This enzyme-linked immunosorbent assay (ELISA) is indicated for the detection and measurement of circulating IgA antibodies to ß₂ Glycoprotein I in human serum. The presence of these antibodies, in combination with clinical observations and other laboratory findings, is intended to aid in the diagnosis of Antiphospholipid Syndrome.

Device Description

An enzyme-linked immunosorbent assay (ELISA) designed for the detection and measurement of IgA antibodies to ß₂ Glycoprotein I in human serum. The ELISA methodology is commonly used for serum antibody evaluations. Purified human ß₂ glycoprotein has been attached to the inner surfaces of the microwell plate. During the initial incubation step, specific antibodies in patient serum will bind to the antigen and are immobilized on the surface. After incubation and a wash step, a peroxidase labeled anti human IgA (α chain specific) second antibody is added to the wells to react with the immobilized anti beta 2 GP1 antibodies. After incubation and another wash step, the substrate is added. In the wells where the specific antigen-antibody-HRP complex remains bound, the peroxidase enzyme catalyzes a color change in the substrate. After the enzymatic reaction is stopped, the colored product is read in an EIA plate reader at a specified wavelength.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the VIRGO® β₂ Glycoprotein I IgA Antibody Kit, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria for the performance studies in terms of specific sensitivity, specificity, or agreement thresholds. Instead, it presents the results of these performance tests as evidence of the device's effectiveness.

Performance Metric	Reported Device Performance (%)
Comparative Study vs. aCL IgA EIA Assay
Relative Sensitivity	92.6% (95% CI: 84.7% to 96.6%)
Relative Specificity	79.2% (95% CI: 69.1% to 86.7%)
Relative Agreement	83.8% (95% CI: 74.2% to 90.3%)

Performance Testing (Clinical Samples)
APS (Primary)	69.8% positive (30/43)
SLE + APS	71.4% positive (5/7)
Total APS Group	70.0% positive (35/50)
SLE (No APS)	35.0% positive (7/20)
Scl-70 (No APS)	5.0% positive (1/20)
Infectious	15.0% positive (12/80)
Normals	5.0% positive (6/120)

2. Sample Sizes and Data Provenance

Test Set Sample Size:
- Performance Testing (Clinical Samples):
  - APS (Primary): 43
  - SLE + APS: 7
  - SLE (No APS): 20
  - Scl-70 (No APS): 20
  - Infectious: 80
  - Normals: 120
  - Total for Clinical Samples: 290
- Comparative Study (vs. aCL IgA):
  - Clinically characterized serum samples: 50
  - Apparently healthy donors: 30
  - Total for Comparative Study: 80
Data Provenance: The document states "clinically characterized serum specimens from individuals diagnosed with Primary APS and other diseases" and "serum samples from apparently healthy donors." It does not specify the country of origin. The data is retrospective, as the samples were "characterized" prior to being tested with the device.

3. Number of Experts and Qualifications

The document does not provide information on the number of experts used to establish the ground truth for the test set or their qualifications. It only states that the samples were "clinically characterized" or from individuals "diagnosed" with specific conditions.

4. Adjudication Method

The document does not mention any adjudication method (e.g., 2+1, 3+1, none) for establishing the ground truth of the test set.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not conducted. This device is an in vitro diagnostic (IVD) assay, not an imaging or diagnostic AI requiring human interpretation. Therefore, the concept of "human readers improve with AI vs. without AI assistance" does not apply.

6. Standalone Performance

Yes, a standalone (algorithm only) performance study was done. The precision studies (Inter Assay and Intra Assay) and the performance testing with clinically characterized serum samples (reporting percentages positive in various patient groups) demonstrate the device's performance in a standalone capacity. The comparative study against a commercial aCL IgA EIA assay also reflects the device's standalone performance relative to another diagnostic method.

7. Type of Ground Truth Used

The ground truth for the clinical samples was based on clinical diagnosis ("individuals diagnosed with Primary APS and other diseases") and status for healthy individuals ("30 serum samples from apparently healthy donors"). The "infectious group consisted of samples with positive syphilis serology."

8. Sample Size for the Training Set

The document does not provide information on the sample size for a training set. This is typical for an IVD device of this nature from this era, where the "training" (development and calibration) would often involve different types of internal analytical studies rather than a distinct, separate "training set" as understood in modern machine learning.

