CORTISOL ELISA

K062626 · Ibl-Hamburg GmbH · CGR · Dec 20, 2006 · Clinical Chemistry

Device Facts

• Record ID: K062626
• Device Name: CORTISOL ELISA
• Applicant: Ibl-Hamburg GmbH
• Product Code: CGR · Clinical Chemistry
• Decision Date: Dec 20, 2006
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1205
• Device Class: Class 2

Indications for Use

The IBL Cortisol enzyme linked immunosorbent assay is for the in-vitro-diagnostic quantitative determination of cortisol in human serum and saliva. The Cortisol ELISA kit is useful as an aid in the differential diagnosis of Cushing syndrome and Addison’s disease.

Device Story

The IBL Cortisol ELISA is an in-vitro diagnostic test for quantitative determination of cortisol in human saliva and diluted serum. It utilizes a solid-phase enzyme-linked immunosorbent assay (ELISA) based on a competitive binding principle. Patient samples and enzyme-labeled cortisol compete for binding sites on antibody-coated microtiter wells. After incubation and washing, a substrate reaction occurs; the resulting color intensity is inversely proportional to the cortisol concentration in the sample. Results are determined via a standard curve. The device is intended for use by clinical laboratory professionals to aid in the assessment of Cushing Syndrome and Addison's Disease. Healthcare providers use the quantitative results to support clinical diagnosis. The device provides a standardized method for measuring cortisol levels, facilitating clinical decision-making regarding adrenal function.

Clinical Evidence

No clinical studies performed. Evidence consists of analytical performance data: intra-assay precision (mean CV 5.7% saliva, 8.8% serum), inter-assay precision (mean CV 7.4% saliva, 11.2% serum), and linearity (mean recovery 97% saliva, 105% serum). Method comparison against predicate using 129 serum and 130 saliva samples showed high correlation (R² ≥ 0.990).

Technological Characteristics

Solid-phase competitive ELISA. Components: antibody-coated microtiter strips, enzyme conjugate, standards, controls, wash buffer, TMB substrate, and stop solution. Connectivity: standalone. Energy source: none (manual/automated plate reader). Sterilization: N/A. Software: N/A (manual calculation/plate reader processing).

Indications for Use

Indicated for the in-vitro diagnostic quantitative determination of cortisol in human serum and saliva as an aid in the differential diagnosis of Cushing syndrome and Addison's disease.

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.

Predicate Devices

• IBL Cortisol LIA TEST KIT (k052359)

Submission Summary (Full Text)

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

A. 510(k) Number:
k062626

B. Purpose for Submission:
New device

C. Measurand:
Cortisol

D. Type of Test:
Quantitative ELISA

E. Applicant:
IBL-Hamburg

F. Proprietary and Established Names:
Cortisol ELISA

G. Regulatory Information:

1. Regulation section:
21 CFR § 862.1205 Cortisol (hydrocortisone and hydroxycorticosterone) test system

2. Classification:
Class II

3. Product code:
CGR – Radioimmunoassay, cortisol

4. Panel:

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75 – Clinical chemistry

## H. Intended Use:

1. Intended use(s):
See indications for use below.

2. Indication(s) for use:
The IBL Cortisol enzyme linked immunosorbent assay is for the in-vitro-diagnostic quantitative determination of cortisol in human serum and saliva. The Cortisol ELISA kit is useful as an aid in the differential diagnosis of Cushing syndrome and Addison’s disease.

3. Special conditions for use statement(s):
For prescription use only.

4. Special instrument requirements:
Calibrated EIA reader adjusted to read at 450nm.

## I. Device Description:

The kit contains the components below.

Microtiter Plate – Break-apart strips coated with anti-cortisol antibodies.

Enzyme Conjugate – Ready to use. Contains: Cortisol (chromatographically purified) conjugated to HRP and stabilizers.

Standard A-G – previously cleared (k052359)

Controls – previously cleared (k052359)

TMB Substrate Solution – Ready to use, TMB, Buffer, stabilizers.

TMB Stop Solution – Ready to use, 1 M H₂SO₄.

Wash Buffer Concentrate (10x) – phosphate buffer, Tween, stabilizers.

