IGFBP-1 PROTOCOL



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[pic] CORTISOL EIA

DSL-10-2000

Revision date: December 5, 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@

Website:

I. INTENDED USE

The DSL-10-2000 ACTIVE® Cortisol Enzyme Immunoassay (EIA) Kit provides materials for the quantitative measurement of cortisol in serum or plasma. 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 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 have been performed [8-13]. Cortisol response characteristics for each of these procedures have been reported.

The DSL-10-2000 ACTIVE® Cortisol EIA Kit uses a specific rabbit anti-cortisol antibody, and does not require prior sample extraction of serum or plasma. Cross-reactivity to other naturally-occurring steroids is low.

III. PRINCIPLE OF THE TEST

The procedure follows the basic principle of enzyme immunoassay where there is competition between an unlabeled antigen and an enzyme-labeled antigen for a fixed number of antibody binding sites. The amount of enzyme-labeled antigen bound to the antibody is inversely proportional to the concentration of the unlabeled analyte present. Unbound materials are removed by decanting and washing the wells.

IV. REAGENTS

The DSL-10-2000 ACTIVE® Cortisol EIA Kit contains sufficient reagents for 96 wells. Each kit contains the following reagents:

A. GARG-Coated Microtitration Strips:

One stripholder, containing 96 polystyrene microtiter wells with goat anti-rabbit globulin serum. Store at 2-8°C until expiration date in the resealable pouch with a desiccant to protect from moisture.

B. Cortisol Antiserum: (BLUE)

One vial, 11 mL, containing rabbit anti-cortisol serum in a protein-based (BSA) buffer with a non-mercury preservative. Store at 2-8 °C until expiration date.

C. Cortisol Standards:

One vial, 0.5 mL, labeled A, containing 0 μg/dL cortisol and 7 vials, 0.5 mL each, labeled B-H, containing concentrations of approximately 0.5, 1.5, 4.0, 10.0, 20.0, 40.0, and 60.0 μg/dL cortisol in protein-based (BSA) buffer with a non-mercury preservative. Refer to vial labels for exact concentrations. Store unopened at 2-8°C until kit expiration date. Store opened vials at 2-8°C for up to 3 weeks. For longer periods, store at -20°C or lower until expiration date.

D. Cortisol Controls:

Two vials, 0.5 mL each, Levels I and II, containing low and high concentrations of cortisol in protein-based (BSA) buffer with a non-mercury preservative. Store unopened at 2-8°C until kit expiration date. Store opened vials at 2-8°C for up to 3 weeks. For longer periods, store at -20°C or lower until expiration date.

E. Cortisol Enzyme Conjugate Concentrate:

One vial, containing 0.3 mL of a solution of cortisol conjugated to horseradish peroxidase in a protein-based buffer (BSA) with a non-mercury preservative. Dilute prior to use in Conjugate Diluent. Store at 2-8°C until expiration date.

F. Conjugate Diluent:

One bottle, 11 mL, containing a protein-based buffer (BSA) with a non-mercury preservative. Store at 2-8°C until expiration date.

G. TMB Chromogen Solution:

One bottle, 11 mL, containing a solution of tetramethylbenzidine (TMB) in citrate buffer with hydrogen peroxide. Store at 2-8°C until expiration date.

H. Wash Concentrate:

One bottle, 100 mL, containing buffered saline with a nonionic detergent. Dilute 10-fold with deionized water prior to use. Store at room temperature until expiration date.

I. Stopping Solution:

One vial, 11mL, containing 0.2M sulfuric acid. Store at 2-8°C until expiration date.

NOTE: All reagents and samples must be allowed to reach room temperature (~25( C) and mixed thoroughly by gentle inversion before use.

V. PRECAUTIONS

For in vitro diagnostic use.

