of luminometers (“glow” or “integrating”) allow for integration of light produced by the cleavage of the substrate luciferin by luciferase. We attempted to define conditions that would allow a “glow” luminometer to accurately measure hGH levels using a chemiluminescence assay that is based on the emission of light at 420–430 nm, accompanying the conversion of an acridinium ester (covalently linked to a secondary antihGH antibody), into N-methyl acridone. We used the Turner Model 20e luminometer (Turner Designs, Sunnyvale, CA, USA), which detects light at a peak of 420 nm and which has been shown to afford superior sensitivity among commercially available integrating luminometers (3). hGH standards provided in the chemiluminescence assay kit were dissolved in distilled water and used to develop a standard curve using the Model 20e luminometer. Fifty microliters of each of the standards were incubated with glass beads containing an antihGH antibody directed against one hGH epitope, and a soluble anti-hGH antibody directed against a second hGH epitope. In the kit, an acridinium ester is covalently attached to the soluble anti-hGH antibody. After an incubation period of 1 h, the glass beads were washed as per the chemiluminescence protocol instructions and then transferred to a borosilicate glass culture tube with a plain end (10 × 50 mm, Cat. No. 14-962-22; Fisher Scientific, Pittsburgh, PA, USA). Other brands of borosilicate tubes yielded unacceptable background readings. The Turner Model 20e luminometer was then linked to a Cavro Model 2000 dispenser (Turner Designs) according to the manufacturer’s instructions. Just prior to reading the samples, equal volumes of trigger 1 and trigger 2 solutions were mixed together. The parameter settings for the luminometer were then set as follows: Pre-Delay, 0 s; Delay, 5 s; Integrate, 0 s; Display, fixed range of 0.000. A borosilicate tube containing a glass bead was placed into the sample chamber, and the dispenser was set to deliver 150 μL of the mixture of the two trigger solutions. After delivery of the solution, the peak (P) value of the relative light units (RLU) was recorded. Prior to plotting the data, the background value, representing the RLU of a glass bead plus 0 ng/mL hGH, was subtracted from each peak value. These corrected peak values were then plotted on logarithmic/logarithmic graph paper vs. hGH concentration (ng/mL). As can be seen in Figure 1, there is a linear relationship between the amount of hGH present in a sample and the amount of RLU detected. Upon the addition of a new sample into the sample chamber, 15 s were allowed to pass after the injector stand had been lowered onto the sample chamber prior to subsequent addition of the trigger mixture. To demonstrate the utility of this assay system, a plasmid construct, consisting of -523 bp of the rat osteocalcin 5′-flanking region linked in tandem to the hGH gene (7), was stably transfected into the rat osteoblastic osteosarcoma cell line ROS 17/2.8 (4) using the calcium phosphate precipitation method (2). A 1,25-dihydroxyvitamin D3 (1,25-D3) response element is found within this 5′-flanking region, such that when exposed to 1,25-D3, stimulation of gene expression is observed. The transfected cells were plated into Falcon® 12-well plates (4 cm2/well) in 1 mL of medium contain-