ENZYME IMMUNOASSAY FOR THE DETECTION OF SALIVARY CORTISOL

K051733 · Drg Intl., Inc. · NHG · Dec 7, 2005 · Clinical Chemistry

Device Facts

• Record ID: K051733
• Device Name: ENZYME IMMUNOASSAY FOR THE DETECTION OF SALIVARY CORTISOL
• Applicant: Drg Intl., Inc.
• Product Code: NHG · Clinical Chemistry
• Decision Date: Dec 7, 2005
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1205
• Device Class: Class 2

Indications for Use

The DRG Salivary Cortisol ELISA Test is 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.

Device Story

The DRG Salivary Cortisol ELISA is an enzyme immunoassay used in clinical laboratories to measure free cortisol in human saliva. The device utilizes a competitive binding principle: cortisol in the patient sample competes with horseradish peroxidase-conjugated cortisol for binding sites on mouse polyclonal cortisol-antiserum coated onto microplate wells. After incubation and washing, a TMB substrate is added; the resulting optical density is inversely proportional to the cortisol concentration in the sample. Healthcare providers use these quantitative results to assist in diagnosing and managing adrenal gland disorders. The test provides a diagnostic tool for clinicians to assess adrenal function via non-invasive salivary sampling.

Clinical Evidence

No clinical trials were performed. Evidence consists of analytical bench testing and method comparison studies. Method comparison against a commercial LIA method (n=41, range 3.10–13.01 ng/mL) showed correlation r=0.9876. Comparison against a commercial EIA method (n=30, range <0.5–11.86 ng/mL) showed correlation r=0.95742. Precision studies (intra-assay CV 5.55–6.96%; inter-assay CV 5.82–7.47%) and linearity studies (1.04–77 ng/mL) support analytical performance. Reference range established using 109 healthy adult samples (0.12–1.47 µg/dL).

Technological Characteristics

Enzyme-linked immunosorbent assay (ELISA) using competitive binding principle. Microplate wells coated with mouse polyclonal cortisol-antiserum. Detection via horseradish peroxidase conjugate and TMB substrate. Optical density measurement at 450 nm. Usable range: 0.537–77 ng/mL. Manual or automated plate reader required.

Indications for Use

Indicated for the quantitative measurement of active free cortisol in saliva for the diagnosis and treatment of adrenal gland disorders in adult patients.

Regulatory Classification

Identification

A cortisol (hydrocortisone and hydroxycorticosterone) test system is a device intended to measure the cortisol hormones secreted by the adrenal gland in plasma and urine. Measurements of cortisol are used in the diagnosis and treatment of disorders of the adrenal gland.

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY TEMPLATE

A. 510(k) Number:
k051733

B. Purpose for Submission:
New Device

C. Measurand:
Cortisol, Salivary

D. Type of Test:
Quantitative Enzyme Immunoassay

E. Applicant:
DRG International, Inc.

F. Proprietary and Established Names:
DRG Salivary Cortisol ELISA KIT

G. Regulatory Information:
1. Regulation section:
21 CFR §862.1205, Enzyme Immunoassay Cortisol, Salivary
2. Classification:
Class II
3. Product code:
NHG
4. Panel:
75 (Chemistry)

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H. Intended Use:

1. Intended use(s):

See Indications for Use below.

2. Indication(s) for use:

The DRG Salivary Cortisol ELISA Test is 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.

3. Special conditions for use statement(s):

For prescription use.

4. Special instrument requirements:

Calibrated EIA reader adjusted to read at 450 nm, Precision pipettes (100 and 200 µl), Distilled or Deionized water, Timer (60 min. range), Reservoirs (disposable), Test tube or micro-tube rack in a microplate configuration, Linear-linear graph paper or software for data reduction.

I. Device Description:

The DRG Salivary Cortisol ELISA Test consists of Microtiter plate, 8 well snap-off strips, 12 strips, coated with rabbit anti-Cortisol antiserum. Reference Standard Set, 1 ml each, 0.0; 2; 5; 10; 20; 40; 80 ng/ml. Enzyme-Conjugate, 26 ml, Cortisol conjugated to horseradish peroxidase. Substrate Solution –TMB, 25 ml. Stop Solution, and Wash Solution.

J. Substantial Equivalence Information:

1. Predicate device name(s):

Salimetrics High Sensitivity Salivary Cortisol EIA

KMI/ IBL Cortisol LIA

2. Predicate 510(k) number(s):

k031348 - Salimetrics

k010790 - KMI/ IBL

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3. Comparison with predicate:

The DRG Salivary Cortisol Test is substantially equivalent to the Salimetrics HS Salivary Cortisol EIA (k031348). An additional comparison study was performed versus the KMI Diagnostics, Inc. Cortisol LIA method (k010790).

