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ACTH
[Adrenocorticotropic Hormone]
ELISA
[Enzyme-Linked ImmunoSorbent
Assay]
Specific quantitative assay for the determination
of adrenocorticotropic hormone in plasma
Catalog # RDI-SDX018
$562.00/kit -10% 2-5 kits -15% 6-9 -25% on 10+
For
Research Use Only
Order
through: Research Diagnostics
Inc
San Jose, 95123 CA Snell ave 658
Flanders NJ
07836
(phone) 973-584-7093
(fax)
973-584-0210
web: http://www.researchd.com
INTENDED
USE
This ACTH ELISA is intended for the quantitative determination
of ACTH (Adrenocorticotropic Hormone) in human plasma.
SUMMARY AND
EXPLANATION
ACTH
(Adrenocorticotropic hormone) or corticotropin is a 39-amino acid peptide
hormone (MW=4500) secreted by the pituitary to regulate the production of
steroid hormones by the adrenal cortex.
ACTH secretion from the anterior pituitary is controlled by both a
classical negative feedback control mechanism and CNS-stress mediated control
system.1 Various types of stress or pain perceived in higher levels
of the brain modulate secretion of the hypothalamic neurosecretory hormone,
corticotropin releasing hormone (CRH), a 41-amino acid
peptide. CRH stimulates pituitary
ACTH secretion. The second peptide
that modulates ACTH secretion is vasopressin
(AVP). AVP secretion is also
stimulated by stress and acts synergistically with CRH to increase ACTH secretion
in the pituitary portal circulation.
ACTH increases the synthesis and release of all adrenal sterioids,
aldosterone, cortisol and adrenal
androgens. It is the principal
modulator of cortisol, the most important glucocorticoid in
man. As the cortisol level in
blood increases, release of ACTH is inhibited directly at the pituitary
level. Through this same mechanism,
decreasing cortisol levels lead to elevated ACTH levels. 2,3,4,5
Biologically
active ACTH results from enzymatic cleavage of a large precursor molecule,
pro-opiomelanocortin (POMC). This
molecule contains within its structure the amino acid sequences of ACTH,
Pro-ACTH, ß-melanocyte stimulating hormone, lipotropin, as well as
endorphin and the enkephalins.
Because the reaction in immunoassays is determined by antigenic structure,
not biological function, the usual ACTH RIA reacts with POMC, Pro-ACTH, ACTH
and some fragments of the
ACTH.5
Like
other pituitary hormones, ACTH is secreted in a pulsatile manner. These small
pulses are superimposed on a characteristic diurnal fluctuation of greater
amplitude. In healthy individuals,
ACTH reaches a peak in the early morning (6:00 - 8:00 hour) and levels become
lowest late in the day and near the beginning of the sleep
period. Because of this diurnal
rhythm it is customary to draw plasma ACTH samples between 8:00 and 10:00
hour. However, differentiation
of patients with Cushing’s disease from normal individuals may be best
achieved on samples obtained in the evening (16:00 - 18:00
hour). In Cushing’s disease
and in ectopic ACTH syndromes, the diurnal pattern of ACTH secretion is generally
absent. Stress may also override
the diurnal variation.
CLINICAL
SIGNIFICANCE
Plasma
ACTH assays are useful in the differential diagnosis of pituitary Cushing’s
disease, Addison’s disease, autonomous ACTH producing pituitary tumors
(e.g. Nelson’s syndrome), hypopituitarism with ACTH deficiency and ectopic
ACTH syndrome. 5,6,7,8,9,10
Cushing’s
syndrome is caused by the effects of excess glucocorticoid actions. All causes
of Cushing’s syndrome, with the exception of glucocorticoid medication,
are associated with incresed 24-hour urinary cortisol. The most common cause
of Cushing’s syndrome is bilateral adrenal hyperplasia, due to pituitary
ACTH hypersecretion (Cushing’s disease) from a pituitary adenoma or
corticotroph hyperplasia.5,6,7,8,9,10 Laboratory diagnosis of
Cushing’s disease is supported by the following: (1) suppression of
plasma ACTH and cortisol concentrations,
by high-dose (2.0 mg q 6h x
8) dexamethasone administration, (2) absence of ACTH and cortisol suppression
with low-dose (0.5 mg q 6h x 8 or 1 mg given at 23:30 hour) dexamethasone,
(3) larger than normal response to metyrapone (Metopirone ) stimulation and
normal or elevated plasma ACTH
levels.4
When
Cushing’s syndrome is caused by primary adrenal abnormality (adenoma
or carcinoma), the adrenal gland acts independently of ACTH and pituitary
ACTH secretion is suppressed. 5,6,7,8,9,10 Hence, there is no
response to dexamethasone suppresion or metyrapone
stimulation. This type of Cushing’s syndrome is characterized
by very low, or undetectable levels of ACTH.
