Description
The
125I-hGH IRMA system provides a direct quantitative in vitro
determination of human growth hormone (hGH) in human serum. hGH can be
assayed in the range of 0-100 µIU/ml using 50 µl serum samples.
Introduction
The
human growth hormone (somatotropin, hGH) is a protein hormone with a
molecular weight of 22000, secreted by the pituitary gland. The
structure of the molecule is similar to that of prolactin (hPRL) and
placental lactogen (hPL).
The
secretion of human growth hormone is under double regulation: in certain
neurones of the hypothalamus a growth hormone releasing hormone (GH-RH)
and growth hormone inhibiting hormone, somatostatin (GH-RIH) are
produced.
Several
effects of hGH have been known. It does not only regulate protein
synthesis, the growth of skeleton, the muscles and the viscera but has
lipolytic and lactogenic effects and influences glycogen storage in the
liver as well. In clinical practice the diabetogenic effect of human
growth hormone has also been well-known.
hGH
mean serum level progressively decreases post natally. Its level
increases again significantly during puberty to further decrease with
ageing. Because human growth hormone secretion is pulsative, a single
measurement of hGH concentration does not reflect endogenous hGH
secretion. About 50% of the population has a very low, sometimes
undetectable growth hormone concentration. Standardised stimulatory
tests are therefore necessary to asses pituitary hGH secretion.
The
measuring of human growth hormone can widely be used in clinical
practice for diagnosing hypo- and hypersecretion.
Principle of method
The technology
uses two high affinity monoclonal antibodies in an immunoradiometric
assay (IRMA) system. It offers an increased level of sensitivity and
specificity compared with conventional RIA methods.
The 125I
labelled signal-antibody binds to an epitope of the hGH molecule
different from that recognised by the unlabelled capture-antibody. The
two antibodies react simultaneously with the hGH molecule forming a
“sandwich”.
Standards and samples are incubated with a mixture of the antibodies at
room temperature. At the end of a two hours incubation period (no need
of a shaker), magnetic immunosorbent (MIS) is added in excess. MIS
particles selectively bind the hGH – signal antibody – capture antibody
complex and settle out in a magnetic field. A wash step is critical to
reducing non-specific binding to a minimum for increased low end
precision. By measuring the radioactivity of the magnetic immunosorbent
pellet in a gamma counter the human growth hormone concentration can be
determined.
Contents of the kit
|
1 bottle |
TRACER, Ready to use.
21 ml, containing about 740 kBq 125I-anti-hGH and
capture anti-hGH in buffer with red dye and 0.1% NaN3. |
|
6 vials |
STANDARDS, Lyophilised.
1.0 ml per vial, containing 0, 0.3, 1.3, 5.5, 23, 100 µIU/ml
(WHO 1 st Is 88/624) in human serum with 0.1% NaN3.
See Preparation of reagents. |
|
1 vial |
CONTROL SERUM.
Lyophilized human serum with 0.1% NaN3
The concentration of the control serum is specified in the
quality certificate enclosed.
See Preparation of reagents |
|
1 bottle |
MAGNETIC IMMUNOSORBENT (MIS).
Ready to use.
55 ml, containing paramagnetic particles in buffer with 0.1 %
NaN3. |
|
1 bottle |
WASH BUFFER CONCENTRATE.
20 ml, containing 0.1% NaN3. See Preparation of
reagents |
|
1 pc |
Quality certificate |
|
1 pc |
Pack leaflet |
Materials, tools and equipment required
Round
bottom polystyrene or polypropylene assay tubes, about 12 x 75 mm
Test tube rack
Precision pipettes with disposable tip (50, 200, 500 and 1000 µl)
Distilled water
Vortex mixer
Shaker
Plastic foil
Gamma counter
Absorbent tissue
Recommended
tools and equipment
Repeating pipettes (e.g. Eppendorf)
Dispenser with 300 ml reservoir (instead of the 1 ml pipette)
Specimen collection and storage
Serum
samples can be prepared according to common procedures used routinely in
clinical laboratory practice. Samples can be stored at 2-8 °C if the
assay is carried out within 24 hours, otherwise aliquots should be
prepared and stored deep frozen (-20 °C). Frozen samples should be
thawed and thoroughly mixed before assaying. Repeated freezing and
thawing should be avoided. Do not use lipemic, hemolyzed or turbid
specimens. Samples with a hGH concentration higher than that of the most
concentrated standard should be diluted and assayed.
Preparation of reagents, storage
Add the
wash buffer concentrate (20 ml) to 200 ml distilled water to obtain 220
ml wash solution. Upon dilution store at 2-8 °C until expiry date.
Add 1000 µl
distilled water to the lyophilized standards and control serum. Mix
gently with shaking or vortexing (foaming should be avoided).
