CORTISOL RIA
DSL-2000
Revision date: February 20, 2001
Diagnostic Systems Laboratories, Inc.
Corporate Headquarters, 445 Medical Center Blvd.
Webster, Texas 77598-4217 USA
General Business: Tel: 281.332.9678
Customer Assistance Center: Tel: 800.231.7970 Fax: 281. 338.1895
e-mail: mktg@dslabs.com
Website: http://www.dslabs.com
I.
INTENDED USE
The DSL-2000 Cortisol Radioimmunoassay Kit provides materials for the
quantitative measurement of cortisol in serum, plasma or urine. This assay is
intended for in vitro diagnostic use.
II.
SUMMARY AND EXPLANATION OF THE TEST
Cortisol (hydrocortisone, compound F) is the most potent glucocorticoid produced
by the human adrenal cortex. As with other adrenal steroids, cortisol is
synthesized from cholesterol through a series of enzymatically mediated steps
[reviewed in 1,2]. The first and rate-limiting step in adrenal steroidogenesis,
conversion of cholesterol to pregnenolone, is stimulated by pituitary
adrenocorticotropic hormone (ACTH) which is, in turn, regulated by hypothalamic
corticotropin releasing factor (CRF). ACTH and CRF secretion are inhibited by high
cortisol levels. In plasma, the major portion of cortisol is bound with high affinity
to corticosteroid-binding globulin (CBG, transcortin), with most of the remainder
loosely bound to albumin. Cortisol acts through specific intracellular receptors
and has effects in numerous physiologic systems, including immune function,
glucose-counterregulation, vascular tone, substrate utilization and bone
metabolism [1-3]. Cortisol is excreted primarily in the urine in an unbound (free)
form.
Cortisol production has an ACTH-dependent circadian rhythm with peak levels in
the early morning and a nadir at night. The factors controlling this circadian
rhythm are not completely defined. The circadian rhythm of ACTH/cortisol
secretion matures gradually during early infancy, and is disrupted in a number of
physical and psychological conditions [4]. Furthermore, increased amounts of
ACTH and cortisol are secreted independently of the circadian rhythm in response
to physical and psychological stress [4,5].
Elevated cortisol levels and lack of diurnal variation have been identified in
patients with Cushing's disease (ACTH hypersecretion) [2,6]. Elevated circulating
cortisol levels have also been identified in patients with adrenal tumors [7]. Low
cortisol levels are found in primary adrenal insufficiency (e.g. adrenal hypoplasia,
congenital adrenal hyperplasia, Addison's disease) and in ACTH deficiency
[1,2,8,9]. Due to the normal circadian variation of cortisol levels, distinguishing
normal and abnormally low cortisol levels can be difficult. Therefore, various tests
to evaluate the pituitary-adrenal (ACTH-cortisol) axis, including insulin-induced
hypoglycemia, short- and long-term ACTH stimulation, CRF stimulation and
artificial blockage of cortisol synthesis with metyrapone [8-13] have been
performed. Cortisol response characteristics for each of these procedures have
been reported.
The DSL-2000 Cortisol Radioimmunoassay Kit uses a specific rabbit anti-cortisol
antibody, and does not require prior sample extraction on serum. Crossreactivity to other naturally-occurring steroids is low.
III. PRINCIPLE OF THE TEST
The procedure follows the basic principle of radioimmunoassay where there is
competition between a radioactive and a non-radioactive antigen for a fixed
number of antibody binding sites [14]. The amount of [I-125]-labeled analyte
bound to the antibody is inversely proportional to the concentration of the labeled
analyte present. The separation of free and bound antigen is easily and rapidly
achieved by using a pre-reacted double antibody system.
IV. REAGENTS
The DSL-2000 Cortisol RIA Kit contains sufficient reagents for 100 tubes. Each kit
contains the following reagents:
A
Cortisol Standards:
Seven vials, 0.5 mL each, labeled A-G, containing concentrations of approximately
0.0, 0.5, 2.0, 4.0, 10.0, 20.0 and 60.0 g/dL (13.8-1654.8 nmol/L) of cortisol in
human serum (cortisol free) with sodium azide as a preservative. Refer to vial
labels for exact concentrations. Store at 2-8C for up to three weeks. For longer
periods, store at -20C or lower.
B.
Cortisol [I-125] Reagent: (RED)
One bottle, 55 mL, containing <5 Ci (185 kBq) [I-125]-labeled cortisol in a buffer
with polyethylene glycol as a precipitating aid and sodium azide as a preservative.
Store at 2-8C until expiration date.
C.
Cortisol Diluent:
One vial, 5 mL, containing a buffer with a protein stabilizer and sodium azide as a
preservative. Store at 2-8C until expiration date.
D.
Cortisol Antiserum Complex: (BLUE)
One bottle, 55 mL, containing rabbit anti-cortisol serum in phosphate buffer with
sodium azide as a preservative. Store at 2-8C until expiration date.
E.
Cortisol Controls:
Two vials, 0.5 mL each, Levels I and II, containing low and high levels of cortisol in
human serum with sodium azide as a preservative. Store at 2-8C for up to three
weeks. For longer periods, store at -20C or lower.
VIII. TEST PROCEDURE
A.
Materials Supplied:
Materials supplied in the DSL Cortisol RIA Kit, Catalog No. DSL-2000:
MATERIAL
NOTE: All reagents and samples must be allowed to reach room temperature
(~25°C) and mixed thoroughly by gentle inversion before use.
V.
Cortisol Standard A
Cortisol Standard B
Cortisol Standard C
Cortisol Standard D
Cortisol Standard E
Cortisol Standard F
Cortisol Standard G
Cortisol [I-125] Reagent
Cortisol Diluent
Cortisol Antiserum Complex
Cortisol Serum Control Level I
Cortisol Serum Control Level II
PRECAUTIONS
For in vitro diagnostic use.
Radioactive material -- Not for Internal or External Use in Humans or Animals.
This radioactive material may be received, acquired, possessed and used only by physicians,
clinical laboratories or hospitals and only for in vitro clinical or laboratory tests not involving
internal or external administration of the material, or the radiation therefrom, to human
beings or animals. Its receipt, acquisition, possession, use and transfer are subject to the
regulations and a general license of each country.
The following precautions should be observed in handling radioactive material:
Store radioactive materials in a designated area.
Do not eat, drink, smoke, or apply cosmetics where radioactive materials are being
handled.