Therefore,
measurement of plasma ACTH is helpful in differential diagnosis of pituitary
Cushing’s syndrome. In
patients with adrenal tumors, ACTH levels are
low. High levels of ACTH are
seen in patients with ectopic ACTH
syndrome. Patients with bilateral
adrenal hyperplasia will have ACTH levels inappropriately elevated for their
degree of hypercortisolism, which should suppress
ACTH. However, in most cases
the ACTH concentration will be within the normal range.
Adrenocortical
insufficiency or inadequate cortisol production can be due to destruction
of the adrenal cortex or to abnormalities of the pituitary or hypothalamus,
which result in inadequate ACTH production of release. 5,6,7,8,9,10
Primary adrenocortical insufficiency, Addison’s disease, is characterized
by markedly elevated plasma ACTH levels and adrenal unresponsiveness to
stimulation with exogenous ACTH.
Hypopituitarism with ACTH deficiency, which is secondary adrenocortical
insufficiency, is characterized by low plasma ACTH and cortisol concentrations,
and a subnormal, but usually distinct adrenal response to stimulation with
synthetic ACTH
(Cortrosyn®). If
hypoglycemic stress or metyrapone stimulation is required for diagnosis,
ACTH and cortisol responses are less than
normal.
Aggressive
and invasive ACTH producing pituitary tumors occurring before or following
bilateral adrenalectomy for Cushing’s disease (Nelson’s syndrome)
are characterized by the development of Addisonian pigmentation, often in
an adrenalectomized patient who is taking adequate glucocorticoid replacement
therapy. In these patients,
plasma ACTH levels are markedly elevated and do not respond well to dexamethasone
suppression.
PRINCIPLE OF THE
TEST
This ACTH Immunoassay is a two-site
ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of the biologically
active 39 amino acid chain of ACTH. A goat polyclonal antibody to human ACTH, purified by
affinity chromatography, and a mouse monoclonal antibody to human ACTH are
specific for well defined regions on the ACTH
molecule. One antibody is prepared
to bind only the C-terminal ACTH 34-39 and this antibody is biotinylated.
The other antibody is prepared to bind only the mid-region and N-terminal
ACTH 1-24 and this antibody is labeled with horseradish peroxidase [HRP]
for detection.
|
In this assay, calibrators, controls,
or patient samples are simultaneously incubated with the enzyme labeled antibody
and a biotin coupled antibody in a streptavidin-coated microplate
well. At the end of the assay
incubation, the microwell is washed to remove unbound components and the
enzyme bound to the solid phase is incubated with the substrate,
tetramethylbenzidine (TMB). An
acidic stopping solution is then added to stop the reaction and converts
the color to yellow. The intensity
of the yellow color is directly proportional to the concentration of ACTH
in the sample. A
dose response curve of absorbance unit vs. concentration is generated using
results obtained from the calibrators. Concentrations of ACTH present in
the controls and patient samples are determined directly from this
curve.
