An enzyme-linked immunosorbent assay (ELISA) designed for the detection and measurement of circulating antibodies to native DNA. These autoantibodies are often associated with systemic lupus erythematosus (SLE) and give positive results in screening tests for antinuclear antibodies.

Device Description

An enzyme-linked immunosorbent assay (ELISA) designed for the detection and measurement of antibodies to native DNA in human serum.

The ELISA methodology is commonly used for serum antibody evaluations. Purified native DNA has been attached to the inner surfaces of the microwell plate. During the initial incubation step, antibodies in patient serum bind specifically to the immobilized antigen and remain in place after a wash step.

A second antibody which is conjugated to horseradish peroxidase (HRP) is used to recognize the "heavy + light" chain regions of the patient's DNA antibodies that remain after the wash step. In the wells where the second antibody remains bound. the conjugated HRP catalyzes a color change in the substrate. After the reaction is stopped, the color is read in an EIA Plate reader.

AI/ML Overview

Acceptance Criteria and Study Details for Hemagen® DNA Kit (K962276)

Based on the provided text, the Hemagen® DNA Kit is an enzyme-linked immunosorbent assay (ELISA) designed for the detection and measurement of antibodies to native DNA in human serum, primarily associated with systemic lupus erythematosus (SLE).

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria for this device are not explicitly stated in numerical thresholds but are implied through "comparative studies" demonstrating "substantial equivalence" to the predicate device. The performance data provided focuses on precision and comparative analytical performance.

Acceptance Criteria Category	Specific Criteria (Implied)	Reported Device Performance
Precision (Reproducibility)	Low variability in repeated measurements (not quantified).	Inter-assay (%CV): Range 6.2-12.4% for IU/mL, 7.4-12.3% for O.D. (n=8 samples, 50 readings each).Intra-assay (%CV): Range 2.6-17.0% for IU/mL, 2.2-13.9% for O.D. (n=8 samples, 20 consecutive readings each).
Correlation with WHO Standard	High degree of correlation with the WHO Anti-Double Stranded DNA 1st International Standard.	A study was conducted to demonstrate a high degree of correlation between kit calibrators and the W.H.O. Standard (specific coefficients or metrics are not provided).
Analytical Sensitivity (vs. Predicate)	100% agreement with predicate for positive disease samples.	100% (23/23) for disease state patients (N=100) after adjudication.
Analytical Specificity (vs. Predicate)	High agreement with predicate for negative disease samples.	97.4% (75/77) for disease state patients (N=100) after adjudication.
Agreement with Predicate (Normal Donors)	100% agreement with predicate for healthy normal samples.	100% (40/40) for normal blood donors (N=40).
Interfering Substances	Less than 15% variation from certain levels of interferents.	Variation <15% for: Hemoglobin (≤ 500 mg/dL), Lipid (≤ 3000 mg/dL), Bilirubin (≤ 20 mg/dL).
Prozone Effect	No unexpectedly low values with high-titered samples.	Kit gives appropriately high positive results with high-titered sera.
Substantial Equivalence	Overall performance deemed equivalent to predicate device.	"The results of the comparative studies support the claim that the Hemagen dsDNA Kit is substantially equivalent to the predicate device."

2. Sample Size and Data Provenance for Test Set

Sample Size:
- Disease state patients: 100 serum specimens.
- Apparently healthy blood donors: 40 serum specimens.
- Total Test Set: 140 serum specimens.
Data Provenance: The text states "serum specimens from different patients with known or suspected autoimmune disease" and "apparently healthy blood donors." The country of origin is not specified, but the use of "patient" and "blood donors" suggests these were clinical samples. The study appears to be retrospective as the samples are described as "from different patients with known or suspected autoimmune disease" indicating prior diagnosis or clinical status.

3. Number of Experts and Qualifications for Ground Truth (Test Set)

The text does not explicitly state the number of experts used to establish the ground truth for the test set or their specific qualifications.
However, the predicate device (Hemagen VIRGO® Anti-nDNA IgG IFA Kit) was used as the basis for comparison, implying that the ground truth for the test set was established by the results from this predicate device and clinical information ("known or suspected autoimmune disease").

