Development and Validation of a Fecal Testosterone Biomarker: MATERIALS AND METHODS(2)

MATERIALS AND METHODS(2)Solutions

Stock solutions used in the ELISA included coating buffer (0.05 M sodium bicarbonate, pH 9.6), assay buffer (0.1 sodium phosphate, pH 7.0, containing 0.87% NaCl and 0.1% BSA), wash solution (0.15 M NaCl containing 0.05% Tween 20), and substrate (40 mM 2,2′-azino-bis[3-ethyl-benzthiazoline-6-sulfonic acid] and 0.5 M H2O2 in 0.05 M citrate, pH 4.0). buy cheap antibiotics

Sample Preparation

Feces from several Peromyscus were collected and combined to obtain a single pooled sample for extraction comparisons. Samples were dried overnight under vacuum at 37°C and then ground using a household coffee grinder. Portions (100 mg each) were placed in 13 X 100-mm bo-rosilicate tubes and used to determine extraction efficiency.

When measurements of individual mouse samples were required, the samples were collected, divided into 100-mg portions (3-6 fecal pellets), transferred to 16 X 100-mm borosilicate tubes, and then ground using a 10-ml Teflon (Dupont, Wilmington, DE) tissue grinder (Wheaton, Millville, NJ) and cordless drill at 600 rpm.

Assay

The testosterone was measured colorimetrically using a competitive heterogeneous ELISA with a column-purified polyclonal antibody as described by Munro and Lasley. A 96-well microtiter plate (Immulon I; Dynatec, Chantilly, VA) was coated with antibody, incubated at 4°C for a minimum of 16 h, and washed; 50-^l buffer was then added. Next, 20 ^l of sample was added, followed immediately by the HRP-testosterone conjugate. The competition reaction proceeded at room temperature for 2 h, or 4°C overnight, before the plate was washed to remove the unbound steroid and conjugate. To determine the amount of conjugate-bound antibody in the plate, a substrate solution of H2O2 and ABTS was added, and the absorbance was measured at 405 nm with a Bio-Tek EL 340 microplate reader (Bio-Tek Instruments Inc., Winooski, VT). A standard curve was run from 3 to 384 pg to determine the concentration in unknowns. Unknowns were run within the narrow range representing the most linear portion of the standard curve (typically between 40% and 70% displacement of HRP-testosterone) with r2 > 0.99.

This entry was posted in Testosterone and tagged Biomarker, Mus musculus, Peromyscus maniculatus, Testosterone.