KIT COMPONENTS
Catalog
Number |
Kit
Components |
Description |
Quantity |
||
30-1801
|
Reagent
1 |
Biotinylated
ACTH Antibody [affinity purified goat anti human ACTH] |
1
x 2.5 mL |
||
30-1802 |
Reagent
2 |
Peroxidase
(Enzyme) labeled ACTH Antibody [mouse monoclonal anti human
ACTH] |
1
x 2.5 mL |
||
30-1613 |
ELISA
Reagent
A |
ELISA
Wash Concentrate [Saline with surfactant] |
1
x 30 mL |
||
30-1611 |
ELISA
Reagent
B |
1 x 15 mL |
|||
30-1612 |
ELISA
Reagent
C |
ELISA
Stop Solution [1 N sulfuric acid] |
1 x 10 mL |
||
30-1601 |
Microplate |
One
holder with Streptavidin Coated Strips. |
12 x 8-well
strips |
||
30-1803
30-1804
30-1805
30-1806
30-1807
30-1808 |
Calibrators
A: 0
pg/mL
B:
C:
D:
E:
F: |
Lyophilized
[except zero calibrator] synthetic
h-ACTH. Zero calibrator [BSA/equine
serum solution] is in liquid form, ready to
use. All other calibrators consist
of synthetic
h-ACTH
(1-39) in BSA/equine serum solution |
1
x 4 mL for the
zero
calibrator
1
x 2 mL for all
other
calibrators
|
||
30-1809
30-1810 |
Controls 1 &
2
|
Lyophilized. 2 Levels. Synthetic h-ACTH (1-39)
in BSA/equine serum solution. |
MATERIAL AND
Equipment REQUIRED BUT NOT
PROVIDED
·
Microplate reader.
·
Microplate washer [if washer is unavailable, manual washing
may be acceptable].
·
Precision Pipettors to deliver 25, 200, 100 and 150
µL.
·
(Optional):
A
multi-channel dispenser or a repeating dispenser for 25, 100 and 150 µL.
WARNINGS AND PRECAUTIONS FOR
USERS
Although the reagents
provided in this kit has been specifically designed to contain no human blood
components, the human patient samples, which might be positive for HBsAg,
HBcAg or HIV antibodies, must be treated as potentially infectious
biohazard. Common precautions
in handling should be exercised, as applied to any untested patient
sample.
ELISA Reagent
C, Stop Solution, consists of 1 N Sulfuric
Acid. This is a strong
acid. Although diluted, it still
must be handled with care. It
can cause burns and should be handled with gloves and eye protection, with
appropriate protective clothing.
Any spill should be wiped immediately with copious quantities of
water. Do not breath vapor and
avoid inhalation.
SAMPLE COLLECTION AND STORAGE
The
determination of ACTH should
be performed on EDTA plasma To
assay the specimen in duplicate, 400 µL of EDTA plasma is
required. Collect whole blood
in a lavender [EDTA] tube. After
allowing blood to clot, the plasma should be promptly separated, preferably
in a refrigerated centrifuge, and stored at -20oC or lower. EDTA
plasma samples may be stored up to 8 hours at
2-8°C. EDTA plasma samples
frozen at -20°C are stable for up to 4 months.
REAGENT PREPARATION AND STORAGE
Store all kit components
at 2-8 oC except Wash Concentrate and Stop Solution
1.
All
reagents except the non-zero calibrators, kit controls and the Wash Concentrate
are ready-to-use. Store all
reagents at 2-8 oC, except the Wash Concentrate, which should
be kept at room temperature until dilution to avoid
precipitation.
2.
For
each of the non-zero calibrators (Calibrator B through F) and kit controls
1 and 2, reconstitute each vial with 2 mL of distilled or deionized water
and mix. Allow the vial to stand
for 10 minutes and then mix thoroughly by gentle inversion to insure complete
reconstitution.
Use the calibrators and controls
as soon as possible upon reconstitution.
Freeze (-20oC) the
remaining calibrators and controls as soon as possible after
use. Standards and controls
are stable at
-20
oC for 6 weeks after reconstitution with up to 3
freeze thaw cycles when handled as recommended in “Procedural Notes”
section.
3.
ELISA
Reagent
A: Wash Concentrate: Mix contents of wash concentrate
thoroughly. Add wash concentrate
(30 mL) to 570 mL of distilled or deionized water and
mix.
ASSAY PROCEDURE
1.
Place
sufficient Streptavidin Coated
Strips in a holder to run all six (6) ACTH calibrators, A - F of
the ACTH CALIBRATORS [Exact
concentration is stated on the vial label], Quality Control Plasma and patient
samples.
2.
Pipet
200 µL of sample into the
designated or mapped well.
Freeze (-20oC) the
remaining calibrators and controls as soon as possible after use.
3.
