To know the levels
of the hormones, we check or test them through:
Chemiluminescence Immunoassay for Growth Hormone
Chemiluminescence Immunoaasay has been considered the diagnostic
assay for Growth hormone because it has been shown to be more sensitive than
the conventional colorimetric methods, and does not require long incubations or
the addition of stopping reagents, as is the case in some colorimetric assays.
Principle:
·
Microtiter wells (Solid phase)—Monoclonal anti-HGH Antibody
· Conjugate solution-- Monoclonal anti-HGH Antibody in the antibody
enzyme (Horseradish peroxidase)
Substrate
Substrate
·
Serum specimen—HGH (Antigen)
If there is the presence of HGH in the
serum, after the addition of the Conjugate enzyme solution, it will bind to the
antibody on the well and the conjugate enzyme solution that will result in
Sandwiching of the 2 antibodies and the antigen. To remove unbound labelled antibodies,
the microtiter wells are being washed after the 1 hour incubation. Then, a solution
of chemiluminescent substrate is then added.
The
intensity of the emitting light is directly proportional to the amount of
enzyme present and to the antigen present in the sample.
Procedure:
1.
In a microtiter well, dispense a 50 uL of the specimen. Mix
thoroughly for 10 seconds.
2.
Dispense 100 uL of the conjugate enzyme solution. Mix thoroughly
for 30 seconds.
3.
Incubate for 1 hour.
4.
Remove the incubation mixture by emptying the plate content into a
waste container.
5.
Rinse and flick the wells 5 times with washing buffer.
6.
Dispense 100 uL Chemiluminescence Substrate solution into the
well. Mix for 5 seconds.
7.
Read wells with a chemiluminescence microwell reader 5 minutes
later.
Sensitivity
0.5 ng/ml
Normal
Values
In
most adult subjects at rest, after an overnight fast, the HGH level in serum is
7 ng/ml or less.
Cortisol ELISA
Principle:
Cortisol ELISA
This test is used for the quantitative
determination of cortisol in serum.
·
Microwell titers—Rabbit anti-cortisol antibody coated
·
Conjugate Solution— Antigen Cortisol with Horseradish peroxidise (enzyme)
·
Substrate
·
Serum specimen—Unlabeled antigen (Cortisol)
This test employs competitive binding
assay. Competition occurs between an unlabeled antigen and the enzyme-labelled
antigen which is the conjugate. If the Cortisol is present in the specimen,
during the 45 minutes incubation, it will compete with the enzyme-labelled
antigen for the binding to the antibody on the mirowell. After the washing
step, a substrate is being added. The absorbance is measured on the microtiter
plate reader. The intensity of the color formed is inversely proportional to
the levels of Cortisol in the serum.
Procedure:
1.
Dispense 20 uL of the sample in the microwell plate.
2.
Add 100 uL of the conjugate solution.
3.
Incubate for 45 minutes on room temperature.
4.
Wash the wells 3 times with 300 uL of diluted wash buffer and tap
the plate firmly against absorbent paper.
5.
Add 150 uL of substrate.
6.
Incubate for 15-20 minutes.
7.
Read the plate on the reader for 450 nm filter.
Sensitivity:
0.4 ug/dL
Normal
Values;
3.95-27.23 ug/dL
References:
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