KRONUS TSH RECEPTOR AUTOANTIBODY (TRAB) ELISA ASSAY KIT

K080159 · Kronus Market Development Associates, Inc. · JZO · May 22, 2008 · Immunology

Device Facts

• Record ID: K080159
• Device Name: KRONUS TSH RECEPTOR AUTOANTIBODY (TRAB) ELISA ASSAY KIT
• Applicant: Kronus Market Development Associates, Inc.
• Product Code: JZO · Immunology
• Decision Date: May 22, 2008
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5870
• Device Class: Class 2

Indications for Use

The KRONUS TRAb ELISA Assay Kit is for the semi-quantitative determination of antibodies to the thyroid stimulating hormone receptor in human serum. The KRONUS TRAb ELISA Assay is useful as an aid in the diagnosis of Graves' Disease in conjunction with other clinical and laboratory findings.

Device Story

KRONUS TRAb ELISA Assay Kit is an in vitro diagnostic test for human serum samples. It utilizes enzyme-linked immunosorbent assay (ELISA) technology to detect TSH receptor autoantibodies. The assay is performed in a clinical laboratory setting by trained personnel. The device provides semi-quantitative results, which are interpreted by clinicians alongside patient history and other laboratory data to assist in diagnosing Graves' Disease. The test aids in clinical decision-making by identifying the presence of autoantibodies associated with thyroid dysfunction.

Clinical Evidence

No clinical data provided in the document; bench testing only.

Technological Characteristics

Semi-quantitative ELISA; microtiter wells coated with TSH receptor; M22-peroxidase conjugate; TMB substrate. Requires ELISA plate reader (450 nm) and plate shaker (500 rpm). Standardized against NIBSC 90/672. Measuring range 1-40 IU/L. Shelf life 8 months.

Indications for Use

Indicated for the semi-quantitative determination of TSH receptor autoantibodies in human serum to aid in the diagnosis of Graves' Disease. For prescription use only.

Regulatory Classification

Identification

A thyroid autoantibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the thyroid autoantibodies (antibodies produced against the body's own tissues). Measurement of thyroid autoantibodies may aid in the diagnosis of certain thyroid disorders, such as Hashimoto's disease (chronic lymphocytic thyroiditis), nontoxic goiter (enlargement of thyroid gland), Grave's disease (enlargement of the thyroid gland with protrusion of the eyeballs), and cancer of the thyroid.

Submission Summary (Full Text)

{0}

1

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k080159

B. Purpose for Submission:
New Device

C. Measurand:
Thyroid Stimulating Hormone Receptor (TSHR) Auto-antibody

D. Type of Test:
Semi-quantitative ELISA

E. Applicant:
KRONUS Market Development Associates, INC.

F. Proprietary and Established Names:
KRONUS TSH Receptor Autoantibody (TRAb) ELISA Assay

G. Regulatory Information:
1. Regulation section:
21 CFR § 866.5870, Thyroid autoantibody immunological test system
2. Classification:
Class II
3. Product codes:
JZO, System, test, thyroid autoantibody
4. Panel:
Immunology (82)

H. Intended Use:
1. Intended use(s):
The KRONUS TRAb ELISA Assay Kit is for the semi-quantitative determination of antibodies to the thyroid stimulating hormone receptor in human serum. The KRONUS TRAb ELISA Assay is useful as an aid in the diagnosis of Graves' Disease in conjunction with other clinical and laboratory findings.
2. Indication(s) for use:
Same as Intended use.
3. Special conditions for use statement(s):
For prescription only.
4. Special instrument requirements:
ELISA plate reader, plate shaker

I. Device Description:
The device consists of: ELISA strip wells coated with TSH Receptor (12x8), four levels calibrators (1, 2, 8, and 40 IU/L), M22 peroxidase, positive and negative controls, M22-peroxidase reconstitution buffer, start buffer, concentrated wash solution, peroxidase (TMB) substrate, and stop solution.