The following precautions should be observed in the laboratory:

1. Do not eat, drink, smoke or apply cosmetics where immunodiagnostic materials are being handled.

2. Do not pipet by mouth.

3. Wear gloves when handling immunodiagnostic materials and wash hands thoroughly afterwards.

4. Cover working area with disposable absorbent paper.

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 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

Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB is dissolved in a solution which contains dimethylformamide, an irritant to skin and mucous membranes. In case of contact with any of these reagents, wash thoroughly with water.

TMB is a suspected carcinogen.

VI. SPECIMEN COLLECTION AND PREPARATION

Serum or plasma should 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. For longer periods, store at -20°C or lower [14]. Avoid repeated freezing and thawing of samples.

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 samples.

VII. PROCEDURAL NOTES

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.

Bring all kit reagents and specimens to room temperature (~25(C) before use. Thoroughly mix the reagents and samples before use by gentle inversion.

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.

Incomplete washing will adversely affect the outcome and assay precision.

To minimize potential assay drift due to variation in the substrate incubation time, care should be taken to add the stopping solution into the wells in the same order and speed used to add the TMB Chromogen Solution.

Avoid microbial contamination of reagents, especially of the conjugate concentrate and the conjugate diluent. Avoid contamination of the TMB Chromogen Solution with the Enzyme Conjugate. Use a clean disposable pipette tip for each reagent, Standard, Control or specimen. For dispensing sulfuric acid and TMB Chromogen Solution, avoid pipettes with metal parts.

Containers and semi-automatic pipette tips used for the Enzyme Conjugate Solution and the TMB Chromogen Solution can be reused provided they are thoroughly rinsed with distilled water and dried prior to and after each usage.

The enzyme used as the label is inactivated by oxygen, and is highly sensitive to microbial contamination, sodium azide, hypochlorous acid and aromatic chlorohydrocarbons often found in laboratory water supplies. Use high quality water.

Avoid exposure of the reagents to excessive heat or direct sunlight during storage and incubation.

VIII. TEST PROCEDURE

A. Materials Supplied:

Materials supplied in the DSL ACTIVE® Cortisol EIA Kit, Catalog No. DSL-10-2000:

|MATERIAL |QUANTITY |CATALOG NO. |

|Cortisol Standard A |One Vial |10-2001 |

|Cortisol Standard B |One Vial |10-2002 |

|Cortisol Standard C |One Vial |10-2003 |

|Cortisol Standard D |One Vial |10-2004 |

|Cortisol Standard E |One Vial |10-2005 |

|Cortisol Standard F |One Vial |10-2006 |

|Cortisol Standard G |One Vial |10-2007 |

|Cortisol Standard H |One Vial |10-2008 |

|GARG-Coated Microtitration |One Plate |10-3731 |

|Strips |One Vial |10-2051 |

|Cortisol Control Level I |One Vial |10-2052 |

|Cortisol Control Level II |One Vial |10-2010 |

|Cortisol Antiserum |One Vial |10-2020 |

|Cortisol Enzyme Conjugate |One Bottle |10-2045 |

|Concentrate |One Bottle |10-9755 |

|Conjugate Diluent D |One Bottle |10-4030 |

|TMB Chromogen Solution |One Vial |10-9780 |

|Wash Concentrate I | | |

|Stopping Solution A | | |

B. Materials Required But Not Supplied:

5. Microtitration plate reader capable of absorbance measurement at 450 nm and preferentially capable of dual wavelength correction at 600 or 620 nm

6. Deionized water

7. Precision pipette to deliver 25 μL

8. Semi-automatic pipette to deliver 100 μL and 200 μL

9. Microtitration plate shaker capable of 500-700 orbital revolutions per minute (rpm)

10. Automatic microtitration plate washer

11. Vortex mixer

12. Absorbent materials for blotting the strips

13. Linear-log graph paper for manual data reduction

C. Preparation of Reagents:

1. Wash Solution

Prepare Wash Solution by diluting Wash Concentrate 10-fold with deionized water.

2. Enzyme Conjugate Solution

The Enzyme Conjugate Concentrate should be diluted at a ratio of 1 part Enzyme Conjugate Concentrate into 50 parts Conjugate Diluent, according to the number of wells used. If an entire plate is to be used, pipet exactly 200 μL of the Enzyme Conjugate Concentrate into 10 mL of the Conjugate Diluent. The Enzyme Conjugate Solution should be prepared just prior to use.

