DIAMEDIX IS-ANTI-CARDIOLIPIN IGA TEST SYSTEM

K012450 · Diamedix Corp. · MID · Oct 26, 2001 · Immunology

Device Facts

• Record ID: K012450
• Device Name: DIAMEDIX IS-ANTI-CARDIOLIPIN IGA TEST SYSTEM
• Applicant: Diamedix Corp.
• Product Code: MID · Immunology
• Decision Date: Oct 26, 2001
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5660
• Device Class: Class 2

Intended Use

The Diamedix Is anti-Cardiolipin IgA Test Kit is an indirect enzyme immunoassay (EIA) for the semi-quantitative measurement of IgA antibodies to cardiolipin in human serum as an aid in the assessment of the risk of thrombosis in patients with SLE or SLE-like disorders. The test can be used either manually or in conjunction with the MAGO® Plus Automated EIA Processor.

Device Story

The Is anti-Cardiolipin IgA Test System is an indirect solid-phase enzyme-linked immunosorbent assay (ELISA). It utilizes plastic microwells coated with purified cardiolipin saturated with human β2-Glycoprotein I. Patient serum samples, calibrators, and controls are added to the wells; IgA antibodies bind to the cardiolipin. An anti-human IgA horseradish peroxidase conjugate is added, followed by a substrate solution. The enzyme converts the substrate to a blue product, which turns yellow upon acid addition. The final product is measured spectrophotometrically at 450 nm (reference 600-630 nm). The optical density is directly proportional to the concentration of cardiolipin IgA antibodies. The device is used in clinical laboratories, either manually or with the MAGO® Plus Automated EIA Processor. Results assist clinicians in assessing thrombosis risk in patients with SLE or SLE-like disorders.

Clinical Evidence

Clinical performance was evaluated using 354 frozen, clinically characterized sera (214 normal, 57 APS, 33 SLE, 35 other autoimmune, 15 RPR positive). Analytical sensitivity (limit of detection) is 0.2 APL U/ml. Relative sensitivity vs. a comparative EIA was 85.7% (95% CI 75.3-92.9%) and relative specificity was 99.3% (95% CI 96.2-100.0%). Precision studies (intra-assay and inter-assay) were conducted manually and on the MAGO Plus system.

Technological Characteristics

Indirect solid-phase ELISA; plastic microwells coated with purified cardiolipin and human β2-Glycoprotein I; horseradish peroxidase conjugate; spectrophotometric detection at 450 nm. Supports manual or automated (MAGO Plus) processing. 3-point or 6-point calibration.

Indications for Use

Indicated for semi-quantitative measurement of IgA antibodies to cardiolipin in human serum to aid in assessing thrombosis risk in patients with SLE or SLE-like disorders.

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

• Orgentec Anti-cardiolipin IgA ELISA Assay

Submission Summary (Full Text)

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## OCT 2 6 2001

K012450

## 510(k) Summary of Safety and Effectiveness

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is: K012450

#### Applicant Information:

| Date Prepared: | October 17, 2001 |
|----------------|-----------------------------------------|
| Name: | Diamedix Corporation |
| Address: | 2140 N. Miami Avenue<br>Miami, FL 33127 |

| Contact Person: | Dr. Lynne Stirling |
|-----------------|--------------------|
| Phone Number: | 305-324-2354 |
| Fax Number: | 305-324-2388 |

#### Device Information:

| Trade Name: | Is anti-Cardiolipin IgA Test System |
|----------------------|-------------------------------------------|
| Common Name: | Anti-Cardiolipin ELISA test |
| Classification Name: | Anticardiolipin immunological test system |

#### Equivalent Device:

Orgentec Anti-cardiolipin IgA ELISA Assay

Device Description: The Is anti-Cardiolipin IgA Test System is an enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative measurement of IgA antibodies to cardiolipin in human serum

Intended Use: The assay is intended for the semi-quantitative measurement of IgA antibodies to cardiolipin in human serum. The results of the assay can be used as an aid in the assessment of the risk of thrombosis in patients with SLE or SLE-like disorders.