Adhesive Foil

## J. Substantial Equivalence Information:

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1. Predicate device name(s):

IBL Cortisol LIA TEST KIT

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

k052359

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Cortisol ELISA (IBL) (device) | Cortisol LIA (IBL) Predicate |
| Sample | Serum and saliva | Serum and saliva |
| Test principle | Competitive immunoassay. Competition is between a labeled and non-labeled antigen for a fixed number of antibody binding sites. The amount of labeled analyte bound to the antibody is inversely proportional to the concentration of the analyte present in the sample. | Competitive immunoassay. Competition is between a labeled and non-labeled antigen for a fixed number of antibody binding sites. The amount of labeled analyte bound to the antibody is inversely proportional to the concentration of the analyte present in the sample |
| Calibration | Quantitative from standard curve with 7 calibrators | Quantitative from standard curve with 7 calibrators |
| Quality control | 2 Controls at different levels | 2 Controls at different levels |
| Assay range | Saliva: 0.015 – 4.0 μg/dL Serum: 0.75 – 200 μg/dL | Saliva: 0.015 – 4.0 μg/dL Serum: 0.75 – 200 μg/dL |
| Microtiter plate | Break apart strips coated with anti-cortisol antibodies | Break apart strips coated with anti-cortisol antibodies |
| Differences | | |
| --- | --- | --- |
| Item | Cortisol ELISA (IBL) (device) | Cortisol LIA (IBL) Predicate |
| Method | Enzyme linked immunosorbent assay | Luminescence immunoassay |
| Detection | Colorimetric detection | Luminescence detection |

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

Not applicable

L. Test Principle:

Solid phase enzyme-linked immunosorbent assay (ELISA) based on the competition principle. An unknown amount of antigen present in the sample and a fixed amount of enzyme labeled antigen compete for the binding sites of the antibodies coated onto the wells. After incubation the wells are washed to stop the competition reaction. After the substrate reaction the intensity of the developed color is inversely proportional to the amount of the antigen in the sample. Results of samples can be determined directly using the standard curve.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

Intra assay precision for saliva was determined by repeated measurements of control saliva samples within the same run. The mean CV was 5.7% (range 3.2 – 7.6%). Inter assay precision for saliva was determined by repeated double measurements of control saliva samples in 10 different runs on consecutive days. The mean CV was 7.4% (range 6.2 – 9.1%).

Intra assay variation for serum was determined by repeated measurements of samples after 1:51 dilution with standard A. The mean CV was 8.8% (range 3.8 – 11.8%). Inter assay variation for serum was determined by repeated double measurements of control sera samples after 1:51 dilution in 10 different runs. The mean CV was 11.2% (range 10.8 – 12.0%).

b. Linearity/assay reportable range:

Reportable range:

Saliva: 0.015 – 4 µg/dL Cortisol (anal.Sens. to highest standard- higher samples must be diluted to give results within the standard curve)

Serum: 0.75 – 200 µg/dL Cortisol (anal.Sens. to highest standard multiplied with dilution factor of 50)

Linearity of saliva samples

Saliva samples having different cortisol levels were serially diluted with zero standard. Dilutions covering the full measuring range of the assay were performed for each sample. Each dilution was measured in duplicate in one assay run. No relevant deviation of the expected linearity was observed. The

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mean linearity recovery for saliva was 97% (range was 83 to 114% recovery).

## Linearity of serum samples

Serum samples (already diluted 1:50) having different cortisol levels were serially diluted with zero standard. The measured concentrations were multiplied with 50 to get the serum concentration. Dilutions covering the full measuring range of the assay were performed for each sample. Each dilution was measured in duplicate in one assay run. No relevant deviation of the expected linearity was observed. The mean linearity recovery for serum was 105% (range was 90 to 120% recovery).

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

The complete Kit will have a shelf life of 9 months at 2 – 8°C. Traceability, stability, and expected values of calibrators and controls were previously established (k052359).

d. Detection limit:

The analytical sensitivity was calculated from the mean of the OD of the zero calibrator minus 2 SD of 26 replicate analyses. The lowest detectable level that can be distinguished from the zero standard is 0.015 µg/mL using 4 parameter logistics for curve fit.

e. Analytical specificity:

The cross-reactivity of the cortisol antiserum has been measured against various compounds. The percent of cross-reactivity is expressed as the ratios of cortisol concentration to the concentration of the reacting compound at 50% binding of the zero standard.