3. Microtitration Wells

Select the number of coated wells required for the assay. The remaining unused wells should be placed in the resealable pouch with a desiccant. The pouch must be resealed to protect from moisture.

D. Assay Procedure:

Allow all specimens and reagents to reach room temperature (~25(C) and mix liquid reagents thoroughly by gentle inversion before use. Standards, Controls and unknowns should be assayed in duplicate.

1. Mark the microtitration strips to be used.

2. Prepare the Enzyme Conjugate Solution by diluting the Conjugate in the Conjugate Diluent as described under the Preparation of the Reagents section of this package insert.

3. Pipet 25 μL of each Standard, Control and unknown into the appropriate wells.

4. Add 100 μL of the Enzyme Conjugate Solution to each well using a semi-automatic dispenser. Gently tap the well holder for 5-10 seconds.

5. Add 100 (L of the Cortisol Antiserum to each well using a semi-automatic dispenser.

6. Incubate the wells at room temperature (~25(C) on a shaker set at 500-700 rpm for 45 minutes.

7. Aspirate and wash each well 5 times with the Wash Solution using an automatic microplate washer. Blot dry by inverting plate on absorbent material.

NOTE: Use of an automatic microplate washer is strongly recommended. Incomplete washing will adversely affect assay precision. If a microplate washer is not available, (a) completely aspirate the liquid from each well, (b) dispense 0.35 mL of the Wash Solution into each well, and (c) repeat steps (a) and (b) five times.

8. Add 100 μL of the TMB Chromogen Solution to each well using a semi-automatic dispenser.

9. Incubate the wells at room temperature (~25°C) for 10-15 minutes on a shaker set at 500-700 rpm.

10. Add 100 μL of the Stopping Solution to each well using a semi-automatic dispenser.

11. Shake the plate by hand for 5-10 seconds.

12. Read the absorbance of the solution in the wells within 30 minutes, using a microplate reader set to 450 nm.

NOTE: If wavelength correction is available, set the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 600 or 620 nm.

IX. CALCULATION OF RESULTS

A. Calculate the mean absorbance for each Standard, Control or unknown.

B. Using a linear-log graph paper, plot the mean absorbance readings for each of the standards along the y-axis versus the cortisol concentrations in μg/dL along the x-axis. Alternatively, any data reduction software designed for immunoassays could be used. Four-parameter curve-fit is recommended.

C. Draw the best fitting curve through the mean of the duplicate points.

D. Determine the cortisol concentrations of the Controls and unknowns from the standard curve by matching their mean absorbance readings with the corresponding cortisol concentrations.

E. Any sample reading higher than the highest Standard should be appropriately diluted with the 0 μg/dL Standard and reassayed.

F. Multiply the value by the dilution factor if required.

If the microplate reader is not capable of reading absorbance greater than 2 or greater than the absorbance of the last Standard, a second reading at 490 or 492 nm is needed (reference filter 600 or 620 if available). In this case, proceed to construct a second standard curve as above with the absorbance readings of all standards at 490 nm. The concentration of the off-scale samples at 450 nm are then read from the new standard curve. The readings at 490 nm should not replace the on-scale readings at 450 nm.

X. LIMITATIONS

14. The reagents supplied in this kit are optimized to measure cortisol levels in serum or plasma.

15. Avoid repeated freezing and thawing of reagents and specimens.

16. Grossly hemolyzed, icteric or lipemic specimens should be avoided.

XI. QUALITY CONTROL

17. The DSL Controls or other commercial controls should fall within established confidence limits. The confidence limits for the DSL Controls are printed on the Control vial labels.

18. The TMB Chromogen Solution should be colorless. Development of a blue color may indicate reagent contamination or instability.

