DRG SLV-2930 Salivary Cortisol ELISA Instruction Manual

DRG SLV-2930 Salivary Cortisol ELISA Instruction Manual

Please use only the valid version of the Instructions for Use provided with the kit.

1 INTRODUCTION

1.1 Intended Use
An enzyme immunoassay for the quantitative in vitro diagnostic measurement of active free cortisol (hydrocortisone and hydroxycorticosterone) in saliva. Measurements of cortisol are used in the diagnosis and treatment of disorders of the adrenal gland.

1.2 Summary and Explanation
Cortisol, the most potent glucocorticoid, is produced by the zona fasciculate of the human adrenal cortex (1-3). It is synthesized from cholesterol and its production is stimulated by pituitary adrenocorticotropic hormone (ACTH) in response to corticotropin-releasing hormone (CRH). ACTH and CRH secretions are inhibited by high cortisol levels in a negative feedback loop. Cortisol acts through specific intracellular receptors and affects numerous physiologic systems including immune function, glucose counter regulation, vascular tone, and bone metabolism.

Cortisol release follows a diurnal rhythm with highest concentrations in the morning (about 1 hour after wakening). Thereafter, Cortisol concentration steadily decreases to a very low level 12 hours later (4-7). Cortisol secretion increases in response to any stress in the body, whether physical (such as illness, trauma, surgery, or temperature extremes) or psychological (8-14). After secretion, cortisol causes a breakdown of muscle protein, leading to release of amino acids into the bloodstream. These amino acids are then used by the liver to synthesize glucose for the brain, a process called gluconeogenesis. Cortisol also leads to the release of fatty acids from fat cells and its utilization in muscle cells. Taken together, these energy- directing processes prepare the individual to deal with stressors and ensure that the brain receives adequate energy sources.

Elevated cortisol levels and lack of diurnal variation have been identified with Cushing’s disease (ACTH hypersecretion) (7). CRH is released in a cyclic fashion by the hypothalamus, resulting in diurnal peaks (elevated in the morning) and nadirs (low in the evening) for plasma ACTH and cortisol levels. The diurnal variation is lost in patients with Cushing and these patients have elevated levels of evening plasma cortisol. The measurement of late-night salivary cortisol is an effective and convenient screening test for Cushing syndrome (15). Elevated circulating cortisol levels have also been identified in patients with adrenal tumors (16). Low cortisol levels are found in primary adrenal insufficiency (e.g. adrenal hypoplasia, Addison’s disease) and in ACTH deficiency (17). Due to the normal circadian variation in cortisol levels, distinguishing normal from abnormally low cortisol levels can be difficult, therefore several daily collections are recommended.

Saliva is an excellent medium to measure steroids because it is a natural ultra-filtrate of blood. 90-99% of steroid hormones in the blood are bound to carrier proteins (corticoid-binding globulin, sex-hormone binding globulin and albumin) and are unavailable to target tissues. Only about 1-10% of the steroids in blood are in the unbound or free fraction and can diffuse into target tissues of the body and into saliva. The process of passive diffusion of non-bound steroid hormones is supported by their low molecular weight (less than 400 dalton) and relative lipophilicity, thus enabling them to freely diffuse from blood to saliva (18-21).

2 PRINCIPLE

The DRG Salivary Cortisol ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle of competitive binding.
The microtiter wells are coated with a monoclonal (mouse) antibody directed towards an antigenic site on the cortisol molecule.

Endogenous cortisol of a patient sample competes with a cortisol-horseradish peroxidase conjugate for binding to the coated antibody. After incubation the unbound conjugate is washed off.
The amount of bound peroxidase conjugate is inversely proportional to the concentration of cortisol in the sample. After addition of the substrate solution, the intensity of colour developed is inversely proportional to the concentration of cortisol in the patient sample.

3 WARNINGS AND PRECAUTIONS

1. This kit is for in vitro diagnostic use only. For professional use only.
2. All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood.
4. The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (21 °C to 26 °C) before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiter plate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclin, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Safety Data Sheets. Safety Data Sheets for this product are available upon request directly from DRG Instruments GmbH.

