Hormone Assay Protocols
A radioimmunoassay kit for determination of growth hormone (Amersham Pharmacia
Biotech, Baie d’Urfé, Quebec) used a highly specific125-I tracer, in combination with a
highly specific and sensitive antiserum, and allowed growth hormone measurements
ranging from 1.6-to 100 ng/ mL. The sensitivity of the assay was 17-ng /mL. The RIA
procedure was based on competition between unlabelled rat growth hormone and a fixed
quantity of 125-I labeled rat growth hormone for a limited number of binding sites on the
growth hormone-specific antibody. With fixed amounts of antibody to radioactive
ligand, the amount of radioactive ligand bound by the antibody is inversely proportional
to the concentration of non-radioactive ligand present in the sample. The antibody bound
growth hormone was then reacted with Amerlex-M second antibody reagent, and the
bound fraction obtained by centrifugation of the suspension and decantation of the
supernatant. Measurement of the radioactivity in the remaining pellet enabled the
amount of labeled growth hormone in the bound fraction to be calculated. Using
interpolation from a standard curve obtained from preparations using known quantities of
growth hormone, the concentration of unlabelled growth hormone was determined. There
was very little cross-reactivity of this assay for a number of related compounds: other rat
hormones, including FSH, LH, PRL, TSH, and ACTH consistently produced cross
reactivity less than 0.2%.
Enzyme linked immunosorbent assay (ELISA) was used to quantify prolactin
concentrations in plasma samples. Each well of a 96 well Costar EIA microtitre plate
(#3590, Becton Dickinson, Maryland, U.S.A.) was coated with various dilutions of
reference hormone or collected plasma (100 µL per well) and each sample was assayed in
duplicate. For all subsequent incubation procedures, the plate was covered and placed
into a sealable plastic box with paper towel lining the bottom soaked using distilled
water. The plate was incubated for 8-12 hours at 4 C to allow for the binding of the
antigen to the well. After three washes of three minutes each using 200 µL Tris-buffered
saline (TBS) the microtitre plate was post-coated with bovine serum albumin (BSA,
Sigma-Aldrich Canada Ltd., Oakville, Ontario) in TBS (1 mg/ mL, 150 µL per well) for
two hours at room temperature. The post-coating procedure blocked any remaining
protein binding sites in the well, which had not been bound by the antigen, in order to
decrease non-specific binding. The plate was then washed three times as previously
described using a solution of TBS/Tween.
Each well was then coated with 100 µL of an antibody specific to prolactin (e.g.,
mouse anti-PRL) and incubated for 2 hours at room temperature. The plate was again
washed three times using 200 µL TBS/Tween for three minutes per wash. All wells were
probed (100 µL per well) using an antibody conjugated to alkaline phosphatase. This
antibody was specific to the animal in which the first antibody was produced (e.g., goat
anti-rabbit) and was diluted in 1 mg/ mL BSA in TBS/Tween. After incubation for 2
hours at room temperature, the plate was washed four times using 200 µL TBS/Tween for
three minutes per wash and probed using 100µL substrate specific to the alkaline
phosphatase conjugate (pNPP in diethanolamine). The optical density of all wells in the
plate was measured at a wavelength of 405 nm using an electronic Molecular Devices
ELISA plate reader at 60 and 120 minutes following addition of the substrate. Based on
optical densities of the wells containing reference prolactin, a standard curve was
calculated. Determination of unknown prolactin concentrations in the remainder of the
wells was through extrapolation of the standard curve parameters.