4. Adjudication Method for the Test Set

An adjudication method was used for discrepant samples between the proposed device and the predicate.
Method: "The nine discrepant samples were evaluated by hemagglutination for resolution." This suggests a third, independent method was used to arbitrate differences, effectively acting as a form of "2+1" adjudication for the discrepant samples.

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

No, a MRMC comparative effectiveness study was not done.
This device is an automated in vitro diagnostic (ELISA) kit, not a device requiring human interpretation of images or complex data where reader variability would be a primary concern for comparative effectiveness. The comparison is between two assay methods and their accuracy against clinical status/predicate.

6. Standalone Performance Study

Yes, a standalone study was performed in terms of analytical performance. The "Proposed Device" (Hemagen dsDNA Kit) was evaluated for its precision (inter-assay and intra-assay reproducibility), correlation with the WHO standard, and its performance against interfering substances and prozone effect. These are intrinsic performance characteristics of the algorithm/device itself.
The comparative testing against the predicate device also serves as a standalone performance evaluation for the proposed device's diagnostic accuracy (sensitivity and specificity) relative to an established method.

7. Type of Ground Truth Used (Test Set)

The ground truth for the test set was primarily based on the results of the predicate device (Hemagen VIRGO® Anti-nDNA IgG IFA Kit) combined with the clinical status of the patients ("known or suspected autoimmune disease") or healthy status ("apparently healthy blood donors").
For discrepant samples, hemagglutination was used as an additional, independent method to resolve the ground truth.

8. Sample Size for the Training Set

The document does not explicitly provide information on a separate "training set" for the device. As an ELISA kit, its performance is based on the inherent biochemical properties and cutoff values established during its development. These types of devices typically undergo extensive analytical validation to establish parameters like cutoff points, but this process might not be explicitly described as "training" in the same way an AI model would have a training set.
The data presented (precision, WHO correlation, interfering substances, prozone) are generally part of analytical validation studies, not necessarily "training" data as understood for machine learning.

9. How Ground Truth for the Training Set Was Established

Given that no explicit "training set" is mentioned in the context of an algorithm or machine learning, the concept of "ground truth for the training set" as it applies to AI/ML is not directly applicable here.
For an ELISA kit, the establishment of cutoff values and optimal assay parameters would have occurred during the development phase. This process would involve testing numerous known positive and negative samples, likely characterized by clinical diagnosis, other established laboratory tests, and potentially reference materials (like the WHO standard), to determine the concentration or optical density thresholds that best differentiate between positive and negative results. However, the document does not detail this specific process or the sample sizes involved in establishing these parameters.

Summary

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K962276

510(k) Summary

1. Submitter's Name/Contact Person

DEC - 5 1996

Joseph M. Califano, Manager, Regulatory Affairs

Address

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

Phone: (617) 890-3766 (617) 890-3748 Fax:

14 June 1996 Date Prepared:

2. Device Name

Trade Name: Hemagen ® DNA Kit (EIA method) Common Name: Anti-DNA Classification Name: Anti-DNA Antibody, Antigen and Control

3. Predicate Device

Hemagen VIRGO ® Anti-nDNA IgG IFA Kit {Reference 510 (k) No. K 771376A}

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3. Description of Device

An enzyme-linked immunosorbent assay (ELISA) designed for the detection and measurement of antibodies to native DNA in human serum.

4. Intended Use of Device

5.(A) Technological Characteristics

Proposed Device

The Hemagen dsDNA Kit is an enzyme-linked immunosorbent assay. The device utilizes optical density as a measure of antibody presence, with an established cutoff point between a positive and a negative reaction.

Predicate Device

The Hemagen VIRGO Anti-DNA IgG {heavy + light} IFA Kit is an indirect fluorescent antibody assay. The device utilizes the indirect method of fluorescent antibody staining. The resultant level of observed fluorescence is used to determine the presence or absence of antibodies.

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5.(B) Performance Data

Precision .