#### Principle of the Procedure:

The Is-anti-Cardiolipin IgA Test System is an indirect solid-phase enzyme immunoassay. Highly purified cardiolipin is coated onto plastic microwells and saturated with highly purified human 32-Glycoprotein I. Calibrators, controls and diluted patient samples are added to the wells. Any IgA antibodies in the patient sample bind to the well. Anti-human IgA horseradish peroxidase conjugate is then added After incubation and washing, a substrate solution is then added to each well. In the presence of bound enzyme, the substrate is converted to a blue colored product. After acid additon to stop the reaction, a yellow end product is formed that is read spectrophotometrically at 450 nm (reference 600-630 nm) and is directly proportional to the concentration of cardiolipin IgA antibodies in the patient sample.

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# SUMMARY OF SAFETY AND EFFECTIVENESS

## Performance Characteristics

All non-clinical studies were performed using the manual method and 6-point calibration unless otherwise indicated.

#### A. 3-point vs 6-point calibration

To demonstrate the equivalence of both calibration methods, the results of 190 samples tested using the Is-anti-Cardiolipin IgA Test Kit using either the 3-point callbration systems were subjected to linear regression analysis. A scattergram and regression line of the results obtained with 95% confidence intervals is shown in FIGURE 1. Also included are the regression statistics.

Image /page/1/Figure/6 description: The image is a scatter plot titled "FIGURE 1: Is anti-Cardiolipin IgA 3-point vs 6-point Result Correlation". The x-axis is labeled "Is-anti-Cardiolipin IgA - 6-point calibration", and the y-axis is labeled "Is-anti-Cardiolipin IgA- 3-point calibration". The plot shows a positive correlation between the two variables. The equation of the line of best fit is Y = 1.3963 + 0.8754 X.

| Intercept | 1.3963 (95% CI 0.8699 to 1.9226) |
|------------------------------|----------------------------------|
| Slope | 0.8754 (95% CI 0.8523 to 0.8985) |
| Sample Size | 190 |
| Coefficient of determination | = 0.9675 |
| Correlation coefficient r | = 0.9836 |
| 95% CI for r | = 0.9782 to 0.9877 |

### B. Relative Sensitivity and Specificity

Two hundred and thirty-seven frozen retrospective sera were tested for IgA antibodies using the Is-anti-Cardiolipin IgA Test Kit and a commercially available ELISA kit for detecting cardiolipin IgA antibodies. Based on the results of this testing the relative sensitivity, relative specificity and overall agreement were calculated. The results obtained are shown in TABLE 1. Further resolution of the discordant samples showed that of the ten samples that were negative in the Is anti-Cardiolipin IgA and positive by the other EIA, five were negative and five were positive by a referee ElA method. The sample that was positive in the Is-anti-Cardiolipin IgA and negative in the other EIA was positive by the referee method.

| | | Positive | Negative | *Equivocal |
|------------------------------------------------------------------------------------------------------------------|------------|----------|-----------------------------------------------------|------------|
| Other<br>EIA | Positive | 60 | 10 | 6 |
| | Negative | 1 | 142 | 3 |
| | *Equivocal | 6 | 7 | 2 |
| Relative Sensitivity 60/70 = 85.7 %<br>Relative Specificity 142/143 = 99.3%<br>Overall Agreement 214/216 = 94.8% | | | **95% CI<br>75.3-92.9%<br>96.2-100.0%<br>90.9-97.4% | |

| TABLE 1 |
|-------------------------|
| Is-anti-Cardiolipin IgA |

* Equivocal results were excluded from calculations.

** 95% Confidence Intervals (CI) calculated by the Exact Method.

NOTE : Please be advised that 'relative' refers to the assay's results to that of a similar assay. There was not an attempt to correlate the assay's results with disease presence. No judgement can be made on the comparison's accuracy to predict disease.

Linear regression analysis and a scattergram for the correlation study with the comparative method is shown in FIGURE 2.

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Image /page/2/Figure/0 description: The image is a scatter plot titled "FIGURE 2: Is anti-Cardiolipin IgA Correlation with Comparative Method". The x-axis is labeled "Is-anti-Cardiolipin IgA APL U/ml", and the y-axis is labeled "Comparative Method APL U/ml". The scatter plot shows a positive correlation between the two variables. A line of best fit is drawn through the data, and the equation of the line is given as Y = 0.9002 + 1.0293 X.

