The expression and molecular effects of growth hormone are important in a number of facets of life science research as this protein has roles in numerous cellular pathways from growth and sexual maturity to cancer. Growth hormone deficiency can result in growth disorders manifesting in short stature and immature skeletal systems, whereas an excess of growth hormone can cause gigantism (hypermegaly). The proliferative and anti-apoptotic properties of this protein are well-recognized and it is becoming increasingly accepted that growth hormone expressed in extra-pituitary sites can participate in the oncogenic transformation of cells. Therefore, an assay to measure the expression and secretion of growth hormone in cell lines has numerous applications.
The Human Growth Hormone ELISA is a sandwich-type ELISA in which you are provided with a microtiter plate where each well is coated with an anti-human growth hormone antibody to capture the protein in your samples. Preparation for this assay is simple; all that is required is media (containing serum or serum-free) from confluent cells. I grow my cells (MCF-7 mammary carcinoma cells) in a 6-well dish and take 1 ml of serum-free media that has been on confluent cells for 24 hours. Growth hormone standards (0-500 pg/ml) are provided in the kit, so this eliminates the variation which can be introduced when standards are prepared each time the assay is performed.
Only 20 ul of the standards and your samples are required for this assay. The first stage is to incubate the protein standards and samples in the pre-coated wells for 1 hour (room temperature, with vigorous shaking). This is followed by washing the wells (5 times with kit specific wash buffer), addition of an antibody-enzyme conjugate solution, which contains another hGH detecting antibody and the horseradish peroxidase (HRP) enzyme. After half an hour incubation (same conditions as the first incubation), the wells are washed again. The final stages in this assay involve the addition of a TMB Chromagen Solution which contains tetramethylbenzidine (TMB) in a citrate buffer with hydrogen peroxide. This is incubated with shaking at 500-600 rpm for 10 minutes (covered). Finally, stop solution (0.2 M sulfuric acid) is added and the plate is read at 450 nm (with a background reading at 620 nm). (If need be, this ELISA can be performed without shaking.) This ELISA works on the same premise as standard ELISA protocols whereby the more antibody-enzyme-conjugate solution that is ‘captured,’ the higher the levels of enzymatic activity and therefore, the greater the resulting color development.
This assay is extremely sensitive; growth hormone levels as low as 0.03 ng/ml can be accurately measured. The maximum levels measured by this assay are the ‘normal’ range levels of circulating growth hormone and hence, if you have cells which express high levels of the protein, these samples should be diluted.
The kit is quite expensive, but the benefits of the pre-prepared standards and pre-coated wells seem to outweigh this drawback. I have consistently achieved good results with this kit.
PhD Student
The Liggins Institute
University of Auckland