Enzyme Immunoassay for the Quantitative Determination of Human Growth Hormone (HGH) in Human Serum
Catalogue #KH4005
Contents
Intended Use
For the quantitative measurement of total human growth hormone in human serum. This kit is not licensed for use as a diagnostic test in the United States. It is therefore only sold outside the United States and is marked "for export only."
Outside the United States, the appropriateness of this test kit for research or diagnostic purposes depends on local regulations.
Introduction
Human growth hormone (HGH, somatotropin) is a polypeptide secreted by the anterior pituitary. It is 191 amino acids in length and has a molecular mass of approximately 22,000 daltons. Its metabolic effects are primarily anabolic. HGH promotes protein conservation and is engaged in a wide range of mechanisms for protein synthesis. It also enhances glucose transport and facilitates glycogen storage. Its cascade of growth-promoting action is mediated by another family of peptide hormones, the somatomedins. HGH measurement is primarily of interest in the diagnosis and treatment of various forms of abnormal growth hormone secretion. Disorders caused by hyposecretion include dwarfism and unattained growth potential, and hypersecretion is associated with gigantism and acromegaly. In addition, it has been noted that growth hormone levels change in aging adults and in individuals with AIDS.
Caution must be exercised in the clinical interpretation of growth hormone levels. These vary throughout the day, making it difficult to define a normal range or to judge an individual’s status based on a single determination. Many factors are known to influence the rate of growth hormone secretion, including periods of sleep and wakefulness, exercise, stress, hypoglycemia, estrogens, corticosteroids and L-dopa. Because of its similarity to prolactin and placental lactogen, earlier growth hormone immunoassays were often plagued with falsely high values in pregnant and lactating women.
Because not all acromegalic individuals have elevated baseline levels of growth hormone, suppression tests based on glucose loading are of value in this context. In spite of the induced hyperglycemia, there is rarely a decrease from baseline levels in acromegaly.
Growth hormone-deficient individuals have fasting and resting levels similar to those found in normal individuals. Various challenge tests have therefore been devised to differentiate them. For example, with the onset of deep sleep or after 15 to 20 minutes of vigorous exercise, growth hormone levels normally rise. Other tests of growth hormone responsiveness are based on the administration of L-dopa, arginine and insulin. Propanolol or estrogen are sometimes given in conjunction with the primary stimulus to accentuate the response.
A small number of dwarfism cases have been documented in which both the basal level of HGH and the response to challenge testing were normal. Such cases may involve tissue insensitivity to either growth hormone or the somatomedins, or immunoreactive but biologically inactive growth hormone.
The Human Growth Hormone Enzyme Immunoassay provides a rapid, sensitive and reliable test. There is no cross-reactivity with HCG, TSH, LH, HGH and prolactin.
Principle of the test
The HGH Quantitative Test Kit is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a polyclonal anti-HGH antibody for solid phase (microtiter wells) immobilization and a mouse monoclonal anti-HGH antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test sample is allowed to react simultaneously with the antibodies, resulting in HGH molecules being sandwiched between the solid phase and enzyme-linked antibodies. After a 60 minute incubation at room temperature, the wells are washed with water to remove unbound labeled antibodies. A solution of TMB is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped and the color is changed to yellow with the addition of 2N HCl. The extent of color development is measured spectrophotometrically at 450 nm. The concentration of analyte is directly proportional to the color intensity of the test sample.
Materials
Materials provided with the test kits:
- Antibody coated microtiter plate with 96 wells.
- Reference standard set, contains 0, 1, 2.5, 7.5, 15, and 30ng/ml (WHO, 1st IRP, 66/217) HGH, lyophilized.
- Enzyme Conjugate Reagent.
- Color Reagent A.
- Color Reagent B.
- 2N HCl.
Materials required but not provided:
- Precision pipettes: 50µl, 100µl, 200µl, 1ml, and 5ml.
- Disposable pipette tips.
- Distilled water.
- Glass tubes or flasks to mix Color Reagent A and Color Reagent B.
- Absorbent paper or paper towel.
- Graph paper.
Instrumentation
The following equipment items are required to perform this assay:
- A vortex mixer or equivalent to mix reagents.
- A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength is required for use in the absorbance measurement.
Storage of test kits
Unopened test kits should be stored at 2-8°C upon receipt. The microtiter plate should be stored at 2-8°C in a sealed bag with desiccants. This will minimize its exposure to damp air. Opened test kits will remain stable until the expiration date, provided they are stored as described above.
