ImmuLisa Enhanced Centromere Antibody ELISA

K151559 · Immco Diagnostics, Inc. · LJM · Mar 11, 2016 · Immunology

Device Facts

• Record ID: K151559
• Device Name: ImmuLisa Enhanced Centromere Antibody ELISA
• Applicant: Immco Diagnostics, Inc.
• Product Code: LJM · Immunology
• Decision Date: Mar 11, 2016
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5100
• Device Class: Class 2

Intended Use

An enzyme linked immunoassay (ELISA) for the qualitative or semiquantitative detection of anti-centromere antibodies in human serum or plasma to aid in the diagnosis of limited cutaneous systemic sclerosis / CREST in conjunction with other laboratory tests and clinical findings.

Device Story

ImmuLisa™ Enhanced Centromere Antibody ELISA is a solid-phase immunoassay for detecting anti-centromere IgG antibodies in human serum. Microwells are coated with recombinant purified CENP-A and CENP-B centromere antigens. Patient serum, calibrators, and controls are incubated in wells; specific antibodies bind to antigens. An enzyme-labeled anti-human IgG conjugate is added, followed by TMB substrate. A spectrophotometer reads the resulting color change at 450 nm. Results are expressed in ELISA units per milliliter (EU/ml) and reported as positive or negative. The device is used in clinical laboratory settings by trained laboratory personnel. Healthcare providers use these results as an aid in diagnosing limited cutaneous systemic sclerosis (lcSSc) or CREST syndrome, supporting clinical decision-making alongside other diagnostic findings.

Clinical Evidence

Clinical performance evaluated using 124 lcSSc/CREST samples and 865 autoimmune/infectious disease controls. Sensitivity was 53.2%; specificity was 95.5%. Method comparison against the predicate (n=407) showed 93.2% positive percent agreement, 95.4% negative percent agreement, and 95.1% overall agreement. Cross-reactivity testing on 669 specimens from various autoimmune/infectious conditions showed 2.7% positivity. Precision studies (n=80 replicates) showed total CVs ranging from 3.5% to 7.0%. Limit of Detection (LoD) is 3.9 EU/ml; linear range is 3.9–160 EU/ml.

Technological Characteristics

Solid-phase ELISA; recombinant purified CENP-A and CENP-B antigens; HRP-conjugated anti-human IgG; TMB substrate. Requires spectrophotometer (450 nm) and automated microplate washer. Linear range: 3.9–160 EU/mL. Traceable to in-house standards. No international reference preparation available.

Indications for Use

Indicated for qualitative or semi-quantitative detection of anti-centromere IgG antibodies in human serum to aid in the diagnosis of limited cutaneous systemic sclerosis (lcSSc) or CREST syndrome in patients, in conjunction with other clinical and laboratory findings.

Regulatory Classification

Identification

An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues).

Predicate Devices

• QUANTA Lite™ Centromere ELISA (K003959)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the seal of the U.S. Department of Health and Human Services. The seal features a stylized image of an eagle with three human profiles incorporated into its design. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circular pattern around the eagle.

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

March 11, 2016

IMMCO Diagnostics Inc. Mr. Kevin Lawson Chief Regulatory Officer 60 Pineview Drive Buffalo, NY 14228

Re: K151559

Trade/Device Name: ImmuLisa™ Enhanced Centromere Antibody ELISA Regulation Number: 21 CFR §866.5100 Regulation Name: Antinuclear antibody immunological test system Regulatory Class: Class II Product Code: LJM, antinuclear antibody (enzyme-labeled), antigen, controls Dated: February 5, 2016 Received: February 5, 2016

Dear Mr. Lawson:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809]); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

# Kelly Oliner - S

## For

Leonthena R. Carrington, MBA, MS, MT(ASCP) Director

Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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| Form Approved: | OMB No. 0910-0120 |
|------------------|--------------------------|
| Expiration Date: | January 31, 2017 |
| | See PRA Statement below. |

| 510(k) Number (if known) | K151559 |
|--------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Device Name | ImmuLisa™ Enhanced Centromere Antibody ELISA |
| Indications for Use (Describe) | An enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of anti-centromere IgG antibodies in human serum as an aid in diagnosis of limited cutanteous systemic sclerosis / CREST in conjunction with other laboratory and clinical findings. |

| Type of Use (Select one or both, as applicable) | <div> <label><input checked="checked" type="checkbox"/> Prescription Use (Part 21 CFR 801 Subpart D)</label> </div> <div> <label><input type="checkbox"/> Over-The-Counter Use (21 CFR 801 Subpart C)</label> </div> |
|-------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
|-------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|

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Image /page/3/Picture/0 description: The image shows the logo for Immco Diagnostics. The logo features a cluster of blue and green dots on the left, followed by the company name in blue, with the word "DIAGNOSTICS" in green below. Underneath the company name is the text "A Trinity Biotech Company" in a smaller font.

