MPO ELISA TEST SYSTEM
K961765 · Immunoprobe, Inc. · MOB · Aug 19, 1996 · Immunology
Device Facts
| Record ID | K961765 |
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| Device Name | MPO ELISA TEST SYSTEM |
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| Applicant | Immunoprobe, Inc. |
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| Product Code | MOB · Immunology |
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| Decision Date | Aug 19, 1996 |
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| Decision | SESE |
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| Submission Type | Traditional |
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| Regulation | 21 CFR 866.5660 |
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| Device Class | Class 2 |
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Intended Use
The MPO EIA test is an enzyme-linked immunosorbent assay (EIA) for the detection and semi-quantitation of antibodies to myeloperoxidase 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 microscopic polyangiitis. FOR IN VITRO DIAGNOSTIC USE.
Device Story
MPO EIA test is an enzyme-linked immunosorbent assay (EIA) for detecting antibodies to myeloperoxidase (MPO) in human serum. Purified MPO antigen is immobilized on solid-phase microtiter wells. Patient serum is added; if anti-MPO antibodies are present, they bind to the antigen. After washing, an enzyme-labeled anti-human IgG, IgM, and IgA conjugate is added, binding to the captured antibodies. A substrate solution is introduced, causing a color change proportional to the amount of bound antibody. The reaction is stopped, and color intensity is measured photometrically. The device is used in a laboratory setting by trained personnel. Results provide semi-quantitative data to assist clinicians in diagnosing microscopic polyangiitis.
Clinical Evidence
Clinical evaluation compared MPO ELISA to IFA using 233 serum samples (40 microscopic polyangiitis, 40 Wegener's granulomatosis, 153 normals). Relative sensitivity was 95.0% (95% CI: 85.3-100%) and relative specificity was 100% (95% CI: 98.6-100%). Clinical sensitivity for microscopic polyangiitis was 45.0% (95% CI: 29.3-60.7%). Specificity against other autoimmune sera was 100% and against normals was 99.4%. Precision testing showed inter-assay CVs generally <15% for positive samples. Linearity was confirmed via serial dilutions (r > 0.99). No cross-reactivity observed with other autoimmune antigens (SM, RNP, Ro, La, Scl-70, Jo-1, dsDNA).
Technological Characteristics
Enzyme-linked immunosorbent assay (EIA) utilizing purified MPO antigen immobilized on microtiter wells. Detection via enzyme-labeled anti-human IgG, IgM, and IgA conjugate and photometric measurement of substrate color change. Semi-quantitative output. In vitro diagnostic kit format.
Indications for Use
Indicated for detection and semi-quantitation of IgG, IgM, and IgA antibodies to myeloperoxidase in human serum as an aid in the diagnosis of microscopic polyangiitis.
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 (Indirect Immunofluorescence Assay)
Related Devices
- K983390 — IS-ANTI-MPO IGG ELISA TEST SYSTEM · Columbia Bioscience, Inc. · Nov 18, 1998
- K974166 — WIELISA MPO ANCA TEST SYSTEM · Wieslab AB · Feb 17, 1998
- K041040 — VARELISA MPO ANCA, MODEL 17648/17696 · Sweden Diagnostics (Germany) GmbH · Jun 16, 2004
- K051455 — WIESLAB CAP MPO-ANCA · Eurodiagnostica · Nov 23, 2005
- K091860 — AESKULISA MPO, MODEL 30-7303US · Aesku Diagnostics · Feb 23, 2010
Submission Summary (Full Text)
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AUG 19 1996
K961765
# Summary of Safety and Effectiveness Information
## MPO EIA Test Kit
I. Immuno Probe Inc.
1306 Bailes Lane, Suite F
Frederick, Maryland 21701
Contact person: William Boteler
Telephone: 301-695-7920
Date of preparation: June 28, 1996
II. Description of Device
The MPO EIA test is an enzyme-linked immunosorbent assay (EIA) for the detection and semi-quantitation of antibodies to myeloperoxidase 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 microscopic polyangiitis. FOR IN VITRO DIAGNOSTIC USE.
The MPO EIA test is an enzyme linked immunosorbent assay to detect IgG, IgM, and IgA antibodies to myeloperoxidase. Purified MPO 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 MPO EIA 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 MPO 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 MPO.