To evaluate precision, inter-assay and intra-assay studies were conducted.

A. Inter-assay reproducibility {Between-run}

Eight different serum samples were assayed five times each, twice a day, on five different days (a total of 50 readings)

Sample	Mean IU/mL	Std. Dev.	% CV	Mean O.D.	Std. Dev.	% CV
1	102.1	11.3	11.1	0.617	0.05	8.3
2	102.4	6.4	6.2	0.620	0.05	7.4
3	90.1	6.5	7.2	0.553	0.04	7.7
4	55.3	5.2	9.4	0.356	0.04	9.9
5	< 50	N/A	N/A	0.213	0.03	12.4
6	< 50	N/A	N/A	0.264	0.03	9.8
7	122.7	13.8	11.3	0.731	0.09	12.3
8	142.6	9.2	6.4	0.840	0.07	8.3

B. Intra-assay reproducibility {Within-run}

Eight different samples were assayed 20 consecutive times in a single run:

Sample	Mean IU/mL	Std. Dev.	% CV	Mean O.D.	Std. Dev.	% CV
1	105.2	17.9	17.0	0.604	0.08	13.9
2	97.2	2.8	2.8	0.567	0.01	2.2
3	84.9	2.2	2.6	0.505	0.01	2.2
4	54.3	1.7	2.6	0.355	0.01	2.4
5	< 50	N/A	N/A	0.199	0.02	9.0
6	< 50	N/A	N/A	0.240	0.01	5.5
7	115.4	5.6	4.8	0.688	0.03	4.2
8	129.4	3.8	2.9	0.765	0.02	2.6

11. Verification of the DNA Calibrators

The kit calibrators have been compared to the World Health Organization Standard for Anti-Double Stranded DNA (ANTI-dsDNA) 1st International Standard, Code Wo / 80 A study was conducted to demonstrate the high degree of correlation that exists between the kit calibrators and the W.H.O. Standard.

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Comparison Testing III.

The Hemagen dsDNA Kit and the Hemagen VIRGO Anti-nDNA IgG IFA Kit were used to assay 100 serum specimens from different patients with known or suspected autoimmune disease. Forty (40) specimens from apparently healthy blood donors were also assayed with the Hemagen dsDNA Kit and the Hemagen VIRGO Anti-nDNA IgG IFA Kit.

Summary Tables

Table A: Summary of disease state patients, N = 100 {{nitial testing}

Predicate Device
	Positive	Negative	TOTAL
Proposed Device
Positive	23	21	25
Negative	71	68	75
Totals	30	70	100

The nine discrepant samples were evaluated by hemagglutination for resolution

Table B; Summary of disease state patients, N =100 {Final results}

Predicate Device

	Positive	Negative	TOTAL
Proposed DevicePositive	23	2	25
Negative	0	75	75
Totals	23	77	100

The relative analytical sensitivity was found to be 100 % (23/23). The relative analytical specificity was found to be 97.4 % (75/77).

Table C: Normal blood donors, N = 40

Predicate Device

Proposed Device	Negative	TOTAL
Positive	0	0
Negative	40	40
Totals	40	40

Ill.

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IV. Interfering Substances

Lipemic, hemolytic, and icteric samples were evaluated with the assay in accordance with NCCLS Document EP7-P Proposed Guideline " Interference Testing in Clinical Chemistry." The results indicate that there is no significant effect (<15 % variation) on the assay for samples with:

Hemoglobin concentration:	≤ 500 mg/dL
Lipid concentration:	≤ 3000 mg/dL
Bilirubin concentration:	≤ 20 mg/dL

V. Prozone

The Hemagen dsDNA Kit was used to assay a high titered serum sample to determine if the kit would return unexpectedly low values. The results of this evaluation indicate that the kit gives appropriately high positive results with high titered sera.

Conclusion 6.

The results of the comparative studies support the claim that the Hemagen dsDNA Kit is substantially equivalent to the predicate device.

Regulation Number and Section

§ 866.5100 Antinuclear antibody immunological test system.

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