concentration permits construction of a graph of activity and con- 4.1 Required But Not Provided: 2.

d: 2. Working Substrate Solution – Stable for 1 year

centration. From comparison to the dose response curve, an 1. Pipette capable of delivering 0.025ml (25µl), 0.050ml, (50µl) Pour the contents of the amber vial labeled Solution ‘A’ into
unknown specimen's activity can be correlated with cortisol and 0.100ml (100µl) volumes with a precision of better than the clear vial labeled Solution ‘B’. Place the yellow cap on the
concentration. 1.5%. clear vial for easy identification. Mix and label accordingly.
2. Dispenser(s) for repetitive deliveries of 0.050ml (50µl) 0.100ml Store at 2 - 30°C.
3.0 PRINCIPLE (100µl) and 0.350ml (350µl) volumes with a precision of better Note 1: Do not use the working substrate if it looks blue.
than 1.5%. Note 2: Do not use reagents that are contaminated or have
Competitive Enzyme Immunoassay (TYPE 7): 3. Microplate washer or a squeeze bottle (optional). bacteria growth.
The essential reagents required for an enzyme immunoassay 4. Microplate Reader with 450nm and 620nm wavelength
include antibody, enzyme-antigen conjugate and native antigen. absorbance capability. 9.0 TEST PROCEDURE
Upon mixing biotinylated antibody, enzyme-antigen conjugate and 5. Absorbent Paper for blotting the microplate wells.
a serum containing the native antigen, a competition reaction 6. Plastic wrap or microplate covers for incubation steps. Before proceeding with the assay, bring all reagents, serum
results between the native antigen and the enzyme-antigen 7. Vacuum aspirator (optional) for wash steps. reference calibrators and controls to room temperature (20-27°C).
conjugate for a limited number of antibody binding sites. The 8. Timer. **Test Procedure should be performed by a skilled individual
interaction is illustrated by the followed equation: 9. Quality control materials. or trained professional**
ka
5.0 PRECAUTIONS 1. Format the microplates’ wells for each serum reference,
Enz
Ag + Ag + Ab Btn AgAb Btn + EnzAgAb Btn control and patient specimen to be assayed in duplicate.
k -a For In Vitro Diagnostic Use Replace any unused microwell strips back into the
°
Ab Btn = Biotinylated Antibody (Constant Quantity) Not for Internal or External Use in Humans or Animals
Ag = Native Antigen (Variable Quantity) aluminum bag, seal and store at 2-8 C.
Enz
2. Pipette 0.025 ml (25µL) of the appropriate serum reference,
Cortisol Test System Ag = Enzyme-antigen Conjugate (Constant Quantity)
AgAb Btn = Antigen-Antibody Complex
All products that contain human serum have been found to be
control or specimen into the assigned well.
non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV
Product Code: 3625-300 Enz
AgAb Btn = Enzyme-antigen Conjugate -Antibody Complex Antibodies by FDA required tests. Since no known test can offer 3. Add 0.050 ml (50µl) of the ready to use Cortisol Enzyme
k a = Rate Constant of Association complete assurance that infectious agents are absent, all human Reagent to all wells
k -a = Rate Constant of Disassociation serum products should be handled as potentially hazardous and 4. Swirl the microplate gently for 20-30 seconds to mix.
1.0 INTRODUCTION K = k a / k -a = Equilibrium Constant capable of transmitting disease. Good laboratory procedures for 5. Add 0.050 ml (50µl) of Cortisol Biotin Reagent to all wells.
handling blood products can be found in the Center for Disease 6. Swirl the microplate gently for 20-30 seconds to mix.
Intended Use: The Quantitative Determination of Total A simultaneous reaction between the biotin attached to the Control / National Institute of Health, "Biosafety in Microbiological 7. Cover and incubate for 60 minutes at room temperature.