Add
or dispense 25 µL of Reagent
1 (Biotinylated Antibody) into each of the wells which already contain the
sample.
4.
Add
or dispense 25 µL of Reagent
2 (Enzyme Labeled Antibody) into each of the same wells.
Cover the microplate(s) with
aluminum foil or a tray to avoid exposure to light, And place it on an
orbital shaker or rotator set
at 170 + 10 rpm for 4 hours
+ 30 minutes at room temperature
(22o28oC).
5.
Aspirate
and wash each well five (5) times with the Working Wash Solution (prepared
from
ELISA
Reagent A), using an automatic microplate
washer. Blot dry by inverting
the plate on an absorbent material.
The wash solution volume should be set to dispense 0.35 mL into each
well.
6.
Add
or dispense 150 µL of the
ELISA
Reagent B (TMB Substrate)
into each of the wells.
7.
With
appropriate cover to avoid light exposure, place the microplate(s) on an
orbital shaker or rotator set
at 170 + 10 rpm for 30 +5 minutes
at room temperature
(22o28oC).
8.
Add
or dispense 100 µL of the
ELISA
Reagent C (Stop Solution) into each of the
wells. Mix
gently.
9.
Read
the absorbance of the solution in the wells within 10 minutes, using a microplate
reader set to 450 nm against 250
µL of distilled or deionized water.
Read the plate
again with the reader set to
405 nm against distilled or deionized
water.
Note: The second reading is designed to extend the analytical validity of the calibration curve to the value represented by the highest calibrator, which is approximately 500 pg/mL. Hence, patient samples with ACTH > 150 pg/mL can be quantified against a calibration curve consisting of the readings all the way up to the concentration equivalent to the highest calibrator using the 405 nm reading, away from the wavelength of maximum absorbance. In general, patient and control samples should be read using the 450 nm for ACTH concentrations up to 150 pg/mL. ACTH concentrations above 150 pg/mL should be interpolated using the 405 nm reading.
10.
By
using the final absorbance values obtained in the previous step, construct
a calibration curve via cubic spline, 4 parameter logistics, or point-to-point
interpolation to quantify the concentration of
the ACTH.
PROCEDURAL
NOTES
·
ACTH 1-39 is a
very labile molecule. Set up
the assay immediately upon the reconstitution or the thawing of all calibrators,
controls, and patient samples.
·
It is recommended
that all calibrators, controls, and patient samples are assayed in
duplicate. The average absorbance
units of duplicate sets should then be used for reduction of data and the
calculation of results.
·
The samples should
be pipetted into the well with minimum amount of air-bubble. To achieve this,
“reverse pipet” described in the package insert of the manufacturers
of Pipettors is recommended.
·
Patient samples
with values greater than the highest calibrator (Calibrator F), which is
approximately 500 pg/mL (see exact concentration on vial label), may be diluted
with Calibrator A (Zero Calibrator) and
reassayed. Multiply the result by the dilution
factor.
·
Reagents from different
lot numbers must not be interchanged.
·
If preferred, mix
in equal volumes, in sufficient quantities for the assay, Reagent 1 (Biotinylated
Antibody) and Reagent 2 (Enzyme Labeled Antibody) in a clean amber bottle,
Then use 50 µL of the mixed antibody into each
well. This alternative method should replace Step (3) and (4),
to be followed with the incubation with orbital shaker.
Assay
Protocol Flow-Diagram
Microplate
Well Position |
Sample |
CalibratorsControls
OR
Patients |
Reagent 1
Biotinylated
Antibody
Solution |
Reagent 2
Enzyme Conjugate
Antibody |
Incubate at room
temperature
|
Working Wash Solution
|
ELISA
Reagent B TMB Substrate |
Incubate
At room temp.
|
ELISA Reagent C Stop (Acid)
Solution |
Read Aborbance
At 450 nm and 405
nm |
B1
C1
D1
E1
F1
G1
H1
A2
B2
C2
D2
E2
F2 |
Distilled Water
Calibrator A
Calibrator B
Calibrator C
Calibrator D
Calibrator E
Calibrator F
Control 1
Control 2
Patient 1
Patient 2
ETC.
ETC.