Do not pipet by mouth.

Wear gloves when handling radioactive materials and wash hands thoroughly
afterwards.

Cover working area with disposable absorbent paper.

Wipe up all spills immediately and thoroughly and dispose of the contaminated
materials as radioactive waste.

Dispose of the liquid radioactive waste into the sanitary sewage system if permitted by
the local regulations.
WARNING: POTENTIAL BIOHAZARDOUS MATERIAL


This kit may contain reagents made with human serum or plasma. The serum or plasma
used has been tested by an FDA approved method and found to be non-reactive for both
the HIV-1/2 Antibodies, HCV and HBsAg. Because no method can offer complete assurance
that HIV-1/2, HCV, HBsAg or other infectious agents are absent, these reagents should be
handled at the Biosafety Level 2 as recommended for any potentially infectious human
serum or blood specimen in the Centers for Disease Control/National Institutes of Health
manual "Biosafety in Microbiological and Biomedical Laboratories", 3rd Edition, 1993.
CHEMICAL HAZARD:
As stated in Section IV, some of the reagents in this kit contain sodium azide as a
preservative. For all such reagents, the concentration of sodium azide is ~0.09%. Sodium
azide may react with lead and copper plumbing to form explosive metal azides. Dispose of
all nonradioactive reagents by flushing with large amounts of water through the plumbing
system.
VI. SPECIMEN COLLECTION AND PREPARATION
Serum or Plasma
Serum or plasma may be used and the usual precautions for venipuncture should
be observed. Serum or plasma may be stored at 2-8C for up to 24 hours and
should be frozen at -20C or lower if stored for longer periods.
Because of the diurnal variation in cortisol levels, the time of collection of the
specimen should be noted. Do not use grossly hemolyzed or grossly lipemic
specimens.
Urine
B.