TYPICAL ACTIVE® CORTISOL EIA STANDARD CURVE DATA

|WELL |WELL |MEAN |CONC. |

|NO. |CONTENTS |ABSORBANCE |(μg/dL) |

| |STANDARDS | | |

|A1,B1 |A |2.346 |0 |

|C1,D1 |B |1.565 |0.5 |

|E1,F1 |C |1.170 |1.5 |

|G1,H1 |D |0.751 |4.0 |

|A2,B2 |E |0.465 |10.0 |

|C2,D2 |F |0.273 |20.0 |

|E2,F2 |G |0.154 |40.0 |

|G2,H2 |H |0.148 |60.0 |

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 expected ranges for cortisol. The following data were collected from 62 presumed normal adults using the DSL-10-2000 ACTIVE® Cortisol EIA Kit:

|SPECIMENS |N |MEAN ± 1 SD |ABSOLUTE RANGE |

| | |(μg/dL) |(μg/dL) |

|Normal Sera |62 |12.3 ± 5.4 |4.0 - 27.0 |

XIII. PERFORMANCE CHARACTERISTICS

All performance characteristics are stated in μg/dL. To convert to nmol/L:

μg/dL x 27.6 = nmol/L

A. Sensitivity

The theoretical sensitivity or minimum detection limit, calculated by the interpolation of the mean minus two standard deviations of ten replicates of the 0 μg/dL Cortisol Standard, is 0.1 μg/dL.

B. Precision

The intra-assay precision was determined from the mean of 12 replicates each.

|SAMPLE |MEAN |STANDARD |COEFFICIENT |

| | |DEVIATION |OF VARIATION |

| |(μg/dL) |(μg/dL) |(%) |

|I |8.4 |0.2 |2.4 |

|II |15.9 |0.8 |5.0 |

|III |29.2 |3.0 |10.3 |

The inter-assay precision was determined from the mean of average duplicates for 12 separate runs.

|SAMPLE |MEAN |STANDARD |COEFFICIENT |

| | |DEVIATION |OF VARIATION |

| |(μg/dL) |(μg/dL) |(%) |

|I |5.0 |0.6 |12.0 |

|II |11.4 |0.7 |6.1 |

|III |15.0 |1.2 |8.0 |

C. Recovery

Three serum samples containing different levels of endogenous cortisol were spiked with known amounts of cortisol and assayed.

|SAMPLE |ENDOGENOUS |ADDED |EXPECTED |OBSERVED |RECOVERY |

| |((g/dL) |((g/dL) |(μg/dL) |(μg/dL) |(%) |

|I |5.1 |5.5 |10.6 |9.8 |93 |

|II |13.3 |1.8 |15.1 |18.7 |124 |

|III |20.4 |3.6 |24.0 |24.9 |104 |

D. Linearity

Two serum samples were diluted with the 0 μg/dL Cortisol Standard and assayed.

|SAMPLE |DILUTION |EXPECTED |OBSERVED |RECOVERY |

| |FACTOR |(μg/dL) |(μg/dL) |(%) |

|I |--- |--- |13.0 |--- |

| |1:2 |6.5 |6.9 |106 |

| |1:4 |3.2 |3.8 |119 |

| |1:8 |1.6 |1.8 |113 |

|II |--- |--- |27.2 |--- |

| |1:2 |13.6 |12.4 |91 |

| |1:4 |6.8 |5.5 |81 |

| |1:8 |3.4 |3.4 |100 |

| |1:16 |1.7 |2.0 |118 |

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 μg/dL Cortisol Standard.

|COMPOUND |% CROSS-REACTIVITY |

|Cortisol |100 |

|Prednisolone |58.3 |

|Prednisone |10.9 |

|Cortisone |7.0 |

|11-Deoxycortisol |5.7 |

|21-Deoxycortisol |1.9 |

|17α-Hydroxyprogesterone |0.9 |

|Dexamethasone |0.9 |

|Triamcinolone |0.4 |

|Corticosterone |ND |

|Progesterone |ND |

|DHEA |ND |

ND = non-detectable ( ................
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