4 REAGENTS

4.1 Reagents provided

1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells; Wells coated with a anti-cortisol antibody (monoclonal).
2. Standard (Standard 0-6), 7 vials, 1 mL each, ready to use; Concentrations: 0 – 0.1 – 0.5 – 1.5 – 4 – 10 – 30 ng/mL, Conversion factor: 1 ng/mL = 2.76 nmol/L; Standards are calibrated against mass spectrometry. Contain non-mercury preservative.
3. Control Low & High, 2 vials, 1 mL each, ready to use; For control values and ranges please refer to vial label or QC-Datasheet. Contain non-mercury preservative.
4. Enzyme Conjugate, 1 vial, 26 mL, ready to use; Cortisol conjugated to horseradish peroxidase; Contain non-mercury preservative.
5. Substrate Solution, 1 vial, 25 mL, ready to use; Tetramethylbenzidine (TMB).
6. Stop Solution, 1 vial, 14 mL, ready to use; contains 0.5M H2SO4. Avoid contact with the stop solution. It may cause skin irritations and burns.
7. Wash Solution, 1 vial, 30 mL (40X concentrated); see „Preparation of Reagents“.

Note: Additional Standard 0 for sample dilution is available upon request.

4.2 Materials required but not provided
− A microtiter plate calibrated reader (450 ± 10 nm), (e.g. the DRG Instruments Microtiter Plate Reader).
− Calibrated variable precision micropipettes (100 μL, 200 μL).
− Absorbent paper.
− Distilled or deionized water
− Timer.
− Semilogarithmic graph paper or software for data reduction

4.3 Storage Conditions
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again.

4.4 Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.

Wash Solution
Add deionized water to the 40X concentrated Wash Solution.
Dilute 30 mL of concentrated Wash Solution with 1170 mL deionized water to a final volume of 1200 mL.
The diluted Wash Solution is stable for 1 week at room temperature.

4.5 Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Safety Data Sheet.

4.6 Damaged Test Kits
In case of any severe damage to the test kit or components, DRG has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed according to the official regulations.

5 SPECIMEN COLLECTION AND PREPARATION

Eating, drinking, chewing gums or brushing teeth should be avoided for 30 minutes before sampling. Otherwise, it is recommended to rinse mouth thoroughly with cold water 5 minutes prior to sampling.
Do not collect samples when oral diseases, inflammation or lesions exist (blood contamination).
If there is visible blood contamination the patient specimen, it should be discarded, rinse the sampling device with water, wait for 10 minutes and take a new sample.

Note: Samples containing sodium azide should not be used in the assay.

5.1 Specimen Collection
Saliva samples should be collected only using special saliva sampling devices (vial and straw), e.g. SALI-TUBES 100 (SLV-4158).
Due to the cyclic secretion pattern of steroid hormones it is important to care for a proper timing of the sampling.
In order to avoid arbitrary results we recommend that 5 samples always be taken within a period of 2 – 3 hours (multiple sampling) preferably before a meal.
As food might contain significant amounts of steroid hormones samples preferably should be taken while fasting. If fasting should be a problem the collection period should be timed just before lunch or before dinner.

5.2 Specimen Storage and Preparation
The saliva samples may be stored for 7 days at -20 °C or 7 days at 2 °C – 8 °C and should be frozen at -20 °C for longer periods (12 months); repeated thawing and freezing is no problem.
Each sample has to be frozen, thawed, and centrifuged at least once in order to separate the mucins by centrifugation.
Upon arrival of the samples in the lab the samples have to stay in the deep freeze at least overnight. Next morning the frozen samples are warmed up to room temperature and mixed carefully.
Then the samples have to be centrifuged for 5 to 10 minutes (at 2000 – 3000 × g).
Now the clear colorless supernatant is easy to pipette.
If a set of multiple samples is to be tested, the lab (after at least one freezing, thawing, and centrifugation cycle) has to mix the 5 single samples in a separate sampling device and perform the testing from this mixture.

5.3 Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be diluted with Standard 0 and re-assayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.
Example:
a) Dilution 1:10: 10 μl saliva + 90 μl Standard 0 (mix thoroughly)
b) Dilution 1:100: 10 μl of dilution a) + 90 μl Standard 0 (mix thoroughly).

6 ASSAY PROCEDURE

6.1 General Remarks
− All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.
− Once the test has been started, all steps should be completed without interruption.
− Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination.
− Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
− As a general rule the enzymatic reaction is linearly proportional to time and temperature.
− Respect the incubation times as stated in this instructions for use.