Table 1 Sensitivity and Specificity of the MPO ELISA Kit Relative to IFA
| | MPO ELISA | | | |
| --- | --- | --- | --- | --- |
| | Positive
Index ≥ 1.10 | Equivocal
0.91-1.09 | Negative
≤ 90 | Total |
| IFA | Positive 19* | 0 | 1** | 20 |
| | Negative 0 | 0 | 213*** | 213 |
| | Total 19 | 0 | 214 | 233 |
| Relative Sensitivity = 19/20 = 95.0% | | 95% Confidence interval = 85.3% - 100% | | |
| Relative Specificity = 213/213 = 100% | | 95% Confidence interval = 98.6% - 100% | | |
| Relative Agreement= 232/233 = 99.6% | | 95% Confidence interval = 98.7% - 100% | | |
The 95% confidence intervals were calculated using the normal method.
The 95% confidence interval for Specificity was calculated assuming one false positive
* Thirteen sera were patients diagnosed with microscopic polyangiitis with a p-ANCA pattern. One sera was from a patient diagnosed with Wegeners granulomatosis with a p-ANCA pattern. One sera was from a patient diagnosed with Wegeners granulomatosis with a c-ANCA pattern. Four sera were patients diagnosed with microscopic polyangiitis, that were positive for ANA thus making the p-ANCA pattern impossible to read.
**One serum was from a patient diagnosed with microscopic polyangiitis with a p-ANCA pattern.
***Sixty serum were from patients diagnosed with microscopic polyangiitis or Wegeners granulomatosis that had c-ANCA patterns or negative for ANCA. One hundred fifty three serum were from normals 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 MPO ELISA and ELISA
| | | MPO ELISA | | | |
| --- | --- | --- | --- | --- | --- |
| | | Positive | Equivocal | Negative | Total |
| | Index | ≥1.10 | 0.91-1.09 | ≤ 90 | |
| | Positive | 6 | 0 | 1** | 7 |
| Alternate ELISA | Equivocal | 4 | 0 | 7 | 11 |
| | Negative | 11* | 0 | 204 | 215 |
| | Total | 21 | 0 | 212 | 233 |
| Relative Sensitivity = 6/7 = 85.7% | | | 95% Confidence interval = 59.3% - 100% | | |
| Relative Specificity = 204/215 = 94.9% | | | 95% Confidence interval = 91.9% - 97.9% | | |
| Relative Agreement= 210/222 = 94.6% | | | 95% Confidence interval = 91.6% - 97.6% | | |
The 95% confidence intervals were calculated using the normal method.
* Eight serum were from patients diagnosed with Microscopic polyangiitis. Two serum were from patients diagnosed with Wegeners Granulomatosis. One serum was a normal.
** The serum was a normal.
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The clinical sera and the potentially cross-reactive sera were grouped and the clinical sensitivity and specificity of the MPO ELISA assay was calculated. The data in Table 3 summarizes the data.
Table 3 Clinical Sensitivity and Specificity of MPO ELISA
| MPO ELISA | | | | |
| --- | --- | --- | --- | --- |
| Index | Positive ≥ 1.10 | Equivocal 0.91-1.09 | Negative ≤ 90 | Total |
| Microscopic polyangiitis | 18 | 0 | 22 | 40 |
| Wegener's granulomatosis | 2 | 0 | 38 | 40 |
| Other autoimmune sera | 0 | 0 | 21 | 21 |
| Normals | 1 | 0 | 152 | 153 |
| Total | 20 | 0 | 233 | 254 |
Clinical Sensitivity Microscopic polyangiitis = 18/40 = 45.0%
95% confidence interval = 29.3 - 60.7%
Clinical Specificity Wegener's granulomatosis = 38/40 = 95.0%
95% confidence interval = 88.1 - 100%
Clinical Specificity Other autoimmune sera = 21/21 = 100%
95% confidence interval = 85.9 - 100%
Clinical Specificity Normals = 152/153 = 99.4%
95% confidence interval = 98.0 - 100%
The 95% confidence intervals were calculated using the normal method.
The 95% confidence intervals for the clinical specificity for other autoimmune sera were calculated assuming one false positive.