9. How Ground Truth for Training Set Was Established

As no training set is explicitly mentioned or utilized in the sense of a machine learning algorithm, there is no information on how its ground truth was established. The "training" for such an assay typically involves establishing appropriate assay parameters, calibrator values, and cut-offs, often using a combination of analytical studies and preliminary clinical samples. The "Cal Dil" samples and other control materials mentioned in the precision studies would be part of this process.

Summary

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510(k) Summary

K992241

Submitter's Name/Contact Person 1.

loseph M. Califano Director, Regulatory Affairs

Address

Hemagen Diagnostics, Inc. 34-40 Bear Hill Road Waltham, MA, 02154

(781) 890-3766 x 257 Phone: (781) 890-3748 Fax: email: jcalifano@hemagen.com

Date Prepared

14 June 1999

Date Revised

25 August 1999

2-Device Name

Trade Name:	VIRGO® β₂ Glycoprotein I IgA Antibody Kit
Common Name:	β₂ Glycoprotein I Antibodies Test System
Classification Name:	Multiple Autoantibodies Immunological Test System

Predicates 3.

Hemagen ® Cardiolipin Antibody Kit Trade Name: 510 (k) Docket No. K 932373, SE Date; 16 July 1993

Hemagen ® Cardiolipin IgA Calibrator Trade Name: 510 (k) Docket No. K 941840, SE Date; 30 June 1994

The performance of the VIRGO ® B2Glycoprotein I IgA Antibody Kit was evaluated with a panel of characterized serum specimens from individuals diagnosed with Primary APS and other diseases.

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3. Description of Device

An enzyme-linked immunosorbent assay (ELISA) designed for the detection and measurement of IgA antibodies to ß, Glycoprotein I in human serum.

The ELISA methodology is commonly used for serum antibody evaluations. Purified human ß, glycoprotein has been attached to the inner surfaces of the microwell plate. During the initial incubation step, specific antibodies in patient serum will bind to the antigen and are immobilized on the surface. After incubation and a wash step, a peroxidase labeled anti human IgA (α chain specific) second antibody is added to the wells to react with the immobilized anti beta 2 GP1 antibodies. After incubation and another wash step, the substrate is added. In the wells where the specific antigen-antibody-HRP complex remains bound, the peroxidase enzyme catalyzes a color change in the substrate. After the enzymatic reaction is stopped, the colored product is read in an ElA plate reader at a specified wavelength.

4. Intended Use of Device

This enzyme-linked immunosorbent assay (ELISA) is intended for the detection and measurement of IgA antibodies to ß, Glycoprotein I in human serum.

5. Technological Characteristics

Proposed Device

The VIRGO® ® ß, Glycoprotein I IgA Antibody Kit is an enzyme-linked immunosorbent assay. The device utilizes optical density as a measure of antibody presence, with an established cutoff between a positive and a negative reaction. The device also contains a lgA Calibrator to enable the assignment of arbitrary IgA antibody values to patient samples.

Predicate Devices

The Hemagen ® Cardiolipin IgG/IgM Antibody Kit is an enzyme-linked immunosorbent assay. The device utilizes optical density as a measure of antibody presence, with an established cutoff between a positive and a negative reaction. The device also contains both an IgM Calibrator, and an IgG Calibrator to enable the assignment of MPL or GPL unit values to patient samples. The calibrators have been standardized to the IgM and IgG standards obtained from Louisville APL Diagnostics, Inc.

The Hemagen ® Cardiolipin IgA Calibrator is Iyophilized processed human serum that contains cardiolipin antibodies of the IgA class. The calibrator has been standardized to the IgA standards obtained from Louisville APL Diagnostics, Inc. Also provided with the calibrator is a second antibody, goat anti-human IgA, conjugated to the enzyme horseradish peroxidase. The IgA calibrator is intended for use with the Hemagen ® Cardiolipin IgG/IgM Antibody Kit to enable users to measure IgA activity levels in serum samples.

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5. Performance Data

Precision

To estimate the overall precision of the VIRGO ® ß2 Glycoprotein I IgA Antibody Kit, inter, and intra assay studies were conducted. The results of these studies is summarized in the tables below

Inter Assay

Three serum samples, the Negative, and Positive Controls, and the Calibrator were assayed five times each, at least twice a day, on five different days :

	Mean RAU	Std. Deviation	% C.V.
Sample 1	12.9	1.7	13.2
Sample 2	55.1	7.9	14.3
Sample 3	126.0	13.2	10.5
Neg. Control	< 10	N/A	N/A
Pos. Control	62.7	4.9	7.8
Cal Dil 1	153.9	3.3	2.1
Cal Dil 2	80.9	1.2	1.5
Cal Dil 3	40.1	0.3	0.8
Cal Dil 4	20.1	0.2	1.0
Cal Dil 5	10.6	0.4	4.1