Cortisol Concentration in Human Serum or Plasma by a antibody and the streptavidin immobilized on the microwell occurs. and Biomedical Laboratories," 2nd Edition, 1988, HHS Publication 8. Discard the contents of the microplate by decantation or
Microplate Enzyme Immunoassay, Colorimetric This effects the separation of the antibody bound fraction after No. (CDC) 88-8395. aspiration. If decanting, blot the plate dry with absorbent
decantation or aspiration. paper.
2.0 SUMMARY AND EXPLANATION OF THE TEST AgAb Btn + EnzAgAb Btn + Streptavidin CW ⇒ immobilized complex Safe Disposal of kit components must be according to local 9. Add 0.350ml (350µl) of wash buffer (see Reagent Preparation
Streptavidin CW = Streptavidin immobilized on well regulatory and statutory requirement. Section), decant (tap and blot) or aspirate. Repeat two (2)
Cortisol (hydrocortisone, compound F) is the most potent Immobilized complex = sandwich complex bound to the solid surface additional times for a total of three (3) washes. An automatic
glucocorticoid produced by the human adrenal cortex. As with other 6.0 SPECIMEN COLLECTION AND PREPARATION or manual plate washer can be used. Follow the
adrenal steroids, cortisol is synthesized from cholesterol, through a The enzyme activity in the antibody-bound fraction is inversely manufacturer’s instruction for proper usage. If a squeeze
series of enzymatically mediated steps, by the adrenal cortex.1,2 The proportional to the native antigen concentration. By utilizing The specimens shall be blood; serum or plasma in type and the bottle is employed, fill each well by depressing the
first and rate-limiting step in adrenal steroidogenesis, conversion of several different serum references of known antigen con- usual precautions in the collection of venipuncture samples should container (avoiding air bubbles) to dispense the wash.
cholesterol to pregnenolone, is stimulated by pituitary centration, a dose response curve can be generated from which be observed. For accurate comparison to established normal Decant the wash and repeat two (2) additional times.
adrenocorticotropic hormone (ACTH) which is, in turn, regulated by the antigen concentration of an unknown can be ascertained. values, a fasting morning serum sample should be obtained. The 10. Add 0.100 ml (100µl) of working substrate solution to all wells
hypothalamic corticotropin releasing factor (CRF). ACTH and CRF blood should be collected in a plain redtop venipuncture tube (see Reagent Preparation Section). Always add reagents in
secretion are inhibited by high cortisol levels. In plasma, the major 4.0 REAGENTS without additives or anti-coagulants (for serum) or evacuated the same order to minimize reaction time differences
portion of cortisol is bound with high affinity to corticosteroid-binding tube(s) containing EDTA or heparin. Allow the blood to clot for between wells.
globulin (CBG, transcortin), with most of the remainder loosely bound Materials Provided: DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION
serum samples. Centrifuge the specimen to separate the serum or
to albumin. Physiologically effective in anti-inflammatory activity and A. Cortisol Calibrators – 1ml/vial – Icons A-F plasma from the cells. 11. Incubate at room temperature for fifteen (15) minutes.
blood pressure maintenance, cortisol is also involved in Six (6) vials of serum reference for Cortisol at concentrations 12. Add 0.050ml (50µl) of stop solution to each well and gently mix
gluconeogenesis. Cortisol acts through specific intracellular receptors of 0 (A), 1.0 (B), 4.0 (C), 10.0 (D), 20.0 (E) and 50.0 (F) µg/dl. In patients receiving therapy with high biotin doses (i.e. for 15-20 seconds. Always add reagents in the same order
and has effects in numerous other physiologic systems, including Store at 2-8°C. A preservative has been added. >5mg/day), no sample should be taken until at least 8 hours to minimize reaction time differences between wells.