Specimen collection and preparation
- This kit is for use with serum samples prepared from whole blood. Blood should be drawn using standard venipuncture techniques and the serum should be separated from the red blood cells as soon as practical. Avoid grossly hemolytic, lipemic or turbid samples.
- Plasma samples collected in tubes containing EDTA, heparin, or oxalate may interfere with the test procedures and should be avoided.
- Specimens should be capped and may be stored up to 48 hours at 2-8°C, prior to assaying. Specimens held for a longer time can be frozen at -20°C. Thawed samples must be mixed prior to testing.
Reagent preparation
- All reagents should be brought to room temperature (18-25°C ) and mixed by gently inverting or swirling prior to use. Do not induce foaming.
- Add 1 ml of distilled water to reconstitute the lyophilized standards. Allow the reconstituted materials to stand for at least 20 minutes. Mix gently . The reconstituted standards should be stored sealed at 2-8 °C.
- To prepare TMB substrate reagent, , make a 1:1 dilution of Color Reagent A and Color Reagent B at least 15 minutes before use. Mix gently to ensure complete mixing. The prepared TMB substrate reagent is stable at room temperature, in the dark, for up to 3 hours. Discard excess after use.
Assay procedures
- Secure the desired number of coated wells in the holder.
- Dispense 50µl of standard, specimens, and controls into appropriate wells.
- Dispense 100µl of Enzyme Conjugate Reagent into each well.
- Thoroughly mix for 30 seconds. It is very important to have complete mixing in this setup.
- Incubate at room temperature (18-25°C) for 60 minutes. *Prepare TMB solution 15 minutes before use.
- Remove the incubation mixture by flicking plate content into a waste container.
- Rinse and flick the microtiter wells 5 times with running tap or distilled water.
- Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
- Dispense 200µl of TMB solution into each well. Gently mix for 5 seconds.
- Incubate at room temperature for 20 minutes without shacking.
- Stop the reaction by adding 50µl of 2N HCl to each well.
- Gently mix for 30 seconds to make sure that the blue color changes to yellow color completely.
- Within 30 minutes, read the optical density at 450nm with a microtiter plate reader.
Important Notes
- The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
- It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be competed within 3 minutes. A full plate of 96 well may be used if automated pipetting is available.
- Duplication of all standards and specimens, although not required is recommended.
Calculation of results
Calculate the mean absorbance value (A450) for each set of reference standards, specimens, controls and patient samples. Construct a standard curve by plotting the mean absorbance obtained from each reference standard (Y-axis) against its concentration (X-axis) on graph paper. Use the mean absorbance values for each specimen to determine the corresponding concentration of HGH from the standard curve.
Example of Standard Curve
Results of a typical standard run with the optical density reading at 450nm shown in the Y-axis against the HGH concentrations shown in the X-axis.
Note: This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve.
| HGH (ng/ml) | Absorbance (450nm) |
| 0.0 | 0.052 |
| 1.0 | 0.253 |
| 2.5 | 0.501 |
| 7.5 | 1.158 |
| 15.0 | 2.075 |
| 30.0 | 3.025 |
Expected values and sensitivity
Each laboratory must establish its own normal ranges based on patient population. A normal range for human growth hormone levels is difficult to define because of the normal physiological fluctuations in HGH concentration. In most adult subjects at rest, after an overnight fast, the HGH level in serum is 7 ng/ml or less. Changes in HGH levels in response to various stimuli gives a more accurate assessment of pituitary dysfunction requires provocative tests, either stimulation or suppression.
The minimal sensitivity of the test is 0.5 ng/ml.
References
- Van Wyk, J.J. and Underwood, L.E. Growth Hormone, Somatomedins and Growth Failure. Hospital Practice, 13:57; 1978.
- Fisher, D.A. Evaluation of Anterior Pituitary Function in: Radioimmunoassay Manual. Ed. Nicholas, A.L. and Nelson, J.C.P. 3498 Nichols Institute, 1977.
- Goldfine, I.D. Medical Treatment of Acromegaly. Annual Review of Medicine. 29:407; 1978.
- Reichlin, S. et al. Hypothalamic Hormones. Annual Review of Medicine. 27:359; 1976.
- Rimoin, D.L. and Horton, W.A. Short Stature. J. Pediatrics. 92:523; 1978
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