Comparison table for new device compared to the predicate devices

| Item | Predicate Device | Predicate Device | New Device |
| --- | --- | --- | --- |
| Device Name | Salimetrics HS Salivary Cortisol EIA (k031348) | KMI/ IBL Cortisol LIA (k010790) | DRG Salivary Cortisol ELISA (k051733) |
| Analyte | Active Free Cortisol | Active Free Cortisol | Active Free Cortisol |
| Specimens | Saliva | Saliva and Serum | Saliva |
| Method | Enzyme immunoassay | Luminescent immunoassay | Enzyme immunoassay |
| Test Principle | Cortisol in the sample competes with Cortisol-enzyme conjugate for binding sites to antibody bound to a microwell. Unbound components are washed away and bound cortisol-enzyme is measured by a colored reaction with the TMB substrate. | Same except, cortisol-peroxidase is measured by a chemiluminescent reading. | Same as cortisol-peroxidase measured by a colored reaction with the TMB substrate. |
| Detection | Colorimetric microplate reader | Luminometer | Colorimetric reader |
| Calculation | Quantitative determination with standard curve | Quantitative determination with standard curve | Quantitative determination with standard curve |
| Quality Control | Recommended | Recommended | Recommended |
| Indications for use | Measurements of cortisol are used in the diagnosis and treatment of disorders of the adrenal gland. | Measurements of cortisol are used in the diagnosis and treatment of disorders of the adrenal gland. | Measurements of cortisol are used in the diagnosis and treatment of disorders of the adrenal gland. |
| Expected Values (Normal Range) | 0.094 – 1.551 μg/dL | 0.5 – 1.5 μg/dL | 0.12 – 1.47 μg/dL |
| Detection limit | 0.007 μg/dL | 0.015 μg/dL | 0.104 μg/dL |

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K. Standard/Guidance Document Referenced (if applicable):

None referenced

L. Test Principle:

The DRG Salivary Cortisol ELIA KIT is based on the competitive principle and the microplate separation. An unknown amount of Cortisol present in the sample and a fixed amount of Cortisol conjugated with horse-radish peroxidase compete for the binding sites of rabbit polyclonal Cortisol-antiserum coated onto the wells. After one hour incubation the microplate is washed to stop the competition reaction. After addition of the substrate solution the concentration of Cortisol is inversely proportional to the optical density measured.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

The Intra-Assay variation was determined by replicate measurements of 4 saliva samples using DRG ELISA kit. The within assay variation is shown below:

| | Sample 1 | Sample 2 | Sample 3 | Sample 4 |
| --- | --- | --- | --- | --- |
| Mean (ng/mL) | 3.21 ng/mL | 19.09 ng/mL | 32.51 ng/mL | 1.19 ng/mL |
| SD | 0.188 | 1.085 | 1.806 | 0.083 |
| CV (%) | 5.85 | 5.68 | 5.55 | 6.96 |
| n = | 20 | 20 | 20 | 20 |

The Inter-Assay (between-run) variation was determined by quadruplicate measurements of commercial control samples in three different day runs. The inter-assay variation is shown below:

| Mean | 24.29 ng/mL | 40.85 ng/mL |
| --- | --- | --- |
| SD | 1.81 ng/mL | 2.38 ng/mL |
| CV (%) | 7.47 | 5.82 |
| n = | 12 | 12 |

The Inter-Lot (between-lot) variation was determined by duplicate measurements of five saliva samples in three different kit lots. The between run variability is shown below:

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| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 |
| --- | --- | --- | --- | --- | --- |
| Mean | 2.17 ng/mL | 14.01 ng/mL | 22.85 ng/mL | 1.74 ng/mL | 2.03 ng/mL |
| SD | 0.12 ng/mL | 1.17 ng/mL | 1.44 ng/mL | 0.13 ng/mL | 0.15 ng/mL |
| CV (%) | 5.50 | 8.32 | 6.29 | 7.56 | 7.43 |
| n = | 6 | 6 | 6 | 6 | 6 |

b. Linearity/assay reportable range:

Three samples (saliva) containing different amounts of analyte were serially diluted 1:16 with zero standard and assayed with the DRG ELISA. The percentage recovery was calculated by comparing the expected and measured values for the SLV cortisol. An assay linearity of 1.04 – 77 ng/mL has been identified as the usable range. Samples above this range must be diluted and re-run.

| | | Sample 1 | Sample 2 | Sample 3 |
| --- | --- | --- | --- | --- |
| Concentration | ng/mL | 33.13 | 80.0 | 24.32 |
| Average Recovery % | | 107 | 99.1 | 97.5 |
| Range of | from | 101.1 | 97.8 | 90.8 |
| Recovery % | To | 114.1 | 99.6 | 104.4 |

The Linearity study has been expanded to include additional higher samples to yield an upper detection level. As can be seen in the Table below the test is linear through to the highest Calibrator. Previous data demonstrated the lowest level of detectability.