after the last biotin administration, preferably overnight to 13. Read the absorbance in each well at 450nm (using a reference
immune function, glucose-counter regulation, vascular tone, substrate B. Cortisol Enzyme Reagent – 7.0 ml/vial – Icon E
utilization and bone metabolism.1-3 Cortisol is excreted primarily in ensure fasting sample. wavelength of 620-630nm to minimize well imperfections) in a
One (1) ready to use vial containing Cortisol (Analog)- microplate reader. The results should be read within thirty
urine in an unbound (free) form. horseradish peroxides (HRP) conjugate in a protein stabilizing
Samples may be refrigerated at 2-8°C for a maximum period of (30) minutes of adding the stop solution.
matrix with buffer, red dye, preservative and binding protein
Cortisol production has an ACTH-dependent circadian rhythm with five (5) days. If the specimen(s) cannot be assayed within this
inhibitors. Store at 2-8°C. Note: Dilute the samples suspected of concentrations higher than
peak levels in the early morning and a nadir at night. The factors time, the sample(s) may be stored at temperatures of -20°C for up
controlling this circadian rhythm are not completely defined. The C. Cortisol Biotin Reagent – 7.0 ml – Icon ∇ to 30 days. Avoid use of contaminated devices. Avoid repetitive 50 µg/dl 1:5 and 1:10 with cortisol ‘0’ µg/dl patient serum.
circadian rhythm of ACTH/cortisol secretion matures gradually during One (1) vial containing anti-cortisol biotinylated mIgG freezing and thawing. When assayed in duplicate, 0.050ml (50µl)
early infancy, and is disrupted in a number of physical and conjugate in buffer, dye and preservative. Store at 2-8°C. of the specimen is required. 10.0 CALCULATION OF RESULTS
psychological conditions.4 Furthermore, increased amounts of ACTH D. Streptavidin Coated Plate – 96 wells – Icon ⇓
and cortisol are secreted independently of the circadian rhythm in One 96-well microplate coated with 1.0 µg/ml streptavidin and 7.0 QUALITY CONTROL A dose response curve is used to ascertain the concentration
4,5
response to physical and psychological stress. packaged in an aluminum bag with a drying agent. Store at of cortisol in unknown specimens.
2-8°C. Each laboratory should assay controls at levels in the low, normal 1. Record the absorbance obtained from the printout of the
Elevated cortisol levels and lack of diurnal variation have been E. Wash Solution Concentrate – 20ml/vial – Icon and high range for monitoring assay performance. These controls microplate reader as outlined in Example 1.
identified in patients with Cushing's disease (ACTH hyper One (1) vial containing a surfactant in buffered saline. A should be treated as unknowns and values determined in every 2. Plot the absorbance for each duplicate serum reference versus
2,6
secretion). Elevated circulating cortisol levels have also been preservative has been added. Store at 2-8°C. test procedure performed. Quality control charts should be the corresponding cortisol concentration in µg/dl on linear
identified in patients with adrenal tumors.7 Low cortisol levels are E. Substrate A – 7ml/vial – Icon SA maintained to follow the performance of the supplied reagents. graph paper (do not average the duplicates of the serum
found in primary adrenal insufficiency (e.g. adrenal hypoplasia, One (1) vial containing tetramethylbenzidine (TMB) in buffer. Pertinent statistical methods should be employed to ascertain references before plotting).
congenital adrenal hyperplasia, Addison's disease) and in ACTH trends. The individual laboratory should set acceptable assay 3. Connect the points with a best-fit curve.
Store at 2-8°C.
deficiency.1,2,8,9 Due to the normal circadian variation of cortisol levels, F. Substrate B – 7ml/vial – Icon SB performance limits. In addition, maximum absorbance should be 4. To determine the concentration of cortisol for an unknown,
distinguishing normal and abnormally low cortisol levels can be consistent with past experience. Significant deviation from locate the average absorbance of the duplicates for each
One (1) vial containing hydrogen peroxide (H 2 O 2 ) in buffer.
difficult. Therefore, various tests to evaluate the pituitary-adrenal established performance can indicate unnoticed change in unknown on the vertical axis of the graph, find the intersecting
Store at 2-8°C.
