KRONUS GAD AUTOANTIBODY ELISA ASSAY KIT
Device Facts
| Record ID | K072135 |
|---|---|
| Device Name | KRONUS GAD AUTOANTIBODY ELISA ASSAY KIT |
| Applicant | Kronus Market Development Associates, Inc. |
| Product Code | NWG · Immunology |
| Decision Date | Nov 7, 2007 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5660 |
| Device Class | Class 2 |
| Attributes | Real-World Evidence, Pediatric |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K072135 · Nov 7, 2007 | KRONUS GAD AUTOANTIBODY ELISA ASSAY KIT | Kronus Market Development Associates, Inc. | Clinical serum samples from healthy blood donors; Clinical serum samples from patients with autoimmune diseases (Graves', Hashimoto's, RA); Clinical serum samples from patients with Type I and Type II diabetes | The device performance was evaluated using retrospective clinical serum samples from diverse patient populations to determine clinical sensitivity and specificity for the diagnosis of Type I diabetes. | Clinical sensitivity; Clinical specificity; Autoimmune disease cohorts; Healthy blood donors |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Retrospective cohort analysis | 99 Type I diabetes patients, 40 patients with other autoimmune diseases (Graves', Hashimoto's, RA), and 120 healthy blood donors; Sample Size: 259 | Not applicable for this study | Clinical sensitivity and specificity for GAD autoantibody detection |
Indications for Use
The KRONUS GADAb ELISA Assay Kit is for the semi-quantitative determination of glutamic acid decarboxylase antibody in human serum. The assay is useful as an aid in the diagnosis of Type I diabetes mellitus (autoimmune mediated diabetes).
Device Story
The KRONUS GADAb ELISA Assay Kit is an in vitro diagnostic test for semi-quantitative detection of glutamic acid decarboxylase (GAD) autoantibodies in human serum. The device utilizes an enzyme-linked immunosorbent assay (ELISA) principle; GAD autoantibodies in patient serum form a bridge between GAD65-coated microtiter plate wells and liquid-phase GAD65-biotin. Bound GAD-biotin is quantified via streptavidin-peroxidase and TMB chromogenic substrate, with absorbance measured at 450nm using an ELISA plate reader. Results are interpolated from a calibration curve. The assay is intended for use in clinical laboratories by trained personnel. Clinicians use the results as an aid in diagnosing Type I diabetes mellitus. The device provides a non-radioactive alternative to radioimmunoassay (RIA) methods for detecting GAD autoantibodies.
Clinical Evidence
Clinical performance was evaluated using 99 Type I diabetes patients (aged 7-46 years) and 160 non-diabetic controls (40 with other autoimmune diseases, 120 healthy donors). The assay demonstrated a clinical sensitivity of 83.8% (95% CI: 76-90) and a clinical specificity of 99.4% (95% CI: 97-100). Method comparison against the RIA predicate (n=367) showed 97.1% positive percent agreement and 90.9% negative percent agreement. Analytical precision (intra-assay %CV 3.1-16.2%; inter-assay %CV 5.2-22.7%) and functional sensitivity (4.0 IU/mL) were established.
Technological Characteristics
ELISA-based immunoassay; solid-phase microtiter plate coated with GAD65; liquid-phase GAD65-biotin; streptavidin-peroxidase conjugate; TMB substrate; spectrophotometric detection at 450nm. Standardized against NIBSC 97/550. Requires external ELISA plate reader and shaker/rotator.
Indications for Use
Indicated for use as an aid in the diagnosis of Type I diabetes mellitus in patients suspected of having autoimmune mediated diabetes.
Regulatory Classification
Identification
A multiple autoantibodies immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies (antibodies produced against the body's own tissues) in serum and other body fluids. Measurement of multiple autoantibodies aids in the diagnosis of autoimmune disorders (disease produced when the body's own tissues are injured by autoantibodies).
Predicate Devices
- KRONUS Glutamic Acid Decarboxylase Antibody (GADAb) RIA (k051061)
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE
A. 510(k) Number:
k072135
B. Purpose for Submission:
New device
C. Measurand:
Glutamic acid decarboxylase autoantibodies (GADA)
D. Type of Test:
Quantitative enzyme-linked immunosorbent assay (ELISA)
E. Applicant:
KRONUS Market Development Associates, Inc.