Ensure
that complete dissolution is achieved, and allow the solution to
equilibrate at room temperature for at least 20 minutes. Store at -20 °C
until expiry date.
Store
the rest of reagents between 2-8 °C after opening. At this temperature
each reagent is stable until expiry date. The actual expiry date is
given on the package label and in the quality certificate.
CAUTION!
Equilibrate all reagents and serum samples to room temperature. Mix all
reagents and samples thoroughly before use. Avoid excessive foaming.
Assay procedure
(For a quick
guide, refer to Table 1.)
|
1 |
Equilibrate reagents and samples to room temperature
before use. |
|
2 |
Label tubes in duplicate for total counts (T), each
standard (S1-S6), control serums and samples. |
|
3 |
Homogenize all reagents and samples by gentle mixing to
avoid foaming. |
|
4 |
Pipette 50 µl of standards, controls and samples into the
properly labelled tubes. Use rack to hold the tubes. |
|
5 |
Pipette 200 µl of tracer into each tube.
|
|
6 |
Thoroughly vortex mix all tubes except T for 2-5 seconds.
When having an orbital shaker, leave all tubes in the rack
holder, fix the holder onto the plate of the shaker, and shake
it gently for a few seconds. |
|
7 |
Incubate tubes for 2 hours, at room temperature. |
|
8 |
Place T tubes on a separate tube rack. Gently shake and
swirl the bottle containing magnetic immunosorbent until
homogeneity is achieved. Add 500 µl MIS to each tube except T.
When using a single pipette, swirl the bottle of MIS after every
15-20 tubes. With the use of a repeating pipette (e.g.
Eppendorf), there is no need for repeated homogenisation of MIS
reagent. |
|
9 |
Thoroughly vortex mix all tubes, and incubate them for 15
minutes at room temperature. |
|
10 |
Attach the rack on to the magnetic separator base and
ensure that every tube is in contact with the base plate. Let
the MIS particles settle for 5 minutes. Do not remove the rack
from the separator base after the separation of the solid and
liquid phases. Pour off and discard the supernatant. Keeping the
separator inverted, place the tubes on a pad of absorbent tissue
and allow to drain for 2 minutes. |
|
11 |
Return the separator to an upright position and add 1.0
ml of washing solution to each tube. For more comfort and
precision, it is recommended to use either a repeating pipette
(e.g. Eppendorf-pipette) or a dispenser with bottle for the
addition of washing solution. |
|
12 |
Vortex mix each tube thoroughly and repeat Step 10.
Intense vortexing is required when working with a singular
pipette, but repipettors will inject the washing solution
efficiently enough to have the pellett resuspended even without
a subsequent vortexing step. |
|
13 |
Count each tube for at least 60 seconds in a gamma
counter. |
|
14 |
Calculate the concentrations of the samples as described
in Calculation of results. |
Table 1.
Assay Protocol, Pipetting Guide (all volumes in microliters)
|
Tubes |
Total |
Standard |
Sample |
Control |
|
Standard |
|
50 |
|
|
|
Sample |
|
|
50 |
|
|
Control |
|
|
|
50 |
|
Tracer |
200 |
200 |
200 |
200 |
|
Vortex & incubate for 2 hs at room temperature |
|
MIS |
|
500 |
500 |
500 |
|
Vortex & incubate for 15 minutes at room temperature |
|
Separate for 5 minutes |
|
Decant the fluid & blot on filter paper |
|
Wash
Buffer |
|
1000 |
1000 |
1000 |
|
Separate for 5 minutes |
|
Decant the fluid & blot on filter paper |
|
Count radioactivity (60 sec/tube) |
|
Calculate the results |
Table 2.
Typical Assay Data
| |
cpm
1 |
cpm
2 |
cpm
mean |
B / T
% |
|
Total |
205303 |
206909 |
206106 |
- |
|
S1 |
78 |
74 |
76 |
0.04 |
|
S2 |
529 |
630 |
580 |
0.21 |
|
S3 |
2030 |
2146 |
2088 |
0.73 |
|
S4 |
8932 |
8790 |
8861 |
3.00 |
|
S5 |
34273 |
30874 |
32574 |
11.56 |
|
S6 |
98824 |
96142 |
97483 |
41.00 |
|
C |
6831 |
6629 |
6730 |
3.75 |
Calculation of results
The calculation is illustrated using
representative data. The assay data collected should be similar to those
shown in Table 2.
Calculate the average CPM for each pair of
assay tubes. Calculate the normalized percent binding for each standard,
control and sample respectively by using the following equation:
| |
S2-6 [C, Mx] (cpm) - S1
(cpm) |
|
|
B / T % = |
——————————— |
x 100 |
| |
T (cpm) |
|
For simplicity, these values are uncorrected
for non-specific binding (NSB). This is enabled by low NSB being less
than 3% of total count.