J. Substantial Equivalence Information:
1. Predicate device name(s):
KRONUS TRAb Coated Tube (CT) RIA Assay
2. Predicate K number(s):

{1}

k032134

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Trade name | KRONUS TSH Receptor Autoantibody (TRAb) ELISA Assay | KRONUS TRAb Coated Tube RIA Assay |
| Intended Use | Measurement of TSH receptor autoantibody | Same |
| Indication for Use | Aid in differential diagnosis of Graves’ Disease | Same |
| Analyte | TSH receptor autoantibodies | Same |
| Assay principle | TSH receptor immuno-inhibition | Same |
| Sample Matrix | Human serum | Same |
| Calibrator Levels | 4 Levels (1, 2, 4, 40 U/L) | Same |
| Controls | Positive and negative | Same |
| Reference Standard Calibration | NIBSC 90/672 | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Test principles | Enzyme-linked immunosorbent assay | Radioimmunoassay |
| Antibody capture | Microwell coated with F(ab)2 fragment of mouse monoclonal anti-TSHR Ab bind to TSHR | 125I- labeled TSHR coated tube |
| Assay Format | Semi-quantitative | Qualitative and quantitative |
| Solid phase | Microtiter wells | Tubes |
| Shaker | Elisa plate shaker (500 shakes/ minute) | Orbital rotator |
| Enzyme/Substrate | TSH human monoclonal autoantibody M22 peroxidase/ TMB | N/A |
| Incubation periods | 60-25-25 minutes | 120-60 minutes |
| Signal | Optical density | Radioactivity |
| Detection instrument | Elisa microplate reader (spectrophotometer) set with 450 nm filter | Gamma counter set for 125I |
| Units of measure | IU/L | U/L |

{2}

3

| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Analytical sensitivity | 0.06 IU/L | 0.3 U/L |
| Cut-off | Positive: >1 IU/L
Negative: ≤ 1 IU/L | Positive: >1.5 U/L (15% inhibition)
Equivocal: 1.1-1.5 U/L (11-15% inhibition)
Negative: ≤1.0 U/L (10% inhibition) |

K. Standard/Guidance Document Referenced (if applicable):
None

L. Test Principle:
In the KRONUS TRAb ELISA, TSH receptor autoantibodies in patient sera, calibrators and controls are allowed to interact with TSH receptor that are bound to ELISA plate wells by . After 1 hour incubation, the samples are discarded leaving TRAb bound to the immobilized TSH receptor. A thyroid stimulating human monoclonal autoantibody conjugated to peroxidase (M22-peroxidase) is added in a second incubation step, where it interacts with immobilized TSH receptors, which have not been blocked by bound TRAb. The M22-peroxidase bound to the plate is then semi-quantitated by the addition of a colorgenic substrate, TMB, with reading of the final absorbance at 450 nm. A lower absorbance indicates the presence of TRAb in the test sample. Calibrator values are plotted on semi-log graph paper and the antibody concentrations of the controls and patient specimens are interpolated from the curve.

M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Intra-assay
The intra-assay precision was determined by testing six serum samples 15-22 times. The serum samples consisted of 2 high anti-TSHR samples (4.93 and 7.14 IU/L), 2 samples close to the cut-off (1.13 and 1.23 IU/L) and 1 negative sample (1.00 IU/L). Results showed %CV ranged from 3.1% to 11.7% (see table below).
Intra-assay Performance of TRAB Elisa
| Sample | A | B | C | D | E | F |
| --- | --- | --- | --- | --- | --- | --- |
| n | 20 | 20 | 20 | 20 | 22 | 15 |
| Mean (IU/L) | 4.93 | 1.96 | 1.00 | 7.14 | 1.23 | 1.13 |
| SD (IU/L) | 0.15 | 0.14 | 0.08 | 0.28 | 0.15 | 0.09 |
| CV % | 3.1 | 7.2 | 7.7 | 3.9 | 11.7 | 8.1 |
Inter-assay
The inter-assay precision was determined by testing seven serum samples 9-25 independent runs. The serum samples consisted of 4 samples with high

{3}

anti-TSHR concentrations (3.51-18.57 IU/L), 2 samples close to the cut-off (1.89-1.99 IU/L) and 1 negative sample (1.04 IU/L). %CV ranged from 3.3% to 8.5% (see table below).

Inter-assay Performance of TRAB Elisa

| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| N | 20 | 20 | 25 | 25 | 10 | 9 | 10 |
| Mean | 4.56 | 18.57 | 3.51 | 5.38 | 1.04 | 1.99 | 1.89 |
| SD | 0.15 | 1.4 | 0.17 | 0.43 | 0.07 | 0.17 | 0.15 |
| CV % | 3.3 | 7.6 | 4.8 | 8 | 7.1 | 8.5 | 7.9 |

b. Linearity/assay reportable range:

Eight serum samples of varying levels of TSHR autoantibodies (from 6.94 to >40 IU/L) were serially diluted and assayed. All diluted in a linear fashion. Due to the nature of the autoantibodies, affinities, and avidities, linearity for each patient sample is variable; therefore, sample dilutions are not advisable. The measuring range for the assay is from 1-40 IU/L. Results above the highest calibrator should be reported as >40 IU/L.