ETC. |
250 µL
200 µL
|
25 µL
|
25 µL
|
4 + ½
hours
@ 170 +
10 rpm
|
350 µL
Wash
5 times
Aspirate
|
150 µL
|
30 + 5
minutes
@ 170 +
10 rpm
|
100 µL
|
Read against distilled or deionized
water |
CALCULATION OF RESULTS
Manual Method
1.
For the 450 nm
readings, construct a dose response curve (calibration curve) using the first
five calibrators provided, i.e. Calibrators A, B, C, D and
E. For the 405 nm readings,
construct a second dose response curve using the three calibrators with the
highest concentrations, i.e. Calibrators D, E and F.
2.
Assign the
concentration for each calibrator stated on the vial in
pg/mL. Plot the data from the
calibration curve on linear graph paper with the concentration on the X-axis
and the corresponding A.U. on the Y-axis.
3.
Draw a straight
line between 2 adjacent points.
This mathematical algorithm is commonly known as the "point-to-point"
calculation. Obtain the
concentration of the sample by locating the absorbance unit on the Y-axis
and finding the corresponding concentration value on the X-axis. Patient
and control samples should be read using the 450 nm for ACTH concentrations
up to 150 pg/mL. ACTH concentrations
above 150 pg/mL should be interpolated using the 405 nm
reading.
4.
Automated
Method:
Computer programs using cubic spline or
4 PL [4 Parameter Logistics] can generally give a good
fit.
Sample
Data
at 450 nm
[raw A.U. readout against distilled
or deionized water]
Microplate
Well |
1st
Reading Absorbance
Unit |
2nd
Reading Absorbance Unit
|
Average Absorbance Unit
|
ACTH pg/mL
|
ACTH
pg/mL –Result to
report |
Calibrator A |
0.020 |
0.018 |
0.019 |
|
0 |
Calibrator B |
0.077 |
0.074 |
0.076 |
|
5 |
Calibrator C |
0.221 |
0.229 |
0.225 |
|
18 |
Calibrator D |
0.624 |
0.692 |
0.685 |
|
55 |
Calibrator E |
1.802 |
1.934 |
1.868 |
|
165 |
Control 1 |
0.417 |
0.398 |
0.408 |
33.5 |
33.5 |
Control 2 |
2.868 |
2.774 |
2.821 |
> 150 |
* |
Patient Sample 1 |
0.072 |
0.078 |
0.075 |
4.9 |
4.9 |
Patient Sample 2 |
0.185 |
0.177 |
0.181 |
14.0 |
14.0 |
Patient Sample 3 |
0.495 |
.491 |
.493 |
40.8 |
40.8 |
Patient Sample 4 |
2.090 |
2.122 |
2.106 |
> 150 |
* |
* Because the concentration readout is >
150 pg/mL, it is recommended to use the data obtained at 405 nm as shown
in Sample Data
at 405 nm in the table below.
Sample
Data
at 405 nm
[raw A.U. readout against distilled
or deionized water]
Microplate
Well |
1st
Reading Absorbance
Unit |
2nd
Reading Absorbance Unit
|
Average Absorbance Unit
|
ACTH pg/mL
|
ACTH
pg/mL –Result to
report |
Calibrator A |
0.011 |
0.008 |
0.0095 |
|
0 |
Calibrator D |
0.032 |
0.032 |
0.032 |
|
55 |
Calibrator E |
0.074 |
0.081 |
0.078 |
|
165 |
Calibrator F |
1.838 |
1.817 |
1.828 |
|
500 |
Control 1 |
0.138 |
0.132 |
0.135 |
< 150 |
¶ |
Control 2 |
0.921 |
0.894 |
0.908 |
256 |
256 |
Patient Sample 1 |
0.030 |
0.032 |
0.031 |
< 150 |
¶ |
Patient Sample 2 |
0.068 |
0.062 |
0.065 |
< 150 |
¶ |
Patient Sample 3 |
0.165 |
0.159 |
0.162 |
< 150 |
¶ |
Patient Sample 4 |
0.663 |
.677 |
0.670 |
188 |
188 |
¶ For samples with readout < 150 pg/mL, it is
recommended to use the data obtained at 450 nm as shown in
Sample Data at 450
nm in the table above. This practice should give the results with
optimum sensitivity of the assay.
