Human Growth Hormone (hGH) ELISA
INTENDED USE
hGH ELISA kit is used for the quantitative measurement of hGH in human serum or plasma.
SUMMARY AND EXPLANATION
Human Growth Hormone (hGH) is a polypeptide chain, composed of 191 aminoacids and with a molecular weight of 21,500. It is released by the anterior pituitary of both men and women. The secretion is stimulated 3-4 hours after a meal, about 1 hour after the beginning of sleep and after physical exercise.
Hyposecretion of hGH becomes apparent in infants a few months after birth and may result in dwarfism. In the opposite case, hypersecretion of hGH results in gigantism and may be due to hypophysic tumors. In adults, when epiphyses are closed, hypersecretion of hGH provokes an increase in volume of soft tissues (hands, feet, lips) and a proliferation of bones (acromegalysyndrome) and a limited tolerance of glucose.
hGH has profound effects on tissue growth and metabolism, which is thought to be mediated through GH-dependent production of Insulin-like Growth Factor (IGF) I and IGF-II, and their associated binding proteins. hGH apparently stimulates IGF production after binding to specific cell surface receptors in the liver. The major target tissues affected by the IGF-1 in combination with the hGH signal are muscle, cartilage, bone, liver, kidney, nerves, skin and lungs. Evaluation of hGH deficiency is complicated by the episodic nature of hGH secretion and low circulating levels. A variety of physiologic and pharmacologic stimuli have been used to stimulate pituitary hGH release during testing and failure to achieve a normal serum hGH level in response to at least 2 hGH stimulation or provocative tests is considered to be a diagnostic of hGH deficiency. The definition of a normal serum hGH response is controversial, although published values generally range from 5 to 10 ng/ml.
PRINCIPLE OF THE TEST
The DA hGH is a solid phase sandwich ELISA method. The samples, and anti-hGH-HRP conjugate are added to the wells coated with hGH MAb. hGH in the patient’s serum binds to anti-hGH MAb on the well and the anti-HGH second antibody then binds to hGH. Unbound protein and HRP conjugate are washed off by wash buffer. Upon the addition of the substrate, the intensity of color is proportional to the concentration of hGH in the samples. A standard curve is prepared relating color intensity to the concentration of the hGH.
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MATERIALS PROVIDED |
48 tests |
96 tests |
|
1. Microwell coated with hGH MAb |
6x8x1 |
12x8x1 |
|
2. hGH Standard: 6 vials ( ready to use) |
0.35 |
0.7ml |
|
3. hGH Enzyme Conjugate: 1 bottle (ready to use) |
6 ml |
12 ml |
|
4. TMB Substrate: 1 bottle (ready to use) |
12ml |
12ml |
|
5. Stop Solution: 1 bottle (ready to use) |
12ml |
12ml |
|
6. 20X Wash concentrate: 1 bottle |
25ml |
25ml |
MATERIALS NOT PROVIDED
1. Distilled or deionized water
2. precision pipettes
3. Disposable pipette tips
4. Micortiter well reader capable of reading absorbance at 450nm
5. absorbance paper or paper towel aper
6. graph paper
WARNINGS AND PRECAUTIONS
1. Potential biohazardous materials:
The calibrator and controls contain human source components which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984.
2. This test kit is designed for "Research Use Only".
3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
4. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed.
5. It is recommended that standards, control and serum samples be run in duplicate.
6. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data.
SPECIMEN COLLECTION HANDLING
1. Collect blood specimens and separate the serum immediately.
2. Specimens may be stored refrigerated at (2-8° C) for 5 days. If storage time exceeds 5 days, store frozen at (-20° C) for up to one month.
3. Avoid multiple freeze-thaw cycles.
4. Prior to assay, frozen sera should be completely thawed and mixed well.
5. Do not use grossly lipemic specimens.
REAGENTS PREPARATION
Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (18-26° C).
ASSAY PROCEDURE
Prior to assay, allow reagents to stand at room temperature.
Gently mix all reagents before use.
1. Place the desired number of coated strips into the holder
2. Pipet 50 µl of hGH standards, control and patient’s sera.
3. Add 100 µl of hGH enzyme conjugate to all wells.
4. Cover the plate and incubate for 30 minutes at room temperature (18-26° C).
5. Remove liquid from all wells. Wash wells three times with 300 µl of 1X wash buffer. Blot on absorbent paper towels.
6. Add 100 µl of TMB substrate to all wells.
7. Incubate for 10 minutes at room temperature.
8. Add 50 µl of stop solution to all wells. Shake the plate gently to mix the solution.
9. Read absorbance on ELISA Reader at 450 nm within 15 minutes after adding the stopping solution.
CALCULATION OF RESULTS
The standard curve is constructed as follows:
1. Check hGH standard value on each standard vial. This value might vary from lot to lot. Make sure you check the value on every kit. See example of the standard attached .
