← Product Code [OIF](/submissions/IM/subpart-f%E2%80%94immunological-test-systems/OIF) · K073590

# KRONUS IA-2 AUTOANTIBODY RIA ASSAY KIT (K073590)

_Kronus Market Development Associates, Inc. · OIF · Apr 10, 2008 · Immunology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/OIF/K073590

## Device Facts

- **Applicant:** Kronus Market Development Associates, Inc.
- **Product Code:** [OIF](/submissions/IM/subpart-f%E2%80%94immunological-test-systems/OIF.md)
- **Decision Date:** Apr 10, 2008
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.5660
- **Device Class:** Class 2
- **Review Panel:** Immunology

## Indications for Use

The KRONUS IA-2 Autoantibody RIA Assay Kit is for the semi-quantitative determination of antibodies to tyrosine phosphatase (IA-2) in human serum. The KRONUS IA-2 Autoantibody RIA Assay is useful as an aid in the diagnosis of Type I diabetes mellitus (autoimmune mediated diabetes).

## Device Story

The KRONUS IA-2 Autoantibody RIA Assay Kit is an in vitro diagnostic test for human serum. It utilizes a radioimmunoassay (RIA) principle to detect autoantibodies to tyrosine phosphatase (IA-2). Patient serum is incubated with human recombinant I¹²⁵-labeled IA-2 tracer; antibodies bind to the tracer. Protein A is added to precipitate the antigen-antibody complexes. After centrifugation and washing, the radioactivity in the pellet is measured using a gamma counter. The amount of radioactivity is directly proportional to the IA-2 autoantibody concentration. Results are determined by interpolating from a standard curve generated by five calibrators. The assay is performed in a laboratory setting by trained personnel. The output provides a semi-quantitative value (U/mL) used by clinicians to support the diagnosis of Type I diabetes mellitus. The device benefits patients by providing an immunological marker to assist in differentiating autoimmune-mediated diabetes from other forms.

## Clinical Evidence

No clinical data provided in the document; substantial equivalence is based on performance characteristics of the RIA assay.

## Technological Characteristics

Radioimmunoassay (RIA) using human recombinant I¹²⁵-labeled IA-2 tracer. Components include lyophilized tracer, assay buffer, Protein A reagent, and calibrators (0.0-50 U/mL). Requires a gamma radiation counter and refrigerated centrifuge (1500 x g). Semi-quantitative measurement via interpolation from a semi-log standard curve. Shelf-life of 8 weeks at 2-8°C.

## Regulatory 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).

## Submission Summary (Full Text)

> This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com.
>
> Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/).

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k073590

B. Purpose for Submission:
New device

C. Measurand:
Autoantibodies to IA-2 (insulinoma-associated protein 2), a tyrosine phosphatase-like protein

D. Type of Test:
Semi-quantitative, radioimmunoassay (RIA)

E. Applicant:
KRONUS Market Development Associates, Inc.

F. Proprietary and Established Names:
KRONUS IA-2 Autoantibody RIA Assay Kit

G. Regulatory Information:

|  Product Code | Classification | Regulation Section | Panel  |
| --- | --- | --- | --- |
|  OIF: Tyrosine phosphatase (IA-2) autoantibody assay | Class II | 21 CFR 866.5660
Multiple autoantibody immunological test system | Immunology (IM82)  |

H. Intended Use:

1. Intended use(s):
The KRONUS IA-2 Autoantibody RIA Assay Kit is for the semi-quantitative determination of antibodies to tyrosine phosphatase (IA-2) in human serum. The KRONUS IA-2 Autoantibody RIA 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):
The device is for prescription use only

4. Special instrument requirements:
Gamma radiation counter set for I¹²⁵ and a refrigerated centrifuge capable of 1500 x g

I. Device Description:
The KRONUS IA-2Ab RIA Assay Kit consists of:
1. Lyophilized I¹²⁵ IA-2 Tracer (human recombinant).
2. Assay buffer
3. IA-2 Autoantibody calibrators: lyophilized, 0.0, 0.75, 2.0, 10, and 50 U/mL
4. Protein A reagent
5. Control sera: Control A (low) and Control B (high)