## 510(k) Summary

| Submitter: | Immco Diagnostics, Inc. |
|------------------------------|--------------------------------------------------------------|
| Address: | 60 Pineview Dr., Buffalo, NY 14228 |
| Phone Number: | 716-691-0091 ext. 110 |
| Contact: | Kevin Lawson |
| Summary Prepared: | 3-6-2016 |
| Device Name: | ImmuLisa™ Enhanced Centromere Antibody ELISA |
| Common Name: | Centromere Antibody ELISA |
| Product Code: | LJM, Antinuclear Antibody (Enzyme-Labeled), Antigen, Control |
| Substantially Equivalent to: | INOVA QUANTA Lite™ Centromere ELISA |

General Description: Antinuclear antibodies (ANA) occur in sera of patients with various connective tissue disorders such as systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), systemic sclerosis / scleroderma (SSc), polymyositis and Siögren's syndrome. These ANA are directed against nucleding DNA, nucleohistone and various extractable nuclear antigens (ENA) such as RNP, Sm, SS-A (Ro), SS-B (La), Centromere, Scl-70 (topoisomerase I) and Jo-1.

The anti-centromere antibody produces a discrete speckled pattern in HEp-2 immunofluorescence assays, and has been observed in 38-64% of patients with the limited cutaneous systemic sclerosis (IcSSc) or CREST syndrome variant of systemic sclerosis or scleroderma. Centromere antibodies bind the kinetochore of mitotic chromosomes and the pre-kinetochore of interphase cells. Immunoblotting analysis indicates that centromere antibodies recognize at least six epitopes on three chromosomal proteins: CENP-A (17-19kD), CENP-B (80kD), and CENP-C (140kD). CENP-A and CENP-B have been cloned and while CENP-B is the primary autoantigen, it appears that assays incorporating both antigens produce results with greater sensitivity and more comparable to immunofluorescence.

This test is performed as a solid phase immunoassay. Microwells are coated with recombinant purified CENP-A centromere antigens. Controls, calibrators and patient sera are incubated in the antigen coated wells to allow specific antibodies present in the serum to bind to the centromere antibodies are detected by adding an enzyme labeled anti-human lgG conjugate. Specific enzyme substrate (TMB) is then added and the presence of antibodies is detected by a color change that is read by a spectrophotometer at 450 nm. Results are expressed in ELISA units per milliliter (EU/ml) and reported as positive or negative.

Intended Use: An enzyme linked immunoassay (EUSA) for the qualitative detection of anti-centromere IgG antibodies in human serum as an aid in diagnosis of limited cutaneous systemic sclerosis / CREST in conjunction with other laboratory and clinical findings.

Similarities and Differences: Both kits use recombinant CENP-A and CENP-B coated on 96 well plates to detect IgG Centromere antibody with HRP anti-human IgG conjugate and TMB substrate. The IMMCO kit utilizes a 5 point calibrator curve with a borderline/indeterminate range of 20-25EU/ml; while the INOVA kit uses a single low positive sample as a cutoff of 20 units with no borderline range.

## Non-clinical Tests:

Method Comparison: Both kits were tested with well-characterized lcSSc / CREST subjects and disease controls.

| | | INOVA Centromere Ab ELISA | | |
|---------------------------------|----------|---------------------------|----------|-------|
| | | Positive | Negative | Total |
| IMMCO<br>Centromere Ab<br>ELISA | Positive | 55 | 16 | 71 |
| | Negative | 4 | 332 | 336 |
| | Total | 59 | 348 | 407 |

60 Pineview Drive ● ● Buffalo, NY 14228-2120 ● ● USA Toll free [phone]  •  tel. [phone]  •  fax [phone]

## www.immco.com

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Image /page/4/Picture/0 description: The image shows the logo for Immco Diagnostics, a Trinity Biotech Company. The logo features a cluster of blue and green circles on the left, followed by the company name "immco" in blue, with "DIAGNOSTICS" in green underneath. Below the company name is the text "A Trinity Biotech Company" in a smaller font size.

| Positive Percent Agreement: | 93.2% (95% Cl 82.7% - 97.8%) |
|-----------------------------|------------------------------|
| Negative Percent Agreement: | 95.4% (95% CI 92.5% - 97.3%) |
| Overall Agreement: | 95.1% (95% CI 92.4% - 96.9%) |

### Cross Reactivity:

A total of 669 potentially co-incident antibody positive specimens selected from individuals suffering from other autoimmune or infectious diseases were tested for centromere antibodies using the ImmuLisa™ assay. Indeterminate samples were considered positive.