Extraction Procedure for Urine Specimens:
1.
8.
9.
Add 250 L of thoroughly mixed urine to an appropriately marked 12 x
75 mm glass tube.
Add 1 mL of methylene chloride.
Vortex for 30 seconds.
Centrifuge at 2000 rpm for 5 minutes OR allow to stand at room
temperature
(~25°C) for 30 minutes.
Aspirate the upper aqueous layer.
Transfer 100 L of methylene chloride extract (lower organic layer) in
duplicate to appropriately labeled 12 x 75 mm plastic tubes.
Evaporate the extract to dryness by using a gentle stream of air or
nitrogen OR by allowing the tube to stand at room temperature.
Add 25 L of Cortisol Diluent to each tube of dried urine extract.
Proceed with step 3 of the assay procedure.
VII.
PROCEDURAL NOTES
2.
3.
4.
5.
6.
7.
A thorough understanding of this package insert is necessary for successful use of the
product. Reliable results will only be obtained by using precise laboratory techniques and
accurately
following the package insert. A standard curve must be included with each assay.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Failure to obtain the appropriate cortisol values for Controls may indicate imprecise
manipulations, improper sample handling or deterioration of reagents. Failure to blot tubes
adequately following decantation may result in poor replication and spurious values.
2001
2002
2003
2004
2005
2006
2007
2020
2030
2010
2051
2052
Materials Required But Not Supplied:
12 x 75 mm plastic test tubes.
12 x 75 mm glass tubes for urine extraction.
Test tube rack for 12 x 75 mm tubes.
Deionized water.
Precision pipette to deliver 25 L.
Precision repeating pipette to deliver 500 L.
Vortex mixer.
Centrifuge (1500 x g, preferably refrigerated).
Sponge test tube rack or similar device for decantation.
Waterbath capable of 37 ± 2C.
Absorbent material for blotting tubes.
Gamma counter.
Semi-log graph paper or computer RIA data analysis program.
Methylene chloride for urine extraction procedure.
Label and arrange tubes in duplicate for Total Count tubes, Standards,
Controls and unknowns.
Add 25 L of the Standards, Controls or unknowns to the appropriate
tubes. Pipet to the bottom of the tubes.
Immediately add 500 L of the Cortisol [I-125] Reagent to each tube.
Add 500 L of the Cortisol Antiserum Complex to all tubes except Total
Count tubes. This reagent should be mixed thoroughly before use.
Vortex all tubes.
Incubate all tubes in a water bath at 37 ± 2C for 45 minutes.
Centrifuge all tubes except Total Count tubes for 15-20 minutes at 1500
x g in a refrigerated centrifuge.
Decant all tubes, except Total Count tubes by simultaneous inversion,
holding an inversion with a sponge rack into a radioactive waste
receptacle. Allow them to drain on absorbent material for 15 - 30
seconds and gently blot the tubes to remove any droplets adhering to the
rim before returning them to the upright position. Failure to blot tubes
adequately may result in poor replication and spurious results.
Count all tubes in a gamma counter for one minute.
IX. RESULTS
The results in this package insert were calculated using log-linear curve fit. Other
data reduction methods may give slightly different results.
A.
Calculate the mean counts per minute (CPM) for each Standard, Control and
unknown. Calculate the % B/T, or %B/Bo for each Standard, Control and
unknown as follows:
% B/T =
% B/Bo =
B.
C.
D.
E.
F.
Do not mix various lots of any kit component within an individual assay. Do not use any
component beyond the expiration date shown on its label.
After removing assay reagents from the refrigerator, allow them to reach room temperature
(~25°C) before pipetting. Unused reagents should be stored as stated in the Reagent
Section. Standards and Controls should be mixed before use by inverting or swirling gently
rather than vortexing. To insure a homogeneous mixture of the reagents in each assay
tube, gentle and thorough shaking or vortexing is essential.
CATALOG NO.
One Vial
One Vial
One Vial
One Vial
One Vial
One Vial
One Vial
One Bottle
One Vial
One Bottle
One Vial
One Vial
C.
Assay Procedure:
Allow all reagents to reach room temperature (~25°C) and mix thoroughly by
gentle inversion before use. Standards, Controls, and unknowns should be
assayed in duplicate.
The total volume of urine excreted during a 24 hour period should be collected
and mixed in a single container. Preservatives are not required; however, 10
grams of boric acid per liter of urine may be added.
Specimens may be stored at room temperature after collection. Urine samples
which are not to be assayed immediately should be stored at 2-8C. Urine should
be stored at -20C or lower if stored longer than one week. Repeated freezing
and thawing of samples should be avoided.
QUANTITY
Mean Sample Counts
Mean Total Counts
Mean Sample Counts
Mean Counts of 0 g/dL
X 100
X 100
Plot a curve of % B/T, or % B/Bo for the Cortisol Standards (y-axis) against
the cortisol concentration (x-axis) on log-linear (semi-log) graph paper.
Draw a standard curve through the mean of the duplicate points.
Determine the cortisol concentration of the means of the duplicate counts of
each Control and unknown sample from the standard curve.
Any sample reading greater than the highest Standard should be diluted
appropriately with the Cortisol Diluent and re-assayed.
Any sample reading lower than the lowest Standard should be reported as
such.
To determine the cortisol concentration in urine, calculate as above and
correct for total volume of urine collected in 24 hours:
g/dL x 24 hour volume (mL) = g Cortisol/24 hour
100
X.