F. Proprietary and Established Names:
KRONUS Glutamic Acid Decarboxylase Antibody (GADAb) ELISA Assay Kit
G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| NWG, Autoantibodies, glutamic acid decarboxylase (GADA) | Class II | 21 CFR 866.5660
Multiple autoantibodies
immunological test system | Immunology 82 |
H. Intended Use:
1. Intended use(s):
The KRONUS GADAb ELISA Assay Kit is for the semi-quantitative determination of glutamic acid decarboxylase antibody in human serum. The assay is useful as an aid in the diagnosis of Type I diabetes mellitus (autoimmune mediated diabetes).
2. Indication(s) for use:
Same as the intended use
3. Special conditions for use statement(s):
Prescription use only
4. Special instrument requirements:
ELISA plate reader, plate shaker or rotator
I. Device Description:
The device consists of: ELISA strip wells coated with GAD$_{65}$, GAD$_{65}$ biotin, streptavidin-peroxidase, positive and negative control materials, 6 levels of calibrators, GAD-biotin reconstitution buffer, streptavidin-peroxidase reconstitution buffer, wash solution, peroxidase substrate (TMB), and stop solution.
J. Substantial Equivalence Information:
1. Predicate device name(s):
KRONUS Glutamic Acid Decarboxylase Antibody (GADAb) RIA
2. Predicate 510(k) number(s):
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k051061
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | KRONUS Glutamic Acid Decarboxylase Antibody (GADAb) ELISA Assay | KRONUS Glutamic Acid Decarboxylase Antibody (GADAb) RIA Assay |
| Intended Use | Semi-quantitative detection of GAD autoantibodies | Semi-quantitative detection of GAD autoantibodies |
| Indications for Use | Aid in the diagnosis of Type I diabetes mellitus (autoimmune mediated diabetes) | Same |
| Matrix | Serum only | Same |
| Controls | Positive and negative | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Test principle | Enzyme-linked immunosorbent assay | Radioimmunoassay |
| Autoantibody capture | GADAb bind to GAD65 coated on solid phase | GADAb react with 125I-labeled GAD65 in liquid phase |
| Solid phase | Microtiter plate | Not applicable |
| Precipitating reagent | Not applicable | Protein A |
| Second capture antigen | GAD65 biotin | Not applicable |
| Enzyme/substrate | Streptavidin peroxidase/TMB | Not applicable |
| Detection instrument | ELISA plate reader (spectrophotometer) | Gamma counter |
| Calibrators | 5 levels: 5, 18, 35, 120, 250 IU/mL | 7 levels: 0, 1, 3, 10, 30, 120, 300 U/mL |
| Standardization | GAD Standard NIBSC 97/550 | Not standardized against a reference material |
K. Standard/Guidance Document Referenced (if applicable):
"Review Criteria for In Vitro Diagnostic Devices for the Assessment of Thyroid Autoantibodies Using Direct Immunofluorescence Assay (IFA), Indirect Hemagglutination (IHA), Radioimmunoassay (RIA), and Enzyme Linked Immunosorbent Assay (ELISA)"
L. Test Principle:
The new device assay depends on the ability of GAD autoantibodies to act divalently and form a bridge between GAD coated on ELISA plate wells and liquid phase GAD-biotin. The GAD-biotin bound is then quantitated by addition of streptavidin
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peroxidase and a chromogenic substrate (TMB) with reading of final absorbance at 450nm. The absorbance of each well is directly proportional to the amount of antibody present. Calibrator values are plotted on semi-log graph paper and the antibody concentrations of the controls and patient samples are interpolated from the curve.