| Condition | n | Centerome Ab Pos |
|-------------------------------------|-----|------------------|
| Diffuse cutaneous scleroderma | 39 | 1 |
| Anti-phospholipid syndrome | 36 | 4 |
| Anti-phospholipid syndrome with SLE | 20 | 0 |
| Celiac Disease | 35 | 2 |
| Churg-Strauss syndrome | 27 | 0 |
| Crohn's disease | 25 | 0 |
| Granulomatosis with polyangitis | 27 | 0 |
| Graves' disease | 20 | 0 |
| Hashimoto's thyroiditis | 20 | 0 |
| Osteoarthritis | 20 | 0 |
| Polymyositis/Dermatomyositis | 30 | 1 |
| Rheumatoid Arthritis | 24 | 1 |
| Sjögren's Syndrome | 37 | 2 |
| Systemic Lupus Erythematosus | 92 | 1 |
| Thrombocytopenia | 15 | 2 |
| Ulcerative Colitis | 25 | 0 |
| Cytomegalovirus | 20 | 0 |
| Hepatitis C | 20 | 0 |
| Herpes simplex virus type 1 | 20 | 3 |
| Herpes simplex virus type 2 | 20 | 0 |
| Lyme disease | 20 | 0 |
| Mononucleosis | 20 | 1 |
| Rubella | 20 | 0 |
| Syphilis | 17 | 0 |
| Toxoplasmosis | 20 | 0 |
| Total | 669 | 18 (2.7%) |

#### Precision

Precision was tested with positive speciment the range of the assay. Seven patients were run in duplicate, twice per day for 20 days (n=80 replicates per sample). Assays were run by two operators on two different sets of equipment.

| S# | Mean<br>EU/ml | Total<br>Imprecision | | Between days | | Between<br>Operator | | Within run<br>(Repeatability) | |
|----|---------------|----------------------|-----|--------------|-----|---------------------|-----|-------------------------------|-----|
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 8.5 | 0.5 | 6.0 | 0.3 | 3.3 | 0.2 | 2.8 | 0.3 | 4.1 |
| 2 | 16.4 | 1.1 | 7.0 | 0.8 | 5.2 | 0.6 | 3.6 | 0.5 | 3.0 |
| 3 | 20.4 | 1.1 | 5.6 | 0.5 | 2.3 | 0.5 | 2.5 | 0.9 | 4.4 |
| 4 | 23.8 | 1.1 | 4.6 | 0.8 | 3.5 | 0.5 | 2.2 | 0.5 | 1.9 |
| 5 | 49.0 | 3.1 | 6.2 | 2.2 | 4.5 | 1.5 | 3.1 | 1.5 | 3.0 |
| 6 | 105.1 | 4.0 | 3.8 | 2.7 | 2.6 | 2.4 | 2.3 | 1.7 | 1.6 |
| 7 | 150.6 | 5.3 | 3.5 | 2.0 | 1.4 | 2.8 | 1.9 | 4.0 | 2.7 |

60 Pineview Drive ● USA Toll free [phone]  •  tel. [phone]  •  fax [phone]

#### www.immco.com

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Image /page/5/Picture/0 description: The image is a logo for Immco Diagnostics, a Trinity Biotech Company. The logo features a cluster of blue and green dots on the left side, followed by the word "immco" in blue, bold letters. Below "immco" is the word "DIAGNOSTICS" in green, and below that is the text "A Trinity Biotech Company" in a smaller font size.

#### Reproducibility

Qualitative reproducibility was tested with 80 runs of samples range, ~20% below cutoff, ~20% above cutoff, in the moderate positive range of the assay and near the qualitative analysis method. Samples were tested in duplicate twice per day for 20 days be two different operators / equipment sets. Assay results for the cutoff specimen produced 62.5% qualitative (positive) agreement. Assay results for the ~20% above cutoff speciment. All other speciment. All other specimens produced 100% qualitative agreement.

#### Limit of Detection

The limit of detection (LoD) was determined based on 60 replicates of the blank and 10 replicates each of 6 low-level (NHS) samples. LDD was determined to be 3.9 EU/ml.

#### Linearity and Recovery

Linearty and recovery were tested by diluting positive specimens through the assay range in equidistant dilutions and comparing actual vs. expected results. The linear range of the assay was determined be 3.9 (LoD) -160 EU/ml. Results are summarized below:

| Test<br>Range<br>(EU/ml) | Slope<br>(95% CI) | Y-intercept<br>(95% CI) | R2 | % recovery |
|--------------------------|---------------------|-------------------------|--------|-------------|
| 3.2 to 46.0 | 1.05 (0.97 to 1.13) | -0.67 (-2.88 to 1.53) | 0.9942 | 92% to 116% |
| 6.3 to 123.1 | 1.02 (0.95 to 1.10) | -0.18 (-5.64 to 5.29) | 0.9947 | 93% to 104% |
| 4.9 to 188.1 | 1.03 (0.95 to 1.11) | 1.42 (-7.15 to 9.98) | 0.9842 | 87% to 102% |

To assess hook effect, dilutions of high positive specimens with results above the 160 EU/ml measuring range were tested. Hook effect was not demonstrated in dilution samples with theoretical levels of 2531.3 EU/ml for testing within OD range of the microplate reader (~3.500).

Clinical Tests: Sets of clinical samples were tested on the IMMCO Centromere ELISA. By testing 124 IcSSc/CREST samples the sensitivity was determined to be 53.2%. By testing 865 autoimmune and infectious disease controls the specificity was determined to be 95.5%.

Kevin J. Lawson VP Regulatory Affairs

> 60 Pineview Drive Buffalo, NY 14228-2120 Toll free [phone]

> > www.immco.com