J. Substantial Equivalence Information:

1. Predicate device name(s):
KRONUS Glutamic Acid Decarboxylase Antibody (GADAb) RIA Assay

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2. Predicate 510(k) number(s): k051061
3. Comparison with predicate:

|  Similarities  |   |   |
| --- | --- | --- |
|  Item | Device | Predicate  |
|   | Tyrosine Phosphatase Autoantibody (IA-2Ab) RIA | Glutamic Acid Decarboxylase Autoantibody (GADAb) RIA  |
|  Intended Use | Semi-quantitative detection of autoantibodies | Same  |
|  Indications for Use | Aid in the diagnosis of Type I diabetes mellitus (autoimmune mediated diabetes) | Same  |
|  Matrix | Serum | Same  |
|  Test principle | Radioimmunoassay | Same  |
|  Test platform | Antibodies bind to labeled antigen in liquid phase (test tube), the antigen-antibody complexes are precipitated and the reaction is measured | Same  |
|  Detection instrument | Gamma counter | Same  |
|  Antigen label | Iodine 125 (I125) | Same  |
|  Calibrators | 5 calibrators: 0.0, 0.75, 2.0, 10, and 50 U/mL | 5 calibrators: 0.0, 0.4, 1.0, 10, and 50 U/mL  |
|  Precipitation reagent | Protein A | Same  |
|  Controls | 2 levels | Same  |
|  Differences  |   |   |
| --- | --- | --- |
|  Item | Device | Predicate  |
|   | Tyrosine Phosphatase Autoantibody (IA-2Ab) RIA | KRONUS Glutamic Acid Decarboxylase Autoantibody (GADAb) RIA  |
|  Analyte | Tyrosine phosphatase autoantibodies | Glutamic acid decarboxylase autoantibodies  |

K. Standard/Guidance Documents Referenced (if applicable):
FDA guidance: Review Criteria for In Vitro Diagnostic Devices for the Assessment of

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Thyroid Autoantibodies Using Direct Immunofluorescence Assay (IFA), Indirect Hemagglutination (IHA), Radioimmunoassay (RIA), and Enzyme Linked Immunosorbent Assay (ELISA)

L. Test Principle:
Calibrators, controls and patient samples are incubated overnight with human recombinant I¹²⁵ IA-2. During this incubation, antibody binds to the tracer. Protein A is added and the tubes are incubated for one hour during which time the antibodies present are bound by Protein A and removed from solution. Assay buffer is then added and the tubes are centrifuged. After centrifugation, the supernatants are decanted or aspirated. The resulting pellets are then washed and centrifuged an additional time followed by decanting or aspiration of the supernatants. The amount of radioactivity in the pellets is directly proportional to the amount of IA-2 autoantibody contained in the samples. Calibrator concentrations are plotted on semi-log graph paper and the concentration of antibody in the unknowns is interpolated from the curve.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:
a. Precision/Reproducibility:
Intra-assay
Eight samples with values ranging from 0.98 to 27.6 U/mL of IA-2 autoantibody were assayed in 20 or 25 independent runs. Four of the samples had values close to the assay cut-off of 1.0 U/mL. Percent CVs ranged from 2.1 to 15.2%.

|  Intra-Assay  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Sample | A | B | C | D | E | F | G | H  |
|  N | 25 | 25 | 25 | 25 | 25 | 25 | 20 | 20  |
|  Mean (U/mL) | 0.98 | 1.72 | 2.61 | 6.4 | 15.1 | 27.6 | 1.91 | 1.33  |
|  SD | 0.15 | 0.11 | 0.05 | 0.20 | 0.40 | 0.70 | 0.10 | 0.12  |
|  %CV | 15.2 | 6.2 | 2.1 | 2.5 | 2.8 | 2.5 | 5.0 | 8.7  |

Inter-assay
Eight samples with values ranging from 0.50 to 25.83 U/mL of IA-2 autoantibody were assayed in 11-25 replicates each. Several of the samples had values close to the assay cut-off. The %CVs ranged from 3.30 to 25.28%.

|  Inter-Assay  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8  |
|  N | 25 | 25 | 25 | 25 | 25 | 25 | 11 | 12  |
|  Mean (U/mL) | 0.50 | 1.21 | 3.74 | 6.10 | 15.0 | 25.83 | 1.47 | 1.55  |
|  SD | 0.13 | 0.26 | 0.15 | 0.20 | 0.80 | 1.34 | 0.22 | 0.16  |
|  %CV | 25.28 | 21.63 | 3.94 | 3.30 | 5.30 | 5.18 | 15.2 | 10.1  |

Lot-to-Lot
Two control samples, one low and one high levels of IA-2Ab were assayed in 11 kit lots. The %CV for the samples ranged from 0.02 to 0.03%.