Upper Linearity Study

In order to identify the upper level of detectability, three (3) native saliva samples, containing different amounts of analyte, were spiked with purified cortisol to obtain a starting level (undiluted) around and above the highest Calibrator. The spiked saliva were serially diluted with zero standard and assayed to determine upper end of detectability with the DRG ELISA. The percentage recovery was calculated by comparing the expected and measured values for SLV cortisol.

Percentage recovery is calculated as follows:

Measured values x 100

Expected values

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| Spiked saliva Sample | Dilution | Measured OD mean of duplicate (450 nm) | Measured Conc. Cortisol Saliva ng/mL | Expected Conc Cortisol Saliva ng/mL | Recovery (%) |
| --- | --- | --- | --- | --- | --- |
| Sample 1 | undil | 0.197 | 33.13 | 33.13 | |
| | 1:2 | 0.320 | 17.04 | 16.57 | 102.9 |
| | 1:4 | 0.518 | 8.37 | 8.28 | 101.1 |
| | 1:8 | 0.744 | 4.43 | 4.14 | 107.0 |
| | 1:16 | 1.016 | 2.14 | 2.07 | 103.4 |
| | 1:32 | 1.227 | 1.12 | 1.04 | 108.2 |
| | 1:64 | 1.380 | 0.59 | 0.52 | 114.1 |
| | 1:128 | 1.486 | 0.29 | 0.26 | 112.3 |
| Sample 2 | undil | 0.066 | 80.00 | | |
| | 1:2 | 0.084 | 80.00 | | |
| | 1:4 | 0.111 | 73.36 | | |
| | 1:8 | 0.183 | 36.47 | 36.68 | 99.4 |
| | 1:16 | 0.305 | 18.22 | 18.34 | 99.3 |
| | 1:32 | 0.490 | 9.13 | 9.17 | 99.6 |
| | 1:64 | 0.740 | 4.48 | 4.58 | 97.8 |
| sample3 | undil | 0.247 | 24.32 | 24.32 | |
| | 1:2 | 0.394 | 12.68 | 12.16 | 104.3 |
| | 1:4 | 0.612 | 6.35 | 6.08 | 104.4 |
| | 1:8 | 0.917 | 2.81 | 3.04 | 92.4 |
| | 1:16 | 1.149 | 1.45 | 1.52 | 95.4 |
| | 1:32 | 1.348 | 0.69 | 0.76 | 90.8 |
| | Sample 1 | Sample 2 | Sample 3 |
| --- | --- | --- | --- |
| Concentration ng/mL | 33.13 | 80.00 | 23.23 |
| Average % Recovery | 107.0 | 99.1 | 97.5 |
| Range of from | 101.1 | 97.8 | 92.4 |
| % Recovery to | 114.0 | 99.6 | 104.4 |

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

The calibrators are buffer based (artificial saliva matrix). The calibrators were prepared by appropriate dilution from the maximum standard (Smax.: $80\mathrm{ng / mL}$ ). The Cortisol for the Calibrators was purchased from a commercially available source and is weighed in to make the $80\mathrm{ng / mL}$ .

The reference values (calibrators/controls) were established using (Gas chromatography-mass spectrophotometry) methods as per the guidelines

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for quality assurance in medical laboratories, Instand E.V. Germany ( L.D. Dikkesche. Et al. 1988: De toepassing van gaschromatografie-massaspectrometrie als referentiemethode in kwaliteitcontroleprogramma's voor progesterone-, cortisol-, testosterone en oestradiolbepalingen in serums. Tijdschr NVKC 13: 148-155).

WHO standard is not available.

The functional quality of the kit lots were tested using the Lyphochek controls from BioRad, but controls are not included in the kit. These controls are commercially available and can be purchased by the customers.

d. Detection limit:

The analytical sensitivity of the DRG ELISA was calculated by subtracting 2 standard deviations from the mean of 20 replicate analyses of the Zero Standard (S₀).