LIMITATIONS
This kit should not be used to measure cortisol levels in samples from
patients treated with prednisolone or prednisone (prednisone is rapidly
converted in vivo to prednisolone [15,16]) because of the high
cross-reactivity of the antibody to prednisolone.
Hemolyzed and lipemic specimens may give false values and must be
avoided.

Avoid freezing and thawing of reagents and specimens.
XI.


QUALITY CONTROL
Maximum binding (% bound), or counts bound in the absence of unlabeled
antigen is approximately 50% when freshly iodinated tracer is used, and
may fall to 25% near the end of expiration date. Maximum binding less
than 25% may indicate reagent deterioration, contamination, or poor
technique.
DSL Controls or other commercial controls should fall within established
confidence limits. The confidence limits for DSL Controls are printed on
the Control vial labels.
TYPICAL CORTISOL STANDARD CURVE DATA
TUBE
NO.
TUBE
LABEL
BOUND
(cpm)
MEAN
(cpm)
1, 2
TOTAL
COUNTS
79112
79819
79466
3, 4
STANDARDS
A
F
45939
45535
37942
39732
30927
30626
24626
24714
17889
17682
12456
12458
G
5, 6
B
7, 8
C
9, 10
D
11, 12
E
13, 14
15, 16
17, 18
19, 20
CONTROLS
Level I
Level II
B/T
(%)
B/Bo
(%)
CORTISOL
(g/dL)
45737
57.6
100.0
0
38837
48.9
84.9
0.5
30777
38.7
67.3
2.0
24670
31.0
53.9
4.0
17786
22.4
38.9
10.0
12457
15.7
27.2
20.0
8282
8590
8436
10.6
18.4
60.0
24311
26049
12090
12823
25180
31.7
55.1
3.77
12457
15.7
27.2
20.00
ED50 = 5.1 g/dL
CAUTION: The above data must not be employed in lieu of data obtained by the
user in the laboratory.
XII. EXPECTED VALUES
It is recommended that each laboratory establish its own range of normal cortisol
values. Results of a normal range study, conducted with the DSL-2000 Cortisol
RIA, are as follows:
TIME
N
NORMAL RANGE
(g/dL)
A.M.
P.M.
100
100
5-25
2-12
Because of diurnal variation [4] in normal subjects, serum or plasma cortisol levels
are highest in the morning and lowest in the evening. Serum cortisol levels after
ACTH stimulation tests normally increase 2-3 times over the basal value.
Dexamethasone or Metyrapone suppression tests lower the basal serum cortisol
value to 75-90% of normal [8-12].
Assay values for plasma samples (heparin or EDTA) may be approximately 5-10%
lower than for serum.
Urine normal range: 25-120 g/24 hours (non-extracted urine)
XIII. PERFORMANCE CHARACTERISTICS
All performance characteristics are stated in g/dL. To convert to nmol/L:
g/dL x 27.6 = nmol/L
A.
Sensitivity:
The theoretical sensitivity, defined as the lowest detectable level of cortisol that
can be distinguished from the 0 g/dL Cortisol Standard, is 0.11 g/dL at the 95%
confidence limit.
B.
Precision:
The intra-assay precision was determined from the mean of 12 replicates each.
SAMPLE
I
II
III
N
12
12
12
(g/dL)
MEAN
STANDARD
DEVIATION
(g/dL)
COEFFICIENT
OF VARIATION
(%)
5.04
18.80
27.32
0.33
0.66
2.26
6.5
3.5
8.3
The inter-assay precision was determined from the mean of average duplicates for
12 separate runs.
SAMPLE
I
II
III
N
12
12
12
(g/dL)
MEAN
STANDARD
DEVIATION
(g/dL)
COEFFICIENT
OF VARIATION
(%)
5.06
18.46
29.68
0.40
1.26
2.76
7.9
6.8
9.3
C.
Recovery:
Three serum samples containing different levels of endogenous cortisol were