(95% Cl -0.0163 to 1.8167) 0.9002 Intercept 1.0293 (95% Cl 0.9923 to 1.0662) Slope Sample Size 237 Coefficient of determination = 0.9277 Correlation coefficient r = 0.9632 95% Cl for r = 0.9527 to 0.9714

#### C. Clinical Sensitivity and Specificity

A total of three hundred and fifty-four frozen retrospective, clinically characterized sera were assayed using the Is anti-Cardiolipin IgA Test Kit in order to assess both the clinical specificity of the assay system. These samples consisted of 214 normal sera, 57 sera from patients with diagnosed antiphospholipid syndrome (APS), 33 sera from patients with systemic lupus erythematosus (SLE), 35 sera from patients with other autoimmune diseases such as Sjogren's Syndrome, scleroderma, polymyositis and theumatoid arthritis and 15 samples from patients with positive RPR titers. Results are summarized in TABLE 2.

Note that the analytical sensitivity, or limit of detection, calculated by assaying Standard A 20 times and taking the mean of these values plus 2 Standard Deviations was determined as being 0.2 APL U/ml.

| Patient Group | Total | Positive | Negative | Equivocal |
|--------------------------------------|-------|-----------------|----------|-----------|
| Normals | 214 | 3 | 210 | 1 |
| APS | 57 | 25 | 31 | 1 |
| SLE | 33 | 12 | 20 | 1 |
| Other Autoimmune<br>Diseases | 35 | 3 | 32 | 0 |
| RPR Positive | 15 | 3 | 12 | 0 |
| Clinical Specificity: # Neg/Total # | | | | |
| Normals | | 210/214 = 98.1% | | |
| RPR Positive | | 12/15 = 80.0% | | |
| Clinical Sensitivity : # Pos/Total # | | | | |

| 1<br>16<br>( | 11 | | |
|--------------|----|--|--|
|--------------|----|--|--|

| Normals | | 210/214 = 98.1% |
|------------------------------|---------------|-----------------|
| RPR Positive | | 12/15 = 80.0% |
| Clinical Sensitivity : | # Pos/Total # | |
| APS | 25/57 | = 43.8% |
| SLE | 12/33 | = 36.4% |
| Other Autoimmune<br>Diseases | 3/35 | = 8.6% |

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#### D. Cross Reactivity

To assess the potential for positive results due to cross reactive antibodies, 36 samples which were reactive to t various autoantibodies (SSA/SSB, Scl-70, Jo-1, dsDNA and RF) were tested using the Is-anti-Cardiolipin In the Ma Test Kit. One sample positive for Jo-1 antibodies and one sample positive for dsDNA were positive in the Isanti-Cardiolipin IgA test. The remaining 34 samples were negative.

#### E. Linearity

To assess the linearity of the Is-anti-Cardiolipin IgA Test Kit several highly positive samples were settally diluted 10 assess the inteanty of the 18-and-eachers and ever and ever regression graph and scattergram with 95% confidence intervals is presented in FIGURE 3.

Image /page/3/Figure/4 description: The image is a graph titled "FIGURE 3: Is anti-Cardiolipin IgA Linearity". The graph shows the relationship between dilution and concentration of APL U/ml. The x-axis represents dilution, ranging from 0.0 to 1.0, while the y-axis represents concentration of APL U/ml, ranging from -20 to 120. The graph includes a line of best fit, represented by the equation Y = -2.7529 + 112.1109 X.

Image /page/3/Picture/5 description: The image shows statistical data including the slope, intercept, coefficient of determination, correlation coefficient, and confidence interval. The slope is 112.11092 and the intercept is -2.75287. The coefficient of determination is 0.9912, the correlation coefficient is 0.9956, and the 95% confidence interval for r is 0.9691 to 0.9994.

## F. Correlation of Manual and MAGO Plus results

The Is-anti-Cardiolipin IgA Test Kit has been developed for automated as well as manual use. To denomate the rivelence of the manual and MAGO Plus procedures, the results of 190 serum samples tested for anti-Cardiolipin IgA antibodies by both the manual and automated methods were provents of more have a treatived in the disconsideres sion lines of the results obtained with 95% confidence intervals are shown in FIGURE 4. The data indicate good correlation with a Correlation Coefficient (r) of 0.9688. Results obtained using either 3 Standards or 6 Standards were comparable.

Image /page/3/Figure/8 description: The image is a scatter plot titled "FIGURE 4: Is anti-Cardiolipin IgA Manual vs MAGO Plus Correlation". The x-axis is labeled "MANUAL APL U/ml", and the y-axis is labeled "MAGO Plus APL U/ml". The plot shows a positive correlation between the two variables, with a regression line of Y = 1.9220 + 1.0807 X. The image also includes statistical information such as the intercept, slope, sample size, coefficient of determination, correlation coefficient, and 95% confidence intervals.