Hook-effect

Three high serum samples (from 3.89 to >40 IU/L) were serially diluted and no hook effect was observed for samples up to >143.0 IU/L.

c. Traceability, Stability, Expected values (controls, calibrators, or methods): The TSHR ELISA calibrators are standardized against the National Institute for Biological Standards and Control (NIBSC) 90/672 Thyroid Stimulating Hormone Antibody reference preparation.

Shelf life

Data demonstrated the assay has a shelf life of 8 months.

d. Detection limit:

The lower detection limit of the assay was determined by sequentially testing the negative control 20 times. A calibration curve of absorbance (450 nm) vs. concentration was constructed. The mean and standard deviation were calculated and the mean +2 SD, +3 SD and +10 SD were read off the calibration curve to give an IU/L value. The lower detection limit was computed to be 0.56 IU/L which is the mean of 20 determinations +10 SD.

e. Analytical specificity:

Interference by endogenous substances: No interference was observed in samples spiked with hemoglobin up to 50 mg/dL, bilirubin up to 20 mg/dL, lipids up to 3000 mg/dL, human chorionic gonadotropin up to 16.3 U/mL, follicle stimulating hormone up to 5 U/mL, luteinizing hormone up to 5 U/mL, and thyroid stimulating hormone up to 0.335 mU/mL.

HAMA interference study: Four blood donor samples containing human antimouse antibodies did not result in falsely elevated levels of TSHR antibodies with the assay. In addition, six different TRAb positive sera were spiked with three HAMA positive blood donor samples and no interferences were observed.

Crossreactivity with other autoantibodies: The TRAb ELISA kit was tested

{4}

with 36 sera on other auto immune diseases: 18 autoimmune hypothyroidism, 8 systemic lupus erythematosus (SLE) and 10 rheumatoid arthritis (RA). All samples were negative with the TRAb ELISA kit.

f. Assay cut-off:

The assay cut-off was established by testing sera from 104 individual healthy blood donors (44 females) in the KRONUS TRAb ELISA. All 104 sera gave values of less than 1 IU/L.

2. Comparison studies:

a. Method comparison with predicate device:

Testing was performed on 182 samples from 82 patients with Graves Disease, 39 non-autoimmune thyroid diseases, 17 autoimmune hypothyroidism, and 44 healthy blood donor samples. The Positive Percent Agreement was 100.0% (64/64); the Negative Percent Agreement was 95% (112/118) and the Overall Agreement was 97% (176/182). Refer to table below:

| | Predicate device: TRAb CT RIA | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| New deviceTRAb ELISA | Positive | 64 | 6 | 70 |
| | Negative | 0 | 112 | 112 |
| | Total | 64 | 118 | 182 |

Positive Percent Agreement: (64/64) = 100% (95% CI: 95-100)

Negative Percent Agreement: (112/118) = 95% (95% CI: 90-98)

Overall Agreement: (176/182) = 97% (95% CI: 93-99)

b. Matrix comparison:

Not applicable.

3. Clinical studies:

a. Clinical Sensitivity and specificity:

The clinical sensitivity and specificity were evaluated on 261 clinically defined samples from patients with the following diagnosis: 82 Graves Disease, 75 disease controls (39 non-autoimmune thyroid diseases, 18 autoimmune hypothyroidism, 8 SLE, 10 RA, and 104 healthy individuals). The TRAb ELISA assay sensitivity and specificity were 85% (70/82) and 100% (179/179) respectively (refer to table below).

| | Diagnosis | | | |
| --- | --- | --- | --- | --- |
| | | Positives (Graves Disease) | Negative (Disease Controls and Healthy Controls) | Totals |
| TRAb ELISA | Positive | 70 | 0 | 70 |
| | Negative | 12 | 179 | 191 |
| | Total | 82 | 179 | 261 |

Sensitivity: (70/82) = 85%

Specificity: (179/179) = 100%

{5}

b. Other clinical supportive data (when a. is not applicable):
Not applicable.

4. Clinical cut-off:
Refer to assay cut-off.

5. Expected values/Reference range:
The expected value for normal population is ≤1 IU/L.

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.

6