spiked with different amounts of an elevated serum sample and assayed.
SAMPLE
ENDOGENOUS
(g/dL)
ADDED
(g/dL)
EXPECTED
(g/dL)
OBSERVED
(g/dL)
RECOVERY
(%)
I
2.27
II
7.94
III
13.11
1.73
3.65
7.49
1.73
3.65
7.49
1.73
3.65
7.49
4.00
5.92
9.76
9.67
11.59
15.43
14.84
16.75
20.60
3.97
6.57
8.95
8.46
11.80
14.26
14.22
14.99
17.94
99
111
92
87
102
92
96
89
87
D.
Linearity of Dilution:
Three serum samples were diluted with 0 g/dL Cortisol Standard and assayed.
SAMPLE
I
II
III
DILUTION
FACTOR
EXPECTED
(g/dL)
OBSERVED
(g/dL)
RECOVERY
(%)
--1:2
1:4
1:8
1:16
--1:2
1:4
1:8
1:16
--9.90
4.95
2.47
1.23
--13.92
6.96
3.48
1.74
19.81
8.68
4.23
1.86
0.95
27.84
13.85
6.54
3.71
1.87
--88
85
75
77
--99
94
107
107
--1:2
1:4
1:8
1:16
--9.87
4.93
2.46
1.23
19.74
9.64
4.49
2.08
1.04
--98
91
85
85
E.
Specificity:
The cross-reactivity of the cortisol antiserum has been measured against various
compounds. The percent cross-reactivity is expressed as the ratio of the cortisol
concentration to the concentration of the reacting compound at 50% binding of
the 0 g/dL Standard. In most cases, the interference from these compounds is
insignificant when compared to the much higher levels of circulating cortisol. The
cross-reactivity of the antibody with prednisolone precludes the use of this kit for
patients on prednisolone or prednisone therapy (prednisone is rapidly converted
in vivo to prednisolone) [16,17].
STEROID
Cortisol
Prednisolone
Corticosterone
11-Deoxycortisol
Cortisone
Prednisone
17-Hydroxyprogesterone
11-Deoxycorticosterone
Dexamethasone
Testosterone
Progesterone
Epiandrosterone
Dehydroepiandrosterone
Estradiol
XIV. REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
% CROSS REACTIVITY
100
33.33
9.30
3.80
2.22
1.42
1.00
0.61
0.38
0.14
0.12
0.04
0.02
0.02
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Lee PDK, Winter RJ, Green OC: Virilizing adrenocortical tumors in childhood. Eight cases and a review of
the literature. Pediatrics 76:437-444, 1985.
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Stewart PM, Seckl JR, Corrie J, Edwards CRW, Padfield PL: A rational approach for assessing the
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Watts NB, Tindall GT: Rapid assessment of corticotropin reserve after pituitary surgery. JAMA 259:708711, 1988.
Lashansky G, Saenger P, Fishman K, Gautier T, Mayes D, Berg G, Di Martino-Nardi J, Reiter E: Normative
data for adrenal steroidogenesis in a healthy pediatric population: age- and sex-related changes after
adrenocorticotropin stimulation. J Clin Endocrinol Metab 73:674-686, 1991.
Schlaghecke R, Kornely E, Santen RT, Ridderskamp P: The effect of long-term glucocorticoid on
pituitary-adrenal responses to exogenous corticotropin-releasing hormone. New Engl J Med 326:226230, 1992.
Yalow R and Berson S: Introduction and general considerations. IN: Odell WD, Daughday WH (eds):
Principals of Competitive Protein Binnding Assays. J.B. Lippincott Co., Philadelphia, 1971, pp 1-19.
Jenkins J, Sampson P: Conversion of cortisone to cortisol and prednisone to prednisolone. Br Med J
2:205, 1967.
Colburn W, Buller R: Radioimmunoassay for prednisolone. Steroids 21:833, 1973.
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