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# 2. Precision
The precision of the MPO kit was determined by testing nine different sera eleven 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
| | Assay 1 (n=11) | | | | Assay 2 (n=11) | | | Assay 3 (n=11) | | | Inter Assay(n=33) | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | X | S.D. | C.V. | X. | S.D. | C.V. | X | S.D. | C.V. | X | S.D. | C.V. | |
| 1 | 4.60 | 0.137 | 2.99% | 4.39 | 0.222 | 5.06% | 4.53 | 0.347 | 7.67% | 4.5 | 0.258 | 5.73% | |
| 2 | 5.13 | 0.136 | 2.66% | 4.97 | 0.236 | 4.74% | 5.12 | 0.432 | 8.44% | 5.08 | 0.295 | 5.81% | |
| 3 | 3.53 | 0.227 | 6.43% | 3.21 | 0.217 | 6.75% | 3.32 | 0.192 | 5.79% | 3.36 | 0.248 | 7.38% | |
| 4 | 2.76 | 0.218 | 7.90% | 2.51 | 0.338 | 13.45% | 2.74 | 0.282 | 10.29% | 2.67 | 0.297 | 11.13% | |
| 5 | 0.41 | 0.079 | 19.19% | 0.37 | 0.067 | 17.92% | 0.44 | 0.070 | 15.93% | 0.41 | 0.076 | 18.58% | |
| 6 | 0.04 | 0.021 | 46.53% | 0.05 | 0.017 | 35.78% | 0.04 | 0.024 | 59.49% | 0.04 | 0.020 | 46.08% | |
| 7 | 0.05 | 0.17 | 36.14% | 0.05 | 0.018 | 38.94% | 0.05 | 0.028 | 60.79% | 0.05 | 0.021 | 45.24% | |
| 8 | 0.92 | 0.049 | 5.32% | 0.84 | 0.054 | 6.42% | 0.88 | 0.060 | 6.85% | 0.88 | 0.063 | 7.14% | |
| 9 | 1.08 | 0.064 | 5.95% | 0.94 | 0.055 | 5.91% | 0.97 | 0.04 | 4.10% | 0.97 | 0.094 | 9.67% | |
X = Mean MPO Value
S.D. = Standard Deviation
C.V. = Coefficient of Variation
# 3. Linearity
The MPO index values were determined for serial twofold dilutions of five positive sera. The index values were compared to $\log_2$ of dilution by standard linear regression. The data in table #5 indicates that the assay is semi-quantitative.
## Table 5 Linearity
| Serum | Neat | 1:2 | 1:4 | 1:8 | 1:16 | 1:32 | 1:64 | r |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 3.03 | 2.43 | 1.77 | 1.12 | 0.51 | | | 0.999 |
| 2 | 3.57 | 2.67 | 1.83 | 1.05 | 0.52 | | | 0.997 |
| 3 | 2.93 | 2.21 | 1.57 | 0.99 | 0.65 | | | 0.994 |
| 4 | 3.72 | 3.07 | 2.51 | 1.74 | 1.06 | 0.61 | | 0.998 |
| 5 | 4.32 | 3.97 | 3.52 | 2.82 | 2.06 | 1.31 | 0.77 | 0.992 |
Linear regression compared MPO ISR to $\log_2$ of dilution.
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# 4. Cross Reactive Data
Sera containing high level of antibodies to potentially cross reactive antigens were assayed on the MPO ELISA kit. The data in table 6 indicate that antibodies to alternate autoimmune antigens do not cross react with the MPO ELISA kit.
Table 6 Cross Reactive Data
| Serum # | Antibody Specificity | MPO Index Value | Interpretation |
| --- | --- | --- | --- |
| 1. | SM | 0 15 | - |
| 2. | SM | 0 36 | - |
| 3. | SM | 0 23 | - |
| 4. | RNP | 0 10 | - |
| 5. | RNP | 0 17 | - |
| 6. | RNP | 0 09 | - |
| 7. | Ro | 0 12 | - |
| 8. | Ro | 0 07 | - |
| 9. | Ro | 0 09 | - |
| 10. | La | 0 07 | - |
| 11. | La | 0 07 | - |
| 12. | La | 0 06 | - |
| 13. | Scl-70 | 0 10 | - |
| 14. | Scl-70 | 0 12 | - |
| 15. | Scl-70 | 0 08 | - |
| 16. | Jo-1 | 0 09 | - |
| 17. | Jo-1 | 0 10 | - |
| 18. | Jo-1 | 0 10 | - |
| 19. | dsDNA | 0 22 | - |
| 20. | dsDNA | 0 08 | - |
| 21. | dsDNA | 0 27 | - |
