PR-3 ELISA TEST SYSTEM

K961764 · Immunoprobe, Inc. · MOB · Aug 19, 1996 · Immunology

Device Facts

• Record ID: K961764
• Device Name: PR-3 ELISA TEST SYSTEM
• Applicant: Immunoprobe, Inc.
• Product Code: MOB · Immunology
• Decision Date: Aug 19, 1996
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5660
• Device Class: Class 2

Intended Use

The PR-3 ELISA test is an enzyme-linked immunosorbent assay (ELISA) for the detection and semi-quantitation of antibodies to Proteinase-3 in human sera. The assay is to be used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid to the diagnosis of Wegener’s granulomatosis. FOR IN VITRO DIAGNOSTIC USE.

Device Story

PR-3 ELISA test detects IgG, IgM, and IgA antibodies to Proteinase-3 in human serum; utilizes microtiter wells coated with purified PR-3 antigen; patient serum added to wells; bound antibodies detected via enzyme-labeled anti-human IgG, M, A conjugate; substrate addition triggers color change proportional to antibody concentration; color intensity measured photometrically; provides indirect semi-quantitation of specific antibodies; used in clinical laboratory settings; results aid physicians in diagnosing Wegener’s granulomatosis.

Clinical Evidence

Clinical evaluation compared PR-3 ELISA to IFA and an alternate ELISA using 256 samples (40 Wegener's, 40 microscopic polyangiitis, 21 other autoimmune, 155 normals). Relative sensitivity vs IFA was 98.2% (95% CI: 94.5-100%); relative specificity was 95.6% (95% CI: 92.5-98.6%). Clinical sensitivity for Wegener's was 97.5% and for microscopic polyangiitis was 55.0%. Clinical specificity for other autoimmune sera and normals was 100%. Precision testing showed CVs < 15%. Linearity confirmed via serial dilutions (r > 0.98). No cross-reactivity observed with other autoimmune antigens (Ro, La, Sm, RNP, Jo-1, Scl-70, dsDNA).

Technological Characteristics

Enzyme-linked immunosorbent assay (ELISA); solid phase microtiter well coated with purified PR-3 antigen; photometric measurement of colorimetric substrate reaction; semi-quantitative output; manual or automated plate reader processing.

Indications for Use

Indicated for detection and semi-quantitation of IgG, IgM, and IgA antibodies to Proteinase-3 in human serum specimens to aid in the diagnosis of Wegener’s granulomatosis.

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

• IFA

Submission Summary (Full Text)

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K961764

AUG 19 1996

Summary of Safety and Effectiveness Information

PR-3 ELISA Test Kit

I. Immuno Probe Inc.
1306 Bailes Lane, Suite F
Frederick, Maryland 21701
Contact person: William Boteler
Telephone: [phone]
Date of preparation: June 28, 1996

II. Description of Device

The PR-3 ELISA test is an enzyme-linked immunosorbent assay (ELISA) for the detection and semi-quantitation of antibodies to Proteinase-3 in human sera. The assay is to be used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid to the diagnosis of Wegener’s granulomatosis. FOR IN VITRO DIAGNOSTIC USE.

The PR-3 ELISA test is an enzyme linked immunosorbent assay to detect IgG, IgM, and IgA antibodies to Proteinase-3. Purified PR-3 is attached to a solid phase microtiter well. Diluted test sera is added to each well. If the antibodies are present that recognize the antigen they will bind to the antigen in the well. After incubation the wells are washed to remove unbound antibody. An enzyme labeled anti-human IgG, M, A is added to each well. If antibody is present it will bind to the antibody attached to the antigen on the well. After incubation the wells are washed to remove unbound conjugate. A substrate solution is added to each well. If enzyme is present the substrate will undergo a color change. After an incubation period the reaction is stopped and the color intensity is measured photometrically, producing an indirect measurement of specific antibody in the patient specimen.

III. Predicate Device

The PR-3 ELISA test is substantially equivalent to IFA. Equivalence is demonstrated by the following comparative results:

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# Performance Characteristics

1. Sensitivity and Specificity - The PR-3 ELISA kit was evaluated relative to IFA for ANCA. Forty sera were from patients diagnosed with Wegener's granulomatosis. Forty sera were from patients diagnosed with microscopic polyangiitis. One hundred and fifty five sera were from normals with various ages, gender, and geographical areas. The data in Table 1 summarizes the data. Note: the sensitivity and specificity relative to IFA will not be as high if random IFA positive sera are selected due to other disease states causing ANCA patterns not associated with PR-3.