(ACTH-cortisol) axis, including insulin-induced hypoglycemia, short- experimental conditions or degradation of kit reagents. Fresh point on the curve, and read the concentration (in µg/dl) from
STOP
and long-term ACTH stimulation, CRF stimulation and artificial G. Stop Solution – 8ml/vial – Icon reagents should be used to determine the reason for the the horizontal axis of the graph (the duplicates of the unknown
blockage of cortisol synthesis with metronome have been performed.8- One (1) vial containing a strong acid (1N HCl). Store at 2-8°C. variations. may be averaged as indicated). In the following example, the
10
Cortisol response characteristics for each of these procedures have H. Product Instructions. average absorbance (1.071) intersects the dose response
been reported. 8.0 REAGENT PREPARATION curve at (10.2 µg/dl) cortisol concentration (See Figure 1).
Note 1: Do not use reagents beyond the kit expiration date.
The Monobind Cortisol EIA Kit uses a specific monoclonal anti-cortisol Note 2: Avoid extended exposure to heat and light. Opened Note: Computer data reduction software designed for ELISA
1. Wash Buffer
antibody, and does not require prior sample extraction of serum or reagents are stable for sixty (60) days when stored at assays may also be used for the data reduction. If such
plasma. Cross-reactivity to other naturally-occurring steroids is low. Dilute contents of wash solution to 1000ml with distilled or
2-8°C. Kit and component stability are identified on the software is utilized, the validation of the software should be
deionized water in a suitable storage container. Diluted
label. ascertained.
The employment of several serum references of known cortisol reagent can be stored at 2-30°C for up to 60 days.
Note 3: Above reagents are for a single 96-well microplate.
 EXAMPLE 1 Any deviation from Monobind’s IFU may yield inaccurate TABLE 2 6. Hyams JS, Carey DE: ‘Corticosteroids and Growth.’ J of
results. Within Assay Precision (Values in µg/dl ) Pediatrics, 113, 249-254 (1988).
Sample Well Mean Value
I.D. Number
Abs (A)
Abs (B) (µg/dl)
10. All applicable national standards, regulations and laws, Sample N X σ C.V. 7. Kreiger DT, ‘Rhythms of ACTH and corticosteroid secretion
including, but not limited to, good laboratory procedures, must Low 16 3.4 0.28 8.2% in health and disease and their experimental modifications’, J
A1 2.483 be strictly followed to ensure compliance and proper device Normal 16 14.2 0.91 6.4% of Steroid Biochemistry, 6, 785-791 (1975).
Cal A 2.543 0 usage. 8. Leistee S, Ahonen P, Perheentupa J, ‘The diagnosis and
B1 2.575 High 16 36.5 2.23 6.1%
C1 2.150 11. It is important to calibrate all the equipment e.g. Pipettes, staging of hypocortisolism in progressing autoimmune
Cal B 2.194 1.0 Readers, Washers and/or the automated instruments used adrenalitis’, Pedriatics Res, 76, 437 (1985).
D1 2.186 TABLE 3
with this device, and to perform routine preventative Between Assay Precision (Values in µg/dl ) 9. Alsevier RN, Gotlin RW, ‘Handbook of Endocrine Tests in
E1 1.573
Cal C 1.585 4.0 maintenance. Adults and Children’ 2nd Ed Year Book Medical Pub Inc
F1 1.597 Sample N X σ C.V.
12. Risk Analysis- as required by CE Mark IVD Directive 98/79/EC - Chicago. 1978.
G1 1.103 Low 10 3.1 0.30 9.7%
Cal D 1.084 10 for this and other devices, made by Monobind, can be 10. Watts NB, Tindall GT, ‘Rapid assessment of corticotrophin
H1 1.065 requested via email from [email protected]. Normal 10 15.1 1.06 7.0% reserve after pituitary surgery’, JAMA, 259, 708 (1988).