NOTE: The data presented are
for illustration purposes only and must not be used in place of data generated
at the time of the assay.
QUALITY
CONTROL
Control plasma or plasma pools should be analyzed
with each run of calibrators and patient samples. Results generated from
the analysis of the control samples should be evaluated for acceptability
using appropriate statistical methods.
In assays in which one or more of the quality control sample values
lie outside the acceptable limits, the results for the patient sample may
not be valid.
LIMITATIONS
OF THE PROCEDURE
This ACTH ELISA kit has exhibited
no “high dose hook effect” with samples spiked with 20,000 pg/mL
of ACTH. Samples with ACTH levels
greater than the highest calibrator, however, should be diluted and reassayed
for correct values.
Like any analyte used as a diagnostic
adjunct, ACTH results must be interpreted carefully with the overall clinical
presentations and other supportive diagnostic tests.
EXPECTED
VALUES
ACTH levels were measured in eighty-three
(83) apparently normal individuals in the U.S. with this ACTH
ELISA. The values obtained ranged
from 7.9to 66.1 pg/mL. Based
on statistical tests on skewness and kurtosis, the population, when transformed
logarithmically, follows the normal or Gaussian distribution as shown in
the histograms. The geometric
mean + 2 standard deviations of the mean were calculated to be 8.3to
57.8 pg/mL
|
|
|
.
 |
 |
PERFORMANCE
CHARACTERISTICS
Accuracy
One
hundred seventeen (117) patient samples, with ACTH values ranging from 1.5
to 1045
pg/mL
were assayed by this ELISA procedure and the Nichols IRMA (immunoradiometric
assay) ACTH kit. Linear regression
analysis gives the following statistics:
|
Sensitivity
The sensitivity, or minimum detection
limit, of this assay is defined as the smallest single value, which can be
distinguished from zero at the 95% confidence
limit. This ACTH ELISA
has a calculated sensitivity of 0.46 pg/mL.
Precision and
Reproducibility
The
precision (intra-assay variation) of this ACTH ELISA Test was calculated
from 21 replicate determinations on each of the two
samples.
Intra-Assay
Variation
Mean
Value
Coefficient
Sample
(pg/mL)
N
of Variation
%
A
35.7
21
3.1
B
255
21
4.2
The
total precision (inter-assay variation) of this ACTH ELISA Test was calculated
from data on two samples obtained in 35 different assays, by three technicians
on three different lots of reagents, over a nine-week
period
Inter-Assay
Variation
Mean
Value
Coefficient
Sample
(pg/mL)
N
of Variation
%
A
35.2
35
5.8
B
230
35
6.2
Specificity and Cross-Reactivity
Cross-reactivity in the ACTH was studied by the addition of
various materials to an ACTH standard.
The results are as follows:
Cross-reactant |
Concentration |
ACTH |
ACTH |
Change
in |
% |
|
||||||
|
of |
without |
With |
ACTH |
Cross-reactivity |
|
||||||
|
Cross-reactant |
Cross-reactant |
Cross-reactant |
[pg/mL] |
|
|
||||||
|
|
[pg/mL] |
[pg/mL] |
|
|
|
||||||
ACTH
(1-24) |
100,000
pg/mL |
74.5 |
3.1 |
-71.4 |
-0.07% |
|
||||||
|
10,000
pg/mL |
74.5 |
17.1 |
-57.4 |
-0.57% |
|
||||||
|
1,000
pg/mL |
74.5 |
60.9 |
-13.6 |
-1.36% |
|
||||||
|
200
pg/mL |
74.5 |
68 |
-6.5 |
-3.25% |
|
||||||
|
|
|
|
|
|
|
||||||
ACTH