Lab-to-Lab

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One-hundred fifty samples (suspected Type 1 diabetics) were assayed at two different laboratories. The correlation between laboratories was r=0.991. The overall agreement of positive and negative was 99.3% (149/150).

b. Linearity/assay reportable range:

Linearity

The measuring range for the assay is from 0.6 to 50 U/mL. As each patient sample will have a different dilution curve due to the nature of autoantibody affinities and avidities, linearity is variable. KRONUS makes no linearity claims and patient dilutions are not advised. The highest calibrator (50 U/mL) represents the approximate maximum binding of the tracer in the assay and allows for most samples to be read off the curve without need of dilution. Samples with results above the highest calibrator should be reported as >50 U/mL. Samples below 0.6 (Limit of Quantitation) should be reported as <0.6 U/mL.

Recovery

The IA-2 RIA calibrators were spiked with 3 serum samples of varying IA-2 autoantibody levels, were diluted and measured in the assay. The recoveries ranged from 110% to 157% with a mean recovery across all samples of 134%. The studies showed varying results for low, moderate and high sample dilutions across all five samples. KRONUS included a description of problems relating to dilution of patient samples in the Dilution Recovery section of the labeling: "As the dynamics of antibody-antigen interactions are affected by both antibody concentration and affinity each patient sample will have a different dilution profile and KRONUS does NOT recommend that patient samples are diluted for use in this assay."

Hook effect

Four serum samples ranging from 7.5 to >50 U/mL were diluted in kit zero calibrator. No hook effect was observed.

|  Hook Effect  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  1 | U/mL | 2 | U/mL | 3 | U/mL | 4 | U/mL  |
|  Neat | 7.5 | Neat | 22.8 | Neat | 36.6 | Neat | >50  |
|  1:2 | 6.2 | 1:2 | 14.4 | 1:2 | 29.2 | 1:2 | >50  |
|  1:4 | 4.9 | 1:4 | 8.5 | 1:4 | 22.5 | 1:4 | 29.3  |
|  1:8 | 3.9 | 1:8 | 5.1 | 1:8 | 15.9 | 1:8 | 13.2  |
|  1:16 | 2.9 | 1:16 | 3.2 | 1:16 | 10.6 | 1:16 | 7.0  |
|  1:32 | 2.1 | 1:32 | 2.1 | 1:32 | 7.3 | 1:32 | 3.9  |
|  1:64 | 1.4 | 1:64 | 1.3 | 1:64 | 5.3 | 1:64 | 2.2  |
|  1:128 | 0.8 | 1:128 | 0.7 | 1:128 | 3.7 | 1:128 | 1.0  |
|  1:256 | 0.6 | 1:256 | 0.5 | 1:256 | 2.5 | 1:256 | 0.5  |

c. Traceability, Stability, Expected values (controls, calibrators, or methods): There is no reference standard available for IA-2 autoantibodies and no claim was made for traceability.

Stability

Stability was established via both real-time at 2-8°C and accelerated stability studies at 37°C on various kit components as well as whole kit studies. Based

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on these studies a shelf-life of 8 weeks was established for the assay.

d. Detection limit:

Limit of Blank (LoB)

The LoD for the assay was determined by sequentially testing the zero calibrator 20 times. A calibration curve of $\% \mathrm{B} / \mathrm{T}$ (binding of tracer) versus concentration was constructed. The mean and standard deviation were calculated and the mean $+2$ SD were interpreted from the curve. The limit of blank was computed to be $0.19\mathrm{U / ml}$.