Standard curve:

| Standard | Conc. ng/mL | OD_{450} mean of duplicate |
| --- | --- | --- |
| S0 | 0 | 2.05 |
| S1 | 2,0 | 1.24 |
| S2 | 5,0 | 0.85 |
| S3 | 10,0 | 0.55 |
| S4 | 20,0 | 0.33 |
| S5 | 40,0 | 0.20 |
| S6 | 80 | 0.11 |

Controls:

| | Conc. ng/ml | Acc. Range. |
| --- | --- | --- |
| Lyphocheck 142/10 | 21.518 | 15.5 –36.3 |
| Lyphocheck 143/10 | 41.437 | 22.1 – 51.6 |

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| Replicate | OD450 of S0 |
| --- | --- |
| 1 | 1.940 |
| 2 | 1.984 |
| 3 | 1.945 |
| 4 | 1.901 |
| 5 | 1.866 |
| 6 | 1.816 |
| 7 | 1.850 |
| 8 | 1.795 |
| 9 | 1.963 |
| 10 | 1.989 |
| 11 | 1.999 |
| 12 | 1.950 |
| 13 | 1.975 |
| 14 | 1.991 |
| 15 | 1.927 |
| 16 | 1.927 |
| 17 | 2.066 |
| 18 | 2.078 |
| 19 | 1.818 |
| 20 | 1.600 |
| Mean = | 1.919 |
| --- | --- |
| SD = | 0.108 |
| 2xSD = | 0.216 |
| Mean - 2xSD | 1.703 = 0.537 ng/mL |
| N = | 20.00 |

# e. Analytical specificity:

Cross-reactivity was tested with the following compounds whose chemical structure could potentially cause interference with the SLV cortisol ELISA. The specificity of the antiserum used for the ELISA was evaluated by determination of the cross-reactivity at $50\%$ displacement of various compounds listed in the table.

The cross reactivity is defined as:

Concentration of cortisol at $50\%$ B/BO

Concentration of cross-reactant giving $50\%$ B/BO

| Steroid | % Cross Reaction |
| --- | --- |
| Cortisol | 100% |
| Corticosterone | 29% |
| Cortisone | 3.00% |
| 11-Deoxycortisol | < 1,00% |

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| 17-OH Progesterone | < 0,50% |
| --- | --- |
| Prednisone | < 0, 10% |
| Progesterone | < 0, 10% |
| Dexamethasone | < 0, 10% |
| Desoxycorticosterone | < 0, 10% |
| Dehydroepiandrosterone sulfate | < 0, 10% |
| Estradiol | < 0, 10% |
| Estriol | < 0, 10% |
| Estrone | < 0, 10% |
| Testosterone | < 0, 10% |

f. Assay cut-off: Not Applicable for this type of device.

2. Comparison studies:

a. Method comparison with predicate device:

Two studies were performed to evaluate the performance of the SLV Cortisol Saliva ELISA versus two commercially available saliva Cortisol kits.

One study evaluated saliva samples from 41 male and female subjects between ages 40 – 70 years. The samples were run in duplicate on the DRG test and another commercially available LIA method to determine the concentration of Cortisol in the samples. An overall correlation of 0.9876 and a regression formula of $y = 0.9726x + 0.095$ was obtained versus this method. The samples ranged in concentration from 3.10 to $13.01 \, \mathrm{ng/mL}$.

A second study was performed using 30 saliva samples collected from men and women ages 40 – 65 years and run in duplicate on DRG and another commercially available EIA test. A correlation of 0.95742 with a regression formula of $y = 0.9812x + 0.1515$ was observed. The samples ranged in concentration from $&lt; 0.5$ to $11.86 \, \mathrm{ng/mL}$.

b. Matrix comparison:

Not applicable since this assay is for use with saliva samples only.

3. Clinical studies:

a. Clinical Sensitivity:

Not applicable

b. Clinical specificity:

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Not applicable

c. Other clinical supportive data (when a. and b. are not applicable):

4. Clinical cut-off:

Not applicable for this type of device

5. Expected values/Reference range:

109 saliva samples from apparently health adult male and female subjects, ranging in age from 20 to 80 years were collected in the morning and analyzed using the DRG SLV Cortisol ELISA kit. The salivary cortisol concentration did not show any significant differences based on age. Hence the normal range was calculated for the entire group. The normal range of salivary cortisol analyzed using DRG SLV Cortisol ELISA Test.

Adults: 0.12 – 1.47 µg/dL or 1.2 – 14.7 ng/mL (AM collection)

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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