(18-39) |
5,000
pg/mL |
67 |
19 |
-48 |
-0.96% |
|
||||||
|
2,000
pg/mL |
67 |
26.8 |
-40.2 |
-2.01% |
|
||||||
|
500
pg/mL |
67 |
43.3 |
-23.7 |
-4.74% |
|
||||||
|
|
|
|
|
|
|
||||||
a-MSH |
100,000
pg/mL |
72.3 |
1.3 |
-71 |
-0.07% |
|
||||||
|
10,000
pg/mL |
72.3 |
9.8 |
-62.5 |
-0.63% |
|
||||||
|
1,000
pg/mL |
72.3 |
44.5 |
-27.8 |
-2.78% |
|
||||||
|
200
pg/mL |
72.3 |
61 |
-11.3 |
-5.65% |
|
||||||
|
|
|
|
|
|
|
||||||
b-ENDORPHIN |
100,000
pg/mL |
76.3 |
69.3 |
-7 |
-0.01% |
|
||||||
|
50,000
pg/mL |
76.3 |
73.5 |
-2.8 |
-0.01% |
|
||||||
Recovery
Various amounts of ACTH were added to four
different patient plasma to determine the
recovery. The results are described in the following
table:
Plasma
Sample |
Endogenous |
ACTH |
Expected |
Measured |
Recovery |
|
ACTH |
Value |
Value |
(%) |
|
(pg/mL) |
(pg/mL) |
(pg/mL) |
(pg/mL) |
|
|
A |
23.3 |
-- |
-- |
-- |
-- |
|
21.0 |
50.0 |
71.0 |
75.0 |
105.6% |
|
18.6 |
100.0 |
118.6 |
126.0 |
106.2% |
B |
28.1 |
-- |
-- |
-- |
-- |
|
25.3 |
50.0 |
75.3 |
80.7 |
107.2% |
|
22.5 |
100.0 |
122.5 |
142.0 |
115.9% |
C |
21.8 |
-- |
-- |
-- |
-- |
|
19.6 |
50.0 |
69.6 |
67.6 |
97.1% |
|
17.4 |
100.0 |
117.4 |
125.0 |
106.4% |
D |
9.8 |
-- |
-- |
-- |
-- |
|
8.8 |
50.0 |
58.8 |
51.6 |
87.7% |
|
7.8 |
100.0 |
107.8 |
96.4 |
89.4% |
Kinetic Effect of the
Assay
To
determine whether there is any systematic kinetic effect between the beginning
of the run and the end of the run, three spiked patient pools, selected to
represent a good cross section of the ACTH concentration, were placed in
sequence throughout the run of one microplate or 96 wells [with twelve 8-well
strips]. The results, displayed
in the following graphs, show no significant assay
drift.
Linearity of Patient Sample
Dilutions:
Parallelism
Five patient plasma
samples were diluted with Calibrator A (Zero
Calibrator). Results in pg/mL
are shown below:
REFERENCES:
1.
Ryan, WG: Endocrine
Disorders – A Pathophysiiologic Approach, 2nd Edition Year Book Medical
Publishers, Inc. 1980.
2.
Watts,
N.B., J.H. Keffer: Practical
Endocrine Diagnosis, Third Edition, Lea and Febioer,
1982.
3.
Ganong, WF. L.D. Alber,
TC Lee: ACTH and the Regulation
of Adrenocorticol Secretion, N. Engl. J. Med. 290 : 1006,
1974.
4.
Tepperman, J: Metabolic
and Endocrine Physiology, 4th Edition, Year Book Medical Publishers, Inc.,
1981.
5.
Odell, W.D., R. Horton,
M.R. Pandian, J. Wong: The Use
of ACTH and Cortisol Assays in the Diagnosis of Endocrine Disorders. Nichols
Institute Publication, 1989.
6.
Radioimmunoassay Manual,
Edited by A.L. Nichols and J.C.
Nelson, 4th Edition Nichols Institute, 1977.
7.
Gold, E.M.: The
Cushing’s Syndromes: Changing Views of Diagnosis and Treatment. Ann
Intern. Med. 90:829, 1979.
8.
Plasma Cortisol, RIA
for Physicians, Edited by J.C. Travis, 1:8, Scientific Newsletter, Inc.
1976.
9.
Krieger, D.T.:
Physiopathology of Cusihing’s Disease, Endocrine Review 4:22-43,
1983.
10.
Krieger, D.T., A.S.
Liotta, T. Suda, A Goodgold,
and E. Condon: Human Plasma Immunoreactive Lipotropin and Adrenocorticotropin
in Normal Subjects and in Patients with Pituitary-Adrenal Disease, J. Clin.
Endocrinol Metab. 48:566-571, 1979.
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