A2 0.726 High 10 37.4 2.71 7.3%
Cal E 0.725 20
B2 0.724 12.2 Interpretation *As measured in ten experiments in duplicate over a ten day Effective Date: 2019-Jul-16 Rev. 4 DCO: 1353
C2 0.347 1. Measurements and interpretation of results must be period. MP3625 Product Code: 3625-300
Cal F 0.350 50
D2 0.353 performed by a skilled individual or trained professional.
E2 1.624 2. Laboratory results alone are only one aspect for determining 14.2 Sensitivity Size 96(A) 192(B)
Ctrl 1 1.617 3.74 The Cortisol AccuBind® ELISA Test System has a sensitivity of
F2 1.611 patient care and should not be the sole basis for therapy, A) 1ml set 1ml set
G2 0.770 particularly if the results conflict with other determinants. 91.5 pg. This is equivalent to a sample containing a concentration
Ctrl 2 0.760 18.57 of 0.366 µg/dl. The sensitivity was ascertained by determining the B) 1 (7ml) 2 (7ml)
H2 0.749 3. The reagents for the procedure have been formulated to

Reagent (fill)
variability of the 0 µg/dl serum calibrator and using the 2σ (95% C) 1 (7ml) 2 (7ml)
A3 1.056 eliminate maximal interference; however, potential interaction
Patient 1.071 10.24 certainty) statistic to calculate the minimum dose. D) 1 plate 2 plates
B3 1.086 between rare serum specimens and test reagents can cause
erroneous results. Heterophilic antibodies often cause these E) 1 (20ml) 1 (20ml)
*The data presented in Example 1 and Figure 1 is for illustration interactions and have been known to be problems for all kinds 14.3 Accuracy F) 1 (7ml) 2 (7ml)
only and should not be used in lieu of a standard curve prepared of immunoassays. (Boscato LM Stuart MC.’Heterophilic The Cortisol AccuBind® ELISA Test System was compared with a
G) 1 (7ml) 2 (7ml)
with each assay. antibodies: a problem for all immunoassays’ Clin. Chem coated tube radioimmunoassay method. Biological specimens
from low, normal and high cortisol level populations were used. H) 1 (8ml) 2 (8ml)
1988:3427-33). For diagnostic purposes the results from this
assay should be used in combination with clinical examination, The values ranged from 0.4 µg/dl – 95µg/dl. The total number of
Figure 1
patient’s history and, all other clinical findings. such specimens was 202. The least square regression equation
4. For valid test results, adequate controls and other parameters and the correlation coefficient were computed for this cortisol EIA
3.000
must be within the listed ranges and assay requirements. in comparison with the reference method. The data obtained is
2.500 5. If test kits are altered, such as by mixing parts of different kits, displayed in Table 4.
which could produce false test results, or if results are
Absorbance(s)

2.000 TABLE 4
incorrectly interpreted, Monobind shall have no liability.
1.500 6. If computer controlled data reduction is used to interpret the Mean Least Square Correlation
Patient results of the test, it is imperative that the predicted values for Method (x) Regression Analysis Coefficient
1.000 Monobind (y) 16.6 y = -0.228+1.0186(x) 0.984
the calibrators fall within 10% of the assigned concentrations.
0.500 7. Total serum cortisol values may be dependent upon conditions Reference (X) 16.8
such as time of the day for sampling or administration of
0.000 prednisolone or prednisone (structurally related to cortisol). Only slight amounts of bias between this method and the
0 10 20 30 40 50 Caution must be exercised while interpreting cortisol levels for reference method are indicated by the closeness of the mean
patients undergoing therapy with these and other structurally values. The least square regression equation and correlation
Cortisol Values in µg/dl coefficient indicates excellent method agreement.
related corticosteroids such as cortisone or corticosterone.
