DBC Growth Hosmone (hGH) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical one-step capture or ‘sandwich’ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for hGH is immobilized onto the microplate and another monoclonal antibody specific for a different region of hGH is conjugated to horse radish peroxidase (HRP). hGH from the sample and standards are allowed to bind simultaneously to the plate and to the HRP conjugate. The washing and decanting steps remove any unbound HRP conjugate. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of hGH in the sample. 
A set of standards is used to plot a standard curve from which the amount of hGH in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Human growth hormone (hGH) is a polypeptide of 191 amino acids secreted by the somatroph cells of the anterior pituitary. Growth hormone is principally a regulator of body growth and its metabolic effects are primarily anabolic. Some of its effects include promotion of protein conservation through its involvement in a wide range of protein synthesis mechanisms, enhancement of glucose transport and facilitation of glycogen storage. In addition, it induces the release of somatomedins (insulin-like growth factors), which further mediate the cascade of growth promoting actions.
Measurement of hGH is primarily of interest in the diagnosis and treatment of various forms of decreased secretion of hGH. Hyposecretion of hGH in children results in growth retardation and hypersecretion leads to gigantism in children and acromegaly in adults. 
The secretion of hGH varies throughout the day under the influence of intricate neurogenic, metabolic and hormonal control. Due to the pulsatile nature of hGH release, it is often inaccurate to define a reference range and status based on single serum measurements. To diagnose disorders of hGH secretion more reliably, dynamic tests are used in which serum hGH levels are measured over a period following suppression or stimulation of hGH secretion.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Calculate the mean optical density of each unknown duplicate. 
3. Subtract the mean absorbance value of the “0” calibrator from the mean absorbance values of the calibrators, controls and serum samples. 
4. Draw a calibrator curve on log-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
5. Read the values of the unknowns directly off the calibrator curve. 
6. If a sample reads more than 50 ng/mL then dilute it with calibrator A at a dilution of no more than 1:10. The result obtained should be multiplied by the dilution factor.