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| | Cortisol LIA | Cortisol ELISA |
| --- | --- | --- |
| Compound | Cross-reactivity (%) | |
| Cortisol, Hydrocortisone | 100 | 100 |
| Prednisolone | 57 | 29 |
| 11-Deoxycortisol | 12 | 16 |
| Corticosterone | 2.5 | 2.4 |
| Cortisone | 2.0 | 3.3 |
| 6β-Hydroxycortisol | 1.6 | 1.4 |
| Prednisone | 1.0 | 2.2 |
| 17 -Hydroxyprogesterone | 0.5 | 1.2 |
| Deoxycorticosterone | 0.3 | 0.5 |
| 6 -Methyl-17 -Hydroxyprogesterone | 0.1 | 0.3 |
| Progesterone | <0.05 | <0.01 |
| Dexamethasone | <0.05 | 0.06 |
| 17 -Hydroxypregnolen | <0.01 | 0.02 |
| Dehydroisoandrosterone | <0.01 | <0.01 |
| Androstenedione | <0.01 | <0.01 |
| Estriol | <0.01 | <0.01 |
| 6 -Methyl-17 -Hydroxyprogesterone-acetate | <0.01 | <0.01 |
| Pregnenolone | <0.01 | <0.01 |
| Estrone | <0.01 | 0.03 |
| Testosterone | <0.01 | 0.02 |
| 17 -Hydroxyprogesteron-17 sulphate | <0.01 | <0.01 |
| Androsterone sulphate | <0.01 | 0.02 |
| Testosterone sulphate | <0.01 | <0.01 |
| Cholesteryl sulphate | <0.01 | <0.01 |
| 17β-Estradiol-17 sulphate | <0.01 | 0.02 |
| DHEA-S | <0.01 | <0.01 |

The following substances do not have a significant effect (+/-20 % of expected) on the test results up to the below stated concentrations:

| | Serum | |
| --- | --- | --- |
| | Conc. | Cortisol (μg/dL) |
| Hemoglobin | 4.0 mg/mL | 0.06; 0.33; 0.62 |
| Bilirubin | 0.5 mg/mL | 0.07; 0.35; 0.63 |
| Triglyceride | 30 mg/mL | 0.07; 0.40; 0.75 |
| | Saliva | |
| | Conc. | Cortisol (μg/dL) |
| Thimerosal | 0.50 % | 0.19; 0.25; 0.34 |
| Blood | 0.125 % | 0.09; 0.26 |
| NaN3 | 0.60 % | 0.23; 0.31 |

f. Assay cut-off:

Not applicable

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2. Comparison studies:

a. Method comparison with predicate device:

The device was compared to the predicate Cortisol LIA from IBL with 129 serum samples (117 patient serum samples supplemented with 12 spiked samples to cover measuring range). Another comparison was performed with the same predicate device using 130 saliva samples (16 patient saliva samples supplemented with 14 spiked samples to cover the measuring range).

| Sample type | Regression equation | Correlation (R²) |
| --- | --- | --- |
| Serum (n=129) | y=1.174x-2.191 | 0.995 |
| Saliva (n=130) | y=0.915x+0.056 | 0.990 |

b. Matrix comparison:

Not applicable

3. Clinical studies:

a. Clinical Sensitivity:

Not applicable

b. Clinical specificity:

Not applicable

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

Not applicable

4. Clinical cut-off:

Not Applicable

5. Expected values/Reference range:

| | n | μg/dL | | nmol/L | |
| --- | --- | --- | --- | --- | --- |
| | | AM | PM | AM | PM |
| Saliva | 725 | 0.5 – 1.5 | 0.03 – 0.3 | 13.8 – 41.4 | 0.83 – 8.3 |
| Serum | 125 | 5 – 25 | 2 – 12 | 138 - 690 | 55.2 – 331.2 |

Reference

(Westermann J, Demir A, Herbst V. Determination of Cortisol in Saliva and Serum by a Luminescence-Enhanced Enzyme Immunoassay. Clin Lab 2004;50:11-24)

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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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