Table 1 Sensitivity and Specificity of the PR-3 ELISA Kit Relative to IFA

| Wampole PR-3 ELISA | | | | | |
| --- | --- | --- | --- | --- | --- |
| | Index | Positive ≥ 1.10 | Equivocal 0.91-1.00 | Negative ≤ 90 | Total |
| IFA | Positive | 53* | 0 | 1*** | 54 |
| | Negative | 8** | 0 | 173*** | 181 |
| ) | Total | 61 | 0 | 174 | 235 |

Relative Sensitivity = 53/54 = 98.2% 95% confidence interval = 94.5 - 100%
Relative Specificity = 173/181 = 95.6% 95% confidence interval = 92.5 - 98.6%
Relative Agreement = 226/235 = 96.2% 95% confidence interval = 93.7 - 98.7%

The 95% confidence intervals were calculated using the normal method.

*Fifty sera were from patients diagnosed with Wegeners granulomatosis or microscopic polyangiitis with a c-ANCA pattern. Two sera were from patients diagnosed with microscopic polyangiitis with a p-ANCA pattern. One sera was from a patient diagnosed with microscopic polyangiitis with ANA thus making the c-ANCA pattern impossible to read.

** Eight sera were from patients diagnosed with Wegeners granulomatosis or microscopic polyangiitis that were negative for ANCA

***One serum was from a patient diagnosed with microscopic polyangiitis with a c-ANCA pattern.

***One hundred and fifty five serum were from normals that were negative for ANCA. Eighteen sera were from patients diagnosed with microscopic polyangiitis with either a p-ANCA pattern or ANA or negative for ANCA.

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The same group of clinical sera were tested on an legally marketed ELISA device to determine the relative sensitivity and specificity to an alternate ELISA. The data in Table 2 summarizes the data.

Table 2 Comparison of PR-3 ELISA and ELISA

| | PR-3 ELISA | | | |
| --- | --- | --- | --- | --- |
| | Positive
Index ≥ 1.10 | Equivocal
0.91-1.00 | Negative
≤ 90 | Total |
| Positive | 58 | 0 | 5** | 63 |
| Alternate Equivocal ELISA | 1 | 0 | 11 | 12 |
| Negative | 2* | 0 | 158 | 160 |
| Total | 61 | 0 | 174 | 235 |

Relative Sensitivity = 58/63 = 92.1% 95% confidence interval = 85.3 - 98.9%
Relative Specificity = 158/160 = 98.8% 95% confidence interval = 97.0 - 1.00%
Relative Agreement = 216/223 = 98.9% 95% confidence interval = 94.5 - 99.2%

The 95% confidence intervals were calculated using the normal method.

* Both serum were from patients diagnosed with Microscopic Polyangiitis
** All five sera were from normals

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The clinical sera and the potentially cross-reactive sera were grouped and the clinical sensitivity and specificity of the PR-3 ELISA assay was calculated. The data in Table 3 summarizes the data.

Table 3 Clinical Sensitivity and Specificity of PR-3 ELISA

| PR-3 ELISA | | | | |
| --- | --- | --- | --- | --- |
| | Positive Index ≥ 1.10 | Equivocal 0.91-1.00 | Negative ≤ 90 | Total |
| Wegener's granulomatosis | 39 | 0 | 1 | 40 |
| Microscopic polyangiitis | 22 | 0 | 18 | 40 |
| Other autoimmune sera | 0 | 0 | 21 | 21 |
| Normals | 0 | 0 | 155 | 155 |
| Total | 61 | 0 | 195 | 256 |

Clinical Sensitivity Wegener's granulomatosis = 39/40 = 97.5%
95% confidence interval = 92.6 - 100%
Clinical Sensitivity Microscopic polyangiitis = 22/40 = 55.0%
95% confidence interval = 39.3 - 70.7%
Clinical Specificity Other autoimmune sera = 21/21 = 100%
95% confidence interval = 85.9 - 100%
Clinical Specificity Normals = 155/155 = 100%
95% confidence interval = 98.1-100%

The 95% confidence intervals were calculated using the normal method.
The 95% confidence intervals for the clinical specificities were calculated assuming one false positive.

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## 2. Precision

The precision of the PR-3 kit was determined by testing nine different sera ten times each on three different assays. The data is summarized in Table 4. With proper technique the user should obtain C.V.'s of less than 15%.