## M. Performance Characteristics (if/when applicable):
### 1. Analytical performance:
#### a. Precision/Reproducibility:
**Intra-assay**
To assess intra-assay precision, 6 samples containing a range of GADAb levels were assayed in 21-26 replicates.
| | Samples (IU/mL) | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | 1 | 2 | 3 | 4 | 5 | 6 |
| N | 25 | 25 | 26 | 26 | 21 | 21 |
| Mean | 27.23 | 19.98 | 7.01 | 7.49 | 4.24 | 4.81 |
| SD | 7.12 | 1.70 | 0.25 | 0.23 | 0.69 | 0.19 |
| %CV | 7.32 | 8.49 | 3.57 | 3.12 | 16.2 | 4.0 |
**Inter-assay**
To assess inter-assay precision, 8 samples containing a range of GADAb levels were assayed in 15-20 runs.
| | Samples (IU/mL) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| N | 20 | 20 | 15 | 20 | 20 | 20 | 20 | 20 |
| Mean | 96.9 | 21.0 | 7.9 | 8.1 | 17.4 | 8.8 | 3.41 | 5.74 |
| SD | 5.5 | 1.1 | 0.9 | 0.8 | 1.1 | 1.2 | 0.77 | 0.37 |
| %CV | 5.7 | 5.2 | 11.5 | 10.1 | 6.2 | 13.2 | 22.7 | 6.5 |
**Lab-to-lab reproducibility**
One hundred fifty samples (suspected Type I diabetics) were assayed at 2 different laboratories. The correlation between laboratories was r = 0.970 and the overall agreement for positive/negative was 98.7% (148/150).
#### b. Linearity/assay reportable range:
**Linearity**
The measuring range for the assay is from 5 to 250 IU/mL. Each patient sample will have a different dilution curve due to the nature of autoantibodies, affinities and avidities. Linearity is variable and sample dilutions are not advisable. Three high positive samples were serially diluted and assayed. All diluted in a linear fashion. Results above the highest calibrator should be reported as >250 IU/mL.
**Hook-effect**
Three high samples were serially diluted in kit negative control. No hook effect was observed for samples up to 253.3 IU/mL (calculated result).
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c. Traceability, Stability, Expected values (controls, calibrators, or methods): The GADAb ELISA calibrators are standardized against the international reference preparation NIBSC Islet Cell Antibody 97/550.
Stability data demonstrated the assay has a shelf-life of 9 months.
d. Detection limit:
The lower detection limit of the assay was determined by sequentially testing the kit negative control 20 times. A calibration curve of absorbance (450nm) versus concentration was constructed. The mean and SD were calculated and the mean +2SD and +3SD were read off the calibration curve to give an IU/mL value. The lower detection limit was computed to be 0.06 IU/mL which is the mean of 20 replicates +2SD.
Functional sensitivity/Limit of Quantitation (LoQ)
The functional sensitivity (defined as the lowest level yielding an average inter-assay CV not greater than 20%) was determined. The means of the samples ran to establish inter-assay reproducibility were plotted against their %CVs and functional sensitivity was determined to be 4.0 IU/mL. KRONUS recommends that results below the functional sensitivity or LoQ of the assay be reported as less than 4.0 IU/mL.
e. Analytical specificity:
Hemoglobin
Samples from patients positive for antibodies to GAD and normal healthy blood donors were spiked with hemoglobin levels up to 500 mg/dL and analyzed. Percent differences ranged from 0.7-19.1% for all positive samples.
Bilirubin
Samples from GAD antibody positive patients and healthy blood donors were spiked with 20 mg/dL of bilirubin and analyzed. The % difference for all positive samples ranged from 0.9-11.1%.
Lipids
Samples positive for GAD antibodies as well as from healthy blood donors were spiked with lipid at approximately 3000 mg/dL and 1000 mg/dL. Percent differences ranged from 0.2-16.1%
The package insert states that lipemic or grossly hemolyzed serum samples are not to be used.
f. Assay cut-off:
Twenty samples from healthy blood donors were assayed. All samples contained 1.5 IU/mL or less of GAD autoantibodies. Given these results, values less than or equal to 5 IU/mL are considered negative for GAD antibodies and values greater than 5 IU/mL are considered positive. In an additional study to validate the appropriateness of the assay cut-off, 100 healthy male blood donor serum specimens were tested. Of the 100 tested, 98 (98%) yielded results less than or equal to 5 IU/mL.