With the 3-point calibration, linear regression showed (automated) = 1.2914 (manual) + 0.1773; r = 0.9794. 95% CI for slope and intercept are 1.2532 to 1.3297 and -0.6184 to 0.9730 respectively.

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## G. Precision

To assess the precision of the Is anti-Cardiolipin IgA Test Kit six serum samples of varying reactivity were ested in triplicate in three separate runs. Precision was assessed both man ally and using the MAGO Plus Automated EIA triplicate in the separate runs. Freelers was assesses of are shown in TABLES 3 and 4.

| SERUM | INTRA-ASSAY DAY 1 | | | INTRA-ASSAY DAY 2 | | | INTRA-ASSAY DAY 3 | | | INTERASSAY (n=9) | | |
|-------|-------------------|-------|-------|-------------------|------|-------|-------------------|------|-------|------------------|-------|-------|
| | MEAN<br>APL | SD | CV% | MEAN<br>APL | SD | CV% | MEAN<br>APL | SD | CV% | MEAN<br>APL | SD | CV% |
| A | 0.9 | 0.06 | 6.19 | 1.8 | 0.12 | 6.54 | 1.2 | 0.17 | 14.43 | 1.3 | 0.38 | 29.54 |
| B | 1.0 | 0.06 | 5.97 | 1.7 | 0.17 | 10.19 | 1.1 | 0.17 | 15.75 | 1.3 | 0.36 | 27.78 |
| C | 12.0 | 0.96 | 8.03 | 16.1 | 0.12 | 0.72 | 11.4 | 1.06 | 9.28 | 13.1 | 2.32 | 17.70 |
| D | 33.0 | 0.86 | 2.62 | 31.4 | 1.44 | 4.57 | 33.0 | 1.89 | 5.71 | 32.5 | 1.49 | 4.57 |
| E | 39.3 | 2.06 | 5.25 | 35.9 | 1.51 | 4.22 | 38.4 | 1.99 | 5.17 | 37.9 | 2.20 | 5.86 |
| F | 83.0 | 18.34 | 22.08 | 94.9 | 5.65 | 5.95 | 77.6 | 7.55 | 9.73 | 85.2 | 12.84 | 15.08 |

TABLE 3 : Manual Intra-Assay and Interassay Precision for Is-anti-Cardiolipin IgA

TABLE 4 : MAGO Plus Intra-Assay and Interassay Precision for Is-anti-Cardiolipin IgA

| SERUM | INTRA-ASSAY DAY 1 | | | INTRA-ASSAY DAY 2 | | | INTRA-ASSAY DAY 3 | | | INTERASSAY (n=9) | | |
|-------|-------------------|-------|-------|-------------------|-------|-------|-------------------|------|-------|------------------|-------|-------|
| | MEAN<br>APL | SD | CV% | MEAN<br>APL | SD | CV% | MEAN<br>APL | SD | CV% | MEAN<br>APL | SD | CV% |
| A | 1.6 | 0.17 | 10.83 | 2.2 | 0.25 | 11.27 | 2.0 | 0.49 | 24.26 | 2.0 | 0.40 | 20.63 |
| B | 1.5 | 0.06 | 3.94 | 1.8 | 0.06 | 3.27 | 1.8 | 0.12 | 6.54 | 1.7 | 0.17 | 9.95 |
| C | 15.1 | 0.51 | 3.41 | 16.5 | 0.93 | 5.64 | 18.1 | 1.88 | 10.39 | 16.5 | 1.69 | 10.22 |
| D | 43.6 | 1.50 | 3.45 | 41.1 | 2.54 | 6.18 | 50.9 | 2.75 | 5.41 | 45.2 | 4.86 | 10.75 |
| E | 38.3 | 4.37 | 11.41 | 46.1 | 3.12 | 6.77 | 54.2 | 3.10 | 5.71 | 46.2 | 7.58 | 16.40 |
| F | 89.8 | 22.48 | 25.04 | 82.9 | 12.78 | 15.42 | 103.8 | 8.90 | 8.58 | 92.2 | 16.50 | 17.91 |

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# Expected Values

The prevalence of anti-cardiolipin IgA antibodies may vary depending on a number of factors with as age, gender, geographical location, race, type of test used and clinical history of individual patients. Anitibution to anti-cardiolipin are generally absent, or have a very low incidence, in the normal health population. Increased incidence can occur in the elderly population. A published study has shown a prevalence of 12% in the elderly population (mean age of 70 years) as opposed to 2% for a younger population. In addition, In the elderly population (incan age of 10 yours) as opposes and were also positive for antinuclear antibodies (13).