Intra Assay

Six serum samples were assayed ten consecutive times in duplicate:

	Mean RAU	Std. Deviation	% C.V.1
Sample 1	13.2	1.8	13.6
Sample 2	13.6	1.3	9.8
Sample 3	24.8	2.4	9.6
Sample 4	53.6	5.3	10.0
Sample 5	57.5	7.7	13.4
Sample 6	116.0	7.5	6.5

These values include the well-to-well variation inherent in the plastic strips, which can range up to 5 %, according to the plastic strip supplier.

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Performance Testing

To demonstrate the effectiveness of the device, a number of clinically characterized serum samples were tested. The results are summarized in the table below:

Patient Group	Number	Number Positive (%)
APS1	43	30 (69.8)
SLE + APS	7	5 (71.4)
TOTAL	50	35 (70.0)
SLE (No APS)	20	7 (35.0)
Scl-70 (No APS)	20	1 (5.0)
Infectious2	80	12 (15.0)
Normals	120	6 (5.0)

Notes

APS - Primary antiphospholipid syndrorne 1 .

The infectious group consisted of samples with positive syphilis serology. 2.

Comparison with aCL IgA

The 50 clinically characterized serum samples and 30 serum samples from apparently healthy donors were evaluated with the VIRGO® By Glycoprotein I IgA Antibody Kit, and a commercially available anti-cardiolipin IgA EIA assay. The results are summarized in the table below:

aCL IgA

		Positive	Negative
IgA β₂ GPI	Positive	25	11
	Negative	2	42
	TOTAL	27	53

Relative Sensitivity:	92.6%: {84.7 to 96.6 %; 0.95 Confidence Interval}
Relative Specificity:	79.2%: {69.1 to 86.7 %; 0.95 Confidence Interval}
Relative Agreement:	83.8%: {74.2 to 90.3 %; 0.95 Confidence Interval}

Conclusion

The results of the both the comparative studies with the predicate device and the performance studies utilizing clinically characterized serum specimens support the claim that the proposed device is capable of effectively detecting IgA antibodies to B, Glycoprotein I in human serum.

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Image /page/4/Picture/0 description: The image is a black and white logo for the Department of Health & Human Services - USA. The logo is circular, with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the top half of the circle. Inside the circle is a stylized image of three human profiles facing to the right, with flowing lines suggesting movement or connection.

DEPARTMENT OF HEALTH & HUMAN SERVICES

AUG 30 1999

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Mr. Joseph M. Califano Director, Regulatory Affairs Hemagen Diagnostics, Inc. 34-40 Bear Hill Road Waltham, Massachusetts 02154

K992241 Re:

Trade Name: VIRGO® ß, Glycoprotein I IgA Antibody Kit Regulatory Class: II Product Code: MSV Dated: June 14, 1999 Received: July 1, 1999

Dear Mr. Califano:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we 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). 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 (Premarket Approval), 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 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Regulation (QS) for Medical Devices: General regulation (21 CFR Part 820) and that, through periodic QS inspections, the Food and Drug Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Register. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770) 488-7655.

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 (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to premarket notification"(21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll-free number (800) 638-2041 or (301) 443-6597, or at its internet address "http://www.fda.gov/cdrh/dsma/dsmamain.html".

Sincerely yours,

Steven Putman

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

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VIRGO ® ß2 Glycoprotein I IgA Antibody Kit Device Name:

Indication(s) For Use

This enzyme-linked immunosorbent assay (ELISA) is indicated for the detection and This enzyme-linked immunosonent assay (CLOSA) indical servan. The measurement of circulating (g) a minodies to py coproleem and other
presence of these antibodies, in combination with clinical observations and other presence of these antibodies, in combination with clinical obber attendent and the managical pictures pholipid Syndrome.

(PLEASE DO NOT WRITE BELOW THIS LINE)
Concurrence of CDRH, Office of Device Evaluation (ODE)

Signature

(Division Sign-Off)
Division of Clinical Laboratory Devices

510(k) Number	K992241
---------------	---------

Prescription Use (Per 21 CFR 801.109)	✓	OR	Over-The-Counter-Use
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Regulation Number and Section

§ 866.5660 Multiple autoantibodies immunological test system.

(a)
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).(b)
Classification. Class II (performance standards).