2. To construct the standard curve, plot the absorbance for the hGH standards (vertical axis) versus the hGH standard concentrations (horizontal axis) on a linear graph paper. Draw the best curve through the points.
3. Read the absorbance for controls and each unknown sample from the curve. Record the value for each control or unknown sample.
4. Value above the highest point of the standard are retested after diluting with "0" standard.
EXPECTED VALUES
It is recommended that each laboratory establish its own normal ranges based on a representative sampling of the local population. The following values for hGH may be used as initial guideline ranges only:
|
Classification |
Normal Range (ng/ml) |
|
Adults |
Less than 10 ng/ml |
|
Children |
Less than 20 ng/ml
7-10 ng/ml on two or more tests = impaired hGH secretion. |
LIMITATIONS OF THE TEST
1. The test results obtained using this kit are for research use only and should be interpreted in relation to the patients history, physical findings and other diagnostic procedures.
2. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.
PERFORMANCE CHARACTERISTICS
1. Correlation with a Reference ELISA kit:
A total of 128 sera were tested by this ELISA and a reference ELISA kit. Results were as follows:
|
Correlation |
Slope |
Intercept |
|
0.94 |
0.93 |
0.38 |
2. Precision Intra-Assay
|
Serum |
No. of Replicates |
Mean
ng/ml |
Standard Deviation |
Coefficient of Variation (%) |
|
1 |
16 |
17 |
0.92 |
5.41 |
|
2 |
16 |
9.7 |
0.51 |
5.25 |
|
3 |
16 |
4.5 |
0.27 |
6.00 |
|
Serum |
No. of Replicates |
Mean
ng/ml |
Standard Deviation |
Coefficient of Variation (%) |
|
1 |
10 |
18.6 |
1.2 |
6.45 |
|
2 |
10 |
10.1 |
0.86 |
8.51 |
|
3 |
10 |
3.7 |
0.33 |
8.91 |
3. Sensitivity The sensitivity was determined by calculating the mean plus 2SD of the standard zero point tested 20 times in the same run.
|
Serum |
No. of Replicates |
Mean
ng/ml |
Standard Deviation |
Mean + 2SD
(Sensitivity) |
|
Zero Std |
20 |
0.08 |
0.06 |
0.2 ng/ml |
4. Recovery Known quantities of hGH were added to a serum that contained a low concentration of hGH.
|
Expected Value(ng/ml) |
Recovered (ng/ml) |
Percentage of Recovery |
|
17.7 |
17.4 |
98.3 |
|
8.9 |
9.3 |
104.4 |
|
4.2 |
4.4 |
95.4 |
5. Linearity Two different patient samples were diluted with the "0" calibrator to 1:2, 1:4 and 1:8. hGH values were assayed and results were corrected with the dilution factor. The results of these dilution tests are as follows:
|
Serum |
Original Value (ng/ml) |
Percentage of Recovery |
|
1
2 |
18
8.6 |
1:2 |
1:4 |
1:8 |
|
94.6
102.0 |
95.2
92.4 |
88.7
85.0 |
REFERENCES:
L´opez-Guajardo CC; Armstrong LS; Jordan L; Staten NR; Krivi GG; Martinez AO; Haro LS. Generation, characterization and utilization of anti-human growth hormone 1-43, (hGH1-43), monoclonal antibodies in an ELISA. J Immunol Methods 1998; 215(1-2): 179-85.
Potter MA; Hymus S; Stockley T; Chang PL. Suppression of immunological response against a transgene product delivered from microencapsulated cells. Hum Gene Ther 1988; 9(9): 1275-82.
Strasburger CJ; Wu Z; Pflaum CD; Dressend¨orfer RA. Immunofunctional assay of human growth hormone (hGH) in serum: a possible consensus for quantitative hGH measurement. J Clin Endocrinol Metab 1996; 81(7): 2613-20.
Tsushima T; Katoh Y; Miyachi Y; Chihara K; Teramoto A; Irie M; Hashimoto Y. Serum concentration of 20K human growth hormone (20K hGH) measured by a specific enzyme-linked immunosorbent assay. Study Group of 20K hGH. J Clin Endocrinol Metab, 1999; 84(1): 317-22.
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