Limit of Quantitation (LoQ)

The LoQ (defined as the lowest level yielding an inter-assay $\% \mathrm{CV}$ not greater than $20\%$ using inter-assay precision) was established by plotting a graph of mean concentration versus $\% \mathrm{CV}$. The 7 samples tested ranged from 0.16-25.83 U/mL. The LoQ was determined to be $0.6\mathrm{U / mL}$ and KRONUS recommends that results below this value be reported as less than $0.6\mathrm{U / mL}$.

![img-0.jpeg](img-0.jpeg)
IA-2Ab RIA Functional Sensitivity

e. Analytical specificity:

Fifteen samples were tested before and after the addition of the potentially interfering substance. This included 9 IA-2 autoantibody positive samples (1.7-20.9 U/mL) and 6 negative samples ($\leq 0.53\mathrm{U / mL}$).

Hemoglobin

Samples were spiked with hemoglobin at a level of $500\mathrm{mg / dL}$ and then analyzed. Percent differences ranged from -11.8 to $4.2\%$ for all positive samples.

Bilirubin

Samples were spiked with $20\mathrm{mg / dL}$ of bilirubin and analyzed. The $\%$ differences for all positive samples ranged from -3.5 to $5.9\%$.

Lipids

Samples were spiked with lipid at approximately $3000\mathrm{mg / dL}$ and 1000

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mg/dL. Percent differences for all positive samples ranged from -4.2 to 0.0% and -5.8 to 1.5%, respectively.

The user is instructed to avoid the use of grossly hemolyzed and lipemic samples.

f. Assay cut-off:

Fifty normal healthy controls with no family history of diabetes mellitus were assayed. There were 33 females and 17 males ranging in age from 20-61 years. All samples (100%) contained less than 1.0 U/mL. In addition, sera from 113 healthy blood donors showed 112 samples contained less than 1.0 U/mL. One sample gave a result of 1.0 U/mL. Given these results, values less than or equal to 1.0 U/mL are considered negative for IA-2 autoantibodies and values greater than 1.0 U/mL are considered positive.

2. Comparison studies:

a. Method comparison with predicate device:

Samples from 50 healthy blood donors and 60 Type 1 diabetic patients were tested on both assays.

|   | KRONUS GADAb RIA  |   |   |
| --- | --- | --- | --- |
|  KRONUS IA-2Ab RIA | + | - | Total  |
|  + | 29 | 4 | 33  |
|  - | 9 | 68 | 77  |
|  Total | 38 | 72 | 110  |

Positive Percent Agreement: 76% (29/38) (95% CI = 62-88)

Negative Percent Agreement: 94% (68/72) (95% CI = 88-98)

Overall Agreement: 88% (97/110) (95% CI = 81-93)

b. Matrix comparison:

Not applicable because both assays use serum as matrix.

3. Clinical studies:

a. Clinical Sensitivity and Specificity:

The clinical studies included 795 subjects: Type 1 diabetes (n=277); Type 2 diabetes (n=204); healthy blood donors (n=163); and 151 patients with other types of autoimmune diseases.

|  Clinical Status | Number of Patients positive for IA-2Ab | % Positive  |
| --- | --- | --- |
|  Graves’ Disease | 1/60 | 2%  |
|  Hashimoto’s Thyroiditis | 0/47 | 0%  |
|  Systemic Lupus Erythematosus | 0/10 | 0%  |
|  Myasthenia Gravis | 0/34 | 0%  |
|  healthy blood donors | 1/163 | 1%  |
|  Type 2 Diabetes | 5/204 | 2%  |
|  Type 1 Diabetes | 136/277 | 49%  |

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Clinical Sensitivity, Specificity and Agreement

|  IA-2Ab Assay | Type 1 Diabetes |   | Total  |
| --- | --- | --- | --- |
|   |  + | -  |   |
|  + | 136 | 7 | 143  |
|  - | 141 | 511 | 652  |
|  Total | 277 | 518 | 795  |

Clinical sensitivity: 49% (136/277) (95% CI 43-55)
Clinical specificity: 98.6% (511/518) (95% CI 97-99)

Published literature included in the submission showed clinical sensitivity for IA-2 autoantibodies in the target population ranging from 25 to 89%.

b. Other clinical supportive data (when a. is 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 <1.0 U/mL

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.

---

**Source:** [https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/OIF/K073590](https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/OIF/K073590)

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**Cite:** Innolitics at https://innolitics.com