6.2 Assay Procedure
Each run must include a standard curve.

1. Secure the desired number of Microtiter wells in the frame holder.
2. Dispense 100 μL of each Standard, Control and samples with new disposable tips into appropriate wells.
3. Dispense 200 μL Enzyme Conjugate into each well. Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.
4. Incubate for 60 minutes at room temperature Note: Incubation on a shaker at 300 rpm is recommended.
5. Briskly shake out the contents of the wells. Rinse the wells 5 times with diluted Wash Solution (400 μL per well). Strike the wells sharply on absorbent paper to remove residual droplets. Important note: The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!
6. Add 200 μL of Substrate Solution to each well.
7. Incubate for 30 minutes at room temperature.
8. Stop the enzymatic reaction by adding 100 μL of Stop Solution to each well.
9. Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter plate reader. It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

6.3 Calculation of Results

1. Calculate the average absorbance values for each set of standards, controls and patient samples.
2. Construct a standard curve by plotting the mean absorbance obtained from each standard against its concentration with absorbance value on the vertical(Y) axis and concentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from the standard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a 4-Parameter curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods.) Other data reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations higher than that of the highest standard have to be further diluted. For the calculation of the concentrations this dilution factor has to be taken into account.

6.3.1 Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time of assay.

7. EXPECTED NORMAL VALUES

In order to determine the normal range of Salivary Cortisol ELISA, samples from adult male and female apparently healthy subjects, were collected in the morning, at noon, and in the evening and analyzed using the DRG ELISA kit.

The following range was calculated from this study.

The results alone should not be the only reason for any therapeutic consequences. The results should be correlated to other clinical observations and diagnostic tests.
Since cortisol levels show diurnal cycles, we recommend that the samples be obtained the same hour each day.

Furthermore, we recommend that each laboratory determine its own range for the population tested.

8 QUALITY CONTROL

Good laboratory practice requires that controls be run with each calibration curve. A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance.

It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels.

The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results.

It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid.

In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or DRG directly.

9 PERFORMANCE CHARACTERISTICS

9.1 Detection Capability
The Limit of Blank (LoB) is 0.046 ng/mL.
The Limit of Detection (LoD) is 0.082 ng/mL.
The Limit of Quantification (LoQ) is 0.114 ng/mL.
The range of the assay is between 0.09 – 30 ng/mL.

9.2 Specificity (Cross Reactivity)
The following substances have been evaluated for cross reactivity.

Substantial cross-reactivity (> +10%) was detected for 11-Desoxycortisone, 17 α-Hydroxyprogesterone, Corticosterone, Hydrocortisone, Prednisolone, and Progesterone.
9.3 Repeatability and Reproducibility
9.3.1 Repeatability (Within-run Precision)
The within-run precision was determined with 6 samples covering the complete measuring range within 20 days in 2 independent runs per day. CV was calculated as mean CV of 40 runs. The within-run variability is shown below:

9.3.2 Reproducibility (Between-run Precision)
The between-run precision was determined with 4 samples covering the complete measuring range within 20 days in 2 independent runs per day and with 2 replicates per run (20 x 2 x 2). CV was calculated from 80 determinations. The Between-run variability is shown below:

9.3.3 Reproducibility (Between-lot Precision)
The between-lot variation of the DRG ELISA was determined by 6 measurements of 3 samples with 3 different kit lots. The between-lot CV % should be found below 15 %.

9.4 Recovery

Recovery of the DRG ELISA was determined by adding increasing amounts of the analyte to 4 different saliva samples containing different amounts of endogenous analyte. Each sample (non-spiked and spiked) was assayed and analyte concentrations of the samples were calculated from the standard curve. The percentage recoveries were determined by comparing expected and measured values of the samples.

9.5 Linearity
Four samples containing different amounts of analyte were serially diluted with standard matrix. The percentage recovery was calculated by comparing the expected and measured values for the analyte. All recovery data must be found within the acceptance range of 85 % – 115 %.

9.6 Method Comparison
A comparison of DRG Salivary Cortisol ELISA SLV-2930 (y) and reference method Salimetrics ELISA (x) using clinical samples gave the following correlation:
n = 156
r = 0.995
y = -0,04272 x – 0.055

10 LIMITATIONS OF PROCEDURE

Reliable and reproducible results will be obtained when the assay procedure is performed with a complete understanding of the package insert instruction and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.

10.1 High-Dose-Hook Effect
“High Dose Hook Effect” is not detected in the range between 0.114 – 600 ng/mL for this assay.

10.2 Drug Interferences
The use of topical creams or medication containing Prednisolone, Hydrocortisone and 11-Deoxycortisol should be avoided as they can cause preanalytical contamination of the saliva sample.

The use of topical creams or medication containing Prednisolone, Hydrocortisone and 11-Deoxycortisol may lead to false positive results of the Salivary Cortisol ELISA.

10.3 Matrix Interference
Increasing amounts of Hemoglobin, Ethanol, Food, Chewing gum, Nicotine and Caffeine were added to 3 saliva samples (low, medium and high Cortisol concentration). After determination of the analyte concentrations in the spiked samples, the results were compared with the acceptance ranges (within 10% recovery of the unspiked control sample).