### Table 4

| Serum# | Assay 1 (n=10) | | | Assay 2 (n=10) | | | Assay 3 (n=10) | | | Inter assay (n=30) | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | X | S.D. | C.V. | X | S.D. | C.V. | X | S.D. | C.V. | X | S.D. | C.V. |
| 1 | 8.70 | 0.634 | 7.29% | 8.92 | 0.479 | 5.37% | 9.37 | 0.465 | 4.96% | 9.00 | 0.586 | 6.52% |
| 2 | 2.99 | 0.345 | 11.54% | 3.03 | 0.211 | 6.96% | 2.92 | 0.201 | 6.89% | 2.98 | 0.256 | 8.59% |
| 3 | 2.68 | 0.289 | 10.81 | 2.76 | 0.145 | 5.25% | 2.70 | 0.144 | 5.32% | 2.71 | 0.200 | 7.38% |
| 4 | 2.52 | 0.177 | 7.04% | 2.52 | 0.163 | 6.47% | 2.53 | 0.242 | 9.54% | 2.52 | 0.190 | 7.54% |
| 5 | 10.33 | 0.344 | 3.34% | 11.95 | 0.293 | 2.45% | 10.29 | 0.200 | 1.95% | 10.86 | 0.837 | 7.71% |
| 6 | 0.17 | 0.074 | 42.79% | 0.15 | 0.056 | 37.79% | 0.010 | 0.076 | 80.36% | 0.14 | 0.074 | 53.76% |
| 7 | 0.08 | 0.037 | 47.16% | 0.06 | 0.039 | 68.22% | 0.04 | 0.037 | 84.50% | 0.06 | 0.039 | 65.51% |
| 8 | 1.28 | 0.079 | 6.13% | 1.31 | 0.071 | 5.37% | 1.21 | 0.079 | 6.53% | 1.27 | 0.086 | 6.74% |
| 9 | 1.08 | 0.059 | 5.45% | 1.18 | 0.105 | 8.92% | 1.06 | 0.083 | 7.91% | 1.10 | 0.097 | 8.82% |

## 3. Linearity

The ISR values were determined for serial twofold dilutions of five positive sera. The ISR values were compared to log2 of dilution by standard linear regression. The data in Table 5 indicates that the assay has a linear relationship with serum dilution.

### Table 5

| Serum | Neat | 1:2 | 1:4 | 1:8 | 1:16 | 1:32 | 1:64 | r |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 7.50 | 6.04 | 4.17 | 2.61 | 1.26 | 0.63 | | 0.991 |
| 2 | 2.96 | 2.09 | 1.47 | 0.97 | 0.37 | | | 0.995 |
| 3 | 8.44 | 6.98 | 5.22 | 3.66 | 2.36 | 1.37 | 0.60 | 0.992 |
| 4 | 2.24 | 1.88 | 1.23 | 0.63 | | | | 0.993 |
| 5 | 3.24 | 1.94 | 0.98 | 0.43 | | | | 0.984 |

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# 4. Cross reactivity.

Sera containing antibodies to potentially cross reactive antigens were assayed on the PR-3 ELISA. The data in Table 6 indicate that antibodies to alternate autoimmune antigens do not cross react with the PR-3 ELISA kit.

Table 6

| Serum # | Antibody Specificity | PR-3 Index Value | Interpretation |
| --- | --- | --- | --- |
| 1. | Ro | 0.09 | - |
| 2. | Ro | 0.11 | - |
| 3. | Ro | 0.14 | - |
| 4. | La | 0.07 | - |
| 5. | La | 0.05 | - |
| 6. | La | 0.08 | - |
| 7. | Sm | 0.24 | - |
| 8. | Sm | 0.30 | - |
| 9. | Sm | 0.36 | - |
| 10. | RNP | 0.14 | - |
| 11. | RNP | 0.09 | - |
| 12. | RNP | 0.16 | - |
| 13. | Jo-1 | 0.05 | - |
| 14. | Jo-1 | 0.20 | - |
| 15. | Jo-1 | 0.12 | - |
| 16. | Scl-70 | 0.13 | - |
| 17. | Scl-70 | 0.07 | - |
| 18. | Scl-70 | 0.11 | - |
| 19. | dsDNA | 0.38 | - |
| 20. | dsDNA | 0.24 | - |
| 21. | dsDNA | 0.36 | - |