Using semi-logarithmic graph paper plot B/T
(%) for each standard versus the corresponding concentration of GH.
Determine the GH concentration of the unknown
samples by interpolation from the standard curve. Do not extrapolate
values beyond the standard curve range.
Out of fitting programs applied for
computerized data processing logit-log, or spline fittings can be used.
Automated data processing systems are also available.
Figure 1
A typical standard curve
(Do not use to calculate unknown samples)
Characterization of the assay
Assay parameters
|
NSB / T |
< 0.05% |
|
Bmax / B0 |
> 800 |
Sensitivity
A
detection limit of 0.03 µIU/ml has been obtained by assaying 20
duplicates of the zero standard. The sensitivity has been determined as
the concentration corresponding to the sum of the mean cpm of the zero
standard and its double standard deviation.
Hook effect
There
is no high dose “hook effect” up to a hGH concentration of 2000 µIU/ml.
Specificity
Cross
reactivities with hPL 0.2% and hPRL 1.0%; no other can be detected in
normal physiological concentration.
Precision
4
patient samples were assayed in 15 replicates to determine intra-assay
precision. Values obtained are shown below.
|
Sample |
Number
of replicates |
Mean value
µIU/ml |
SD
µIU/ml |
CV
% |
|
1 |
15 |
0.74 |
0.046 |
6.2 |
|
2 |
15 |
2.98 |
0.12 |
4.0 |
|
3 |
15 |
8.30 |
0.17 |
2.1 |
|
4 |
15 |
24.44 |
0.81 |
3.3 |
|
5 |
15 |
84.33 |
2.45 |
2.9 |
Reproducibility
To
determine inter-assay precision 4 patient samples were measured in
duplicates in 15 independent assays by 2 operators using different kit
batches. Values obtained are shown below.
|
Sample |
Number
of runs |
Mean value
µIU/ml |
SD
µIU/ml |
CV
% |
|
1 |
15 |
0.75 |
0.042 |
5.6 |
|
2 |
15 |
2.85 |
0.137 |
4.8 |
|
3 |
15 |
8.26 |
0.37 |
4.5 |
|
4 |
15 |
22.58 |
1.06 |
4.7 |
|
5 |
15 |
65.29 |
4.37 |
6.7 |
Recovery
Recovery was defined as the measured increase expressed as percent of
expected increase upon spiking serum samples with known amount of hGH.
The average percent recovery for 10 serum pooles spiked with hGH at 3
levels was: 85.0-100.6%
Expected Values
Healthy
adults: 0-14 µIU/ml (mean 0.79 µIU/ml SD = 1.94 µIU/ml)
It is recommended that each laboratory determine a reference range for
its own patient population.
Procedural notes
Addition of wash buffer. For the addition of wash
buffer the use of a common laboratory dispenser equipped with a 300 ml
glass bottle, and a flexible outlet tubing end is recommended. In lack
of this tool a large volume syringe attached to a repeating pipette can
be used.
Additional information
Components from various lots or kits of different manufacturers should
not be mixed or interchanged.
Precaution
Radioactivity
This
product contains radioactive material. It is the responsibility of the
user to ensure that local regulations or code of practice related to the
handling of radioactive materials are satisfied.
Biohazard
Human
blood products used in the kit have been obtained from healthy human
donors. They were tested individually by using approved methods (EIA,
enzyme immunoassay), and were found to be negative, for the presence of
both Human Immunodeficiency Virus antibody (Anti-HIV-1) and Hepatitis B
surface Antigen (HBsAg).
Care
should always be taken when handling human specimens to be tested with
diagnostic kits. Even if the subject has been tested, no method can
offer complete assurance that Hepatitis B Virus, Human Immunodeficiency
Virus (HIV-1), or other infectious agents are absent. Human blood
samples should therefore be handled as potentially infectious
materials.
Chemical hazard
Components contain sodium azide as an antimicrobial agent. Dispose of
waste by flushing with copious amount of water to avoid build-up of
explosive metallic azides in copper and lead plumbing. The total azide
present in each pack is 74 mg.
 |
Use by |
 |
In vitro diagnostic medical device |
 |
Control |
 |
Batch code |
 |
Manufacturer |
 |
Standard |
 |
Caution, consult accompanying documents |
 |
Radioactive material |
 |
Magnetic immunosorbent |
 |
Biological risk |
 |
Temperature limitation
Store between 2-8 °C |
 |
Tracer |
 |
Consult instructions for use |
 |
Catalogue number |
 |
Wash buffer |
|
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