Hemoglobin (up to 100 μg/mL), Ethanol (up to 10 000 μg/mL), Food (up to 1000 μg/mL), Chewing gum (up to 1000 μg/mL), Nicotine (up to 100 μg/mL) and Caffeine (up to 10 μg/mL) have no influence on the assay results.

Eating, drinking, chewing gums or brushing teeth should be avoided for 30 minutes before sampling. Otherwise, it is recommended to rinse mouth thoroughly with cold water 5 minutes prior to sampling. Do not collect samples when oral diseases, inflammation or lesions exist (blood contamination).

11 LEGAL ASPECTS

11.1 Reliability of Results
The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test.

The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.

11.2 Therapeutic Consequences
Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient.
Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived.
The test result itself should never be the sole determinant for deriving any therapeutic consequences.

11.3 Liability
Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement.
Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2 are also invalid. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

12 REFERENCES

1. Drucker S (1987) New MI: Disorders of adrenal steroidogenesis. Pediatr. Clin. North Am.; 34, 1055-1066
2. Hellhammer DH et al. (1997) Social hierarchy and adrenocortical stress Reactivity in men. Psychoneuroendocrinology, 22; 643-650
3. Van Cauter E (1987) Pulsatile ACTH secretion. In: Wagner T., Filicori M. (eds): Episodic hormone secretion: From basic science to clinical application. Hameln, TM-Verlag, pp 65-75
4. Miller R et al (2016) The CIRCORT database: Reference ranges and seasonal changes in diurnal salivary cortisol derived from a meta-dataset comprised of 15 field studies. Psychoneuroendocrinology. 73; 16-23.
5. Lopes LM et al (2016) Determination of nighttime salivary cortisol during pregnancy: comparison with values in non-pregnancy and Cushing’s disease. Pituitary. 19; 30-8
6. Hucklebridge FH et al. (1999): The awakening of cortisol response and blood glucose levels. Life Sci., 64; 931-937
7. Antonelli G et al (2015) Salivary cortisol and cortisone by LC-MS/MS: validation, reference intervals and diagnostic accuracy in Cushing’s syndrome. Clin. Chim. Acta, 451; 247-51
8. Irwin M et al (1987) Life events, depressive symptoms and immune function. Am. J. Psychiat., 144; 437-441
9. Solomon GF, Moss RH (1964) Emotions, Immunity and disease. A speculative theoretical integration. Arch. Gen. Psychiatry, 11; 657-674
10. Mcgrady A et al (1987) Effect of biofeedback-assisted relaxation in blood pressure and cortisol levels in normotensives and hypertensives. J. Behav. Med., 10; 301-310
11. Chernow B., et al (1987) Hormonal responses to graded surgical stress. Arch. Intern. Med., 147; 1273- 1278
12. Hellhammer DH et al (1987) Measurement of salivary cortisol under psychological stimulation. In: Hingten JN, Hellhammer DH, Huppmann (eds.), Advanced methods in Psychology, Hogrefe, Toronto, pp 281-289
13. Kirchbaum C, Hellhammer DH. (1989) Salivary cortisol in psychobiological Research: An overview. Neuropsychobiology, 22; 150-169
14. Kirchbaum C, Hellhammer DH (1994): Salivary cortisol in psychoneuroendocrine Research: Recent developments and applications. Psychoneuroendocrinology, 19; 313-333
15. Ceccato F et al (2013) Performance of salivary cortisol in the diagnosis of Cushing’s syndrome, adrenal incidentaloma, and adrenal insufficiency. Eur. J. Endocrinol., 169; 31-6
16. Raff H, Raff JL and Findling JW (1998) Late-night salivary cortisol as a screening test for Cushing’s syndrome. J. Clin. Endocrinol. Metab.,83; 2681-2686
17. Restituto P (2008) Advantage of Salivary cortisol measurements in the diagnosis of glucocorticoid related disorders. Clin Biochem. 41; 688-92
18. Riad-Fahny et al (1982), Steroids in saliva for assessing endocrine function. Endocr. Rev., 3, 367-395
19. Robin P et al. (1977) Assay of unbound cortisol in plasma. J. Clin. Endocrinol. Metab., 46; 277-283
20. Vining RF et al (1983) Hormones in saliva: Mode of entry and consequent implications for clinical interpretation, Clin. Chem., 29; 1752-1756
21. Bosch JA (2014) The use of saliva markers in psychobiology: mechanisms and methods. Monogr. Oral Sci., 24; 99-108

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References

• Analyticon® Biotechnologies GmbH
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