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2. Comparison studies:
a. Method comparison with predicate device:
Three hundred sixty seven samples were tested. This group included 99 Type I diabetics (60 newly diagnosed (35 males aged 7 to 46 years and 25 females aged 10 to 35 years) and 39 confirmed Type I diabetes patients); 68 patients suspected of having Type I diabetes mellitus; 40 Type II diabetics; 40 other autoimmune disease patients (20 Graves' disease, 10 Hashimoto's thyroiditis, and 10 rheumatoid arthritis); and 120 samples from healthy blood donors.
The comparison showed the following:
| | Predicate device – GADAb RIA | | | |
| --- | --- | --- | --- | --- |
| | | + | - | Total |
| New device – GADAb ELISA | + | 101 | 24 | 125 |
| | - | 3 | 239 | 242 |
| | Total | 104 | 263 | 367 |
Positive Percent Agreement: (101/104) = 97.1% (95% CI = 93-99)
Negative Percent Agreement: (239/263) = 90.9% (95% CI = 87-94)
Overall Agreement: (340/367) = 92.6% (95% CI = 90-95)
b. Matrix comparison:
Both assays use serum as the recommended matrix.
3. Clinical studies:
a. Clinical Sensitivity and Specificity:
Sixty Type I diabetes serum samples (35 males aged 7 to 46 years and 25 females aged 10 to 35 years) were assayed. Using a cut-off of >5 IU/mL, 44 (73%) were positive. In another study, KRONUS tested samples from an additional 39 confirmed Type I diabetes patients. Of the sera tested, all 39 (100%) were positive for GAD autoantibodies.
KRONUS also submitted data for 40 patients with non-diabetic diseases: 20 Graves' disease, 10 Hashimoto's thyroiditis, and 10 rheumatoid arthritis patients. Combining results from these groups with 99 serum results from Type I diabetes subjects and 120 normal subjects, the following table was submitted:
| | Target = Diabetes present | Non-target = Diabetes absent = Other autoimmunity | Normals |
| --- | --- | --- | --- |
| POSITIVE: | | | |
| | Study 1: 44/60 (73%)
Study 2: 39/39 (100%) | Graves': 0/20 (0%)
Hashimoto's: 0/10 (0%)
RA: 0/10 (0%) | Study A: 0/20 (0%)
Study B: 1/100 (1%) |
| Total positive | 83/99 (83.8%) | 0/40 (0%) | 1/120 (<1%) |
| Combined | 83/99 (83.8%) | | 1/160 (<1%) |
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| | Target = Diabetes present | Non-target = Diabetes absent = Other autoimmunity | Normals |
| --- | --- | --- | --- |
| NEGATIVE: | | | |
| : | Study 1: 16/60 (27%) | Graves’: 20/20 (96%) | Study A: 20/20 (100%) |
| | Study 2: 0/39 (0%) | Hashimoto’s: 10/10 (100%)
RA: 10/10 (100%) | Study B: 99/100 (99%) |
| Total negative | 16/99 (16%) | 40/40 (100%) | 119/120 (99%) |
| Combined | 16/99 (16%) | | 159/160 (99%) |
Calculating the clinical sensitivity and specificity showed the following:
| | Type I Diabetes | | Total | |
| --- | --- | --- | --- | --- |
| | | Present | | Absent |
| GADAb assay result | + | 83 | 1 | 84 |
| | - | 16 | 159 | 175 |
| | Total | 99 | 160 | 259 |
Clinical sensitivity: 83.8% (83/99) (95% CI: 76-90)
Clinical specificity: 99.4% (159/160) (95% CI: 97-100)
Overall agreement: 93.4% (242/259) (95% CI: 90-96)
Published literature included in the submission showed clinical sensitivity for GAD antibodies ranged from 64 to 83% and a clinical specificity ranged from 98-100%.
b. Other clinical supportive data (when a. and b. are not applicable): Not applicable.
4. Clinical cut-off: See assay cut-off
5. Expected values/Reference range: The expected value for the normal population is ≤ 5 IU/mL. However, some patients with other autoimmune diseases and 2-3% of the general population can be positive for GAD autoantibodies.
N. Proposed Labeling: The labeling is sufficient and 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.