In the present study, the expected values for a normal, healthy population were assessed by testing sera from m the prosent study, and exponed varies in the Is-anti-Cardiolipin IgA Test Kit. One hundred and forty-five sera (97.97%) were negative for IgA antibodies, two sera (1.35%) were positive and one serum (0.67%) were equivocal. The age distribution and antibody prevalence for this population are shown in TABLE 5.

The expected values for a clinical population were assessed by testing fifty-seven sera from patients with a diagnosis of anti-phospholipid syndrome (APS) in the Is-anti-Cardiolipin IgA Test Kit. Twenty-five diagliosis of allu-phospionpus syndrone (1118) in the 13 to 13 to 15%) were equivocal for IgA antibodies.

Histograms showing the distribution of values for these normal and clinical populations are shown in FIGURES 5 and 6.

| | Number of Donors | Prevalence |
|-------------------------|---------------------|------------|
| Total Number | 148 | |
| Geographic<br>Location: | South Florida : 148 | 2.03% |
| Age | | |
| 10-19 | 7 | 0.0% |
| 20-29 | 36 | 0.0% |
| 30-39 | 73 | 2.7% |
| 40-49 | 22 | 0.0% |
| 50-59 | 8 | 0.0% |
| 60-69 | 2 | 0.0% |

#### TABLE 5: Age Distribution and Prevalence of anti-Cardiolipin IgA in a Normal S. Florida Population

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FIGURE 5 Distribution of anti-Cardiolipin IgA in a Normal Population

Image /page/6/Figure/1 description: This image is a histogram showing the frequency of APL U/ml values. The x-axis represents APL U/ml, ranging from 0 to 16, while the y-axis represents the frequency, ranging from 0 to 80. The histogram shows a high frequency of values between 0 and 4, with the highest frequency between 0 and 2, and a decreasing frequency as APL U/ml increases.

FIGURE 6 Distribution of anti-Cardiolipin IgA in a Clinical Population

Image /page/6/Figure/3 description: The image is a histogram showing the frequency of APL U/ml. The x-axis represents APL U/ml, ranging from 0 to 160, while the y-axis represents frequency, ranging from 0 to 35. The histogram shows a high frequency of APL U/ml values between 0 and 20, with the frequency decreasing as APL U/ml increases. There are also some small peaks at around 100 and 140 APL U/ml.

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Image /page/7/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized eagle with three tail feathers, representing the department's commitment to health, human services, and well-being. The eagle is encircled by the words "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" in a circular arrangement.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

### Lynne Stirling, Ph.D. Vice President, Regulatory Affairs Diamedix Corporation 2140 North Miami Avenue Miami, Florida 33127

Re: K012450

Trade/Device Name: Diamedix Is-anti-Cardiolipin IgA Test System Regulation Number: 21 CFR § 866.5660 Regulation Name: Multiple Autoantibodies Immunological Test System Regulatory Class: Class II Product Code: MID Dated: September 28, 2001 Received: October 1, 2001

OCT 2 6 2001

Dear Dr. Stirling:

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.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such 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 Part 801); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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#### Page 2

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at [phone]. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at [phone]. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers International and Consumer Assistance at its toll-free number [phone] or [phone] or at its internet address "http://www.fda.gov/cdrh/dsma/dsmamain.html".

Sincerely yours,

Steven Butman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

11 - 1 - 1 - 1 - 1 - 1 - 1

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## Appendix G. Indications for Use Statement

## INDICATIONS FOR USE STATEMENT

510(K) NUMBER : ______________________________________________________________________________________________________________________________________________________________

# DEVICE NAME : Is anti-Cardiolipin IgA Test System

Indications for Use : The Diamedix Is anti-Cardiolipin IgA Test Kit is an indications for USB ? The Eldi (EIA) for the semi-quantitative measureindirect enzyme inimanouceaj (&#%) .
ment of İgA antibodies to cardiolipin in human serum as an aid in the ment of igA antibodios to samebosis in patient with SLE or SLE-like assessment of the next of them be used either manually or in conjunction with the MAGO® Plus Automated EIA Processor.

Sonsan Satterlee
(Division Sign-Off)

**Division of Clinical Laboratory Devices**

510(k) Number K012450

for Prescription Use ✓