11.0 Q.C. PARAMETERS 14.4 Specificity
13.0 EXPECTED RANGES OF VALUES The % cross-reactivity of the cortisol antibody to selected
In order for the assay results to be considered valid the substances was evaluated by adding the interfering substance to
A study of normal adult population was undertaken to determine a serum matrix at various concentrations. The cross-reactivity was
following criteria should be met:
expected values for the Cortisol AccuBind® ELISA Test System. calculated by deriving a ratio between doses of interfering
1. The absorbance (OD) of calibrator 0 µg/dl should be > 1.3.
The mean (R) values, standard deviations (σ) and expected substance to dose of cortisol needed to displace the same amount
2. Four out of six quality control pools should be within the
ranges (±2σ) are presented in Table 1. of labeled analog.
established ranges.
TABLE I Substance Cross Reactivity
12.0 RISK ANALYSIS Expected Values for the cortisol EIA Test System (in µg/dl) Cortisol 1.0000
Population Morning Afternoon Androstenedione 0.0004
The MSDS and Risk Analysis Form for this product are available Adult 5 - 23 µg/dl 3 -13 µg/dl Cortisone 0.2300
on request from Monobind Inc. Child 3 - 21 µg/dl 3 -10 µg/dl Corticosterone 0.1800
Newborn 1 - 24 µg/dl 11-Deoxycortisol 0.0550
12.1 Assay Performance Please note: Normal results may vary from lab to lab Dexamethasone 0.0001
1. It is important that the time of reaction in each well is held
Progesterone 0.0002
constant to achieve reproducible results. It is important to keep in mind that establishment of a range of 17α-OH Progesterone ND
2. Pipetting of samples should not extend beyond ten (10) values which can be expected to be found by a given method for a DHEA ND
minutes to avoid assay drift. population of "normal"-persons is dependent upon a multiplicity of Estradiol ND
3. Highly lipemic, hemolyzed or grossly contaminated factors: the specificity of the method, the population tested and Estrone ND
specimen(s) should not be used. the precision of the method in the hands of the analyst. For these Danazol ND
4. If more than one (1) plate is used, it is recommended to repeat reasons each laboratory should depend upon the range of Testosterone ND
the dose response curve. expected values established by the Manufacturer only until an
5. The addition of substrate solution initiates a kinetic reaction, in-house range can be determined by the analysts using the
which is terminated by the addition of the stop solution. method with a population indigenous to the area in which the 15.0 REFERENCES
Therefore, the substrate and stop solution should be added in laboratory is located.
the same sequence to eliminate any time-deviation during 1. Burtis CA, Ashweed ER: Tietz ‘Textbook of Clinical
reaction. Chemistry’ 2nd Ed. W.B. Saunders Company. Philadelphia,
14.0 PERFORMANCE CHARACTERISTICS
6. Plate readers measure vertically. Do not touch the bottom of 1994. pp 1825-27.
the wells. 2. Foster L, Dunn R, ‘Single antibody technique for
14.1 Precision
7. Failure to remove adhering solution adequately in the radioimmunoassay of cortisol in unextracted serum or
The within and between assay precision of the Cortisol
aspiration or decantation wash step(s) may result in poor plasma’, Clin Chem, 20, 365 (1974).
AccuBind® ELISA Test System were determined by analyses on
replication and spurious results. 3. Wilson JD, Foster DW, (Editors) Williams Textbook of
three different levels of pool control sera. The number, mean th
8. Use components from the same lot. No intermixing of reagents Endocrinology, 7 Ed WB Saunders, Philadelphia (1985).
values, standard deviation and coefficient of variation for each of
from different batches. 4. Ruder H, et al, “Radioimmunoassay for cortisol in plasma
these control sera are presented in Table 2 and Table 3.
9. Accurate and precise pipetting, as well as following the exact and urine”, J Endo and Metab, 35, 219 (1972).
time and temperature requirements prescribed are essential. 5. Crapo L, “Cushing’s syndrome: A review of diagnostic tests”,
Metabolism, 28, 955-977 (1979).