IMMULISA ANCA SCREEN ELISA
Device Facts
| Record ID | K092601 |
|---|---|
| Device Name | IMMULISA ANCA SCREEN ELISA |
| Applicant | Immco Diagnostics, Inc. |
| Product Code | MOB · Immunology |
| Decision Date | Oct 7, 2010 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5660 |
| Device Class | Class 2 |
Intended Use
An enzyme linked immunosorbent assay (ELISA) for the qualitative detection of anti-neutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 (PR3) and myeloperoxidase (MPO) in human serum to aid in the diagnosis of small vessel vasculitis in conjunction with other laboratory and clinical findings.
Device Story
ImmuLisa™ ANCA ELISA is a solid-phase immunoassay for qualitative detection of human IgG antibodies to MPO and PR3 in serum. Device components include microwell strips coated with MPO and PR3, calibrators, HRP-conjugated anti-human IgG, TMB substrate, and controls. Operation involves incubating patient serum in antigen-coated wells; washing to remove unbound proteins; adding HRP-conjugate; washing again; and adding TMB substrate. Color intensity, proportional to antibody concentration, is measured via spectrophotometer at 450 nm. Used in clinical laboratories by trained personnel. Results aid diagnosis of small vessel vasculitis when interpreted alongside clinical and laboratory findings. Benefits include standardized, objective detection of specific ANCA autoantibodies.
Clinical Evidence
Clinical study evaluated 272 samples (confirmed IFA+) including disease groups (glomerulonephritis, Wegener's granulomatosis) and controls (non-ANCA vasculitis, autoimmune disorders). Clinical sensitivity was 98.7% (95% CI 92.2-99.9%) and clinical specificity was 96.4% (95% CI 92.4-98.4%). Analytical performance included precision (total imprecision 5.9-12.2% CV), LoD (3.3 EU/ml), and interference testing for hemoglobin, bilirubin, RF, and triglycerides, showing no significant interference.
Technological Characteristics
Solid-phase ELISA; microwells coated with purified MPO and PR3 antigens. Uses HRP-conjugated anti-human IgG and TMB substrate. Requires microplate reader (450 nm/600-650 nm reference). Reagents include calibrators, controls, serum diluent, wash buffer, and sulfuric acid stop solution. Standardized via relative arbitrary units (EU/ml). Performance validated per CLSI guidelines (EP5-A2, EP7-A2, EP9-A2, EP12-A2, EP17-A).
Indications for Use
Indicated for the qualitative detection of anti-neutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 (PR3) and myeloperoxidase (MPO) in human serum to aid in the diagnosis of small vessel vasculitis in patients suspected of having such conditions. For prescription use only.
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
- Inova Quanta Lite MPO Antibody ELISA (k981330)
- Inova Quanta Lite PR3 Antibody ELISA (k091328)
Related Devices
- K041040 — VARELISA MPO ANCA, MODEL 17648/17696 · Sweden Diagnostics (Germany) GmbH · Jun 16, 2004
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k092601
B. Purpose for Submission:
New Device
C. Measurand:
Antineutrophil Cytoplasmic Antibodies (ANCA)
D. Type of Test:
ELISA (Qualitative)
E. Applicant:
Immco Diagnostics, Inc.
F. Proprietary and Established Names:
ImmuLisa™ ANCA ELISA for PR3 and MPO Antibodies
G. Regulatory Information:
1. Regulation section:
21 CFR § 866.5660, Multiple Autoantibodies Immunological Test System
2. Classification:
Class II
3. Product codes:
MOB, Test System, Antineutrophil Cytoplasmic Antibodies (ANCA)
4. Panel:
Immunology (82)
H. Intended Use:
1. Intended use(s):
An enzyme linked immunosorbent assay (ELISA) for the qualitative detection of anti-neutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 (PR3) and myeloperoxidase (MPO) in human serum to aid in the diagnosis of small vessel vasculitis in conjunction with other laboratory and clinical findings.
2. Indication(s) for use:
Same as intended use
3. Special conditions for use statement(s):
For prescription use only
4. Special instrument requirements:
Microplate reader capable of measuring OD at 450 and a reference wavelength of 600-650 nm.
I. Device Description:
Each device contains the following: microwell strips (12x8) coated with MPO and PR3, one calibrators (30 EU/ml), HRP goat anti-human IgG conjugate, TMB enzyme substrate, positive control, negative control, serum diluent, wash buffer and sulfuric acid stop solution. All reagents are ready to use except for the wash buffer which requires reconstitution.
J. Substantial Equivalence Information:
1. Predicate device name(s):
Inova Quanta Lite MPO Antibody ELISA
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Inova Quanta Lite PR3 Antibody ELISA
2. Predicate K number(s):
k981330 and k091328
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | ImmuLisa ANCA ELISA for PR3 and MPO | Inova Quanta Lite MPO and PR3 ELISA |
| Intended use | An enzyme linked immunosorbent assay (ELISA) for the qualitative detection of anti-neutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 (PR3) and myeloperoxidase (MPO) in human serum | Same |
| Methodology | ELISA | Same |
| Analyte detected | Human IgG antibodies to MPO and PR3 | Same |
| Component set | Includes positive control, negative control, calibrators, conjugate, substrate, diluent, wash buffer, stop solution, microplate | Same |
| Conjugate antibody | HRP | Same |
| Specimen type | Serum | Same |
| Substrate/chromogen | TMB | Same |
| Positive control | MPO and PR3 IgG antibodies | Same |
| Stop solution | H2SO4 | Same |
| Assay dilution | 1:101 | Same |
| Signal detection | 450nm on spectrophotometer | Same |
| Storage | 2-8°C | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | ImmuLisa ANCA ELISA for PR3 and MPO | Inova Quanta Lite MPO and PR3 ELISA |
| Assay type | Qualitative | Semi-Quantitative |
| Capture antigen | MPO and PR3 | MPO or PR3 |
| Cut-off | 20 EU/ml | 20 EU/ml |
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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | <20 EU/ml is negative
≥20 EU/ml is positive | |
| Wash buffer | Powdered or liquid concentrate | Liquid concentrate |
| Positive control | Acceptance range printed on vial | No value/range assigned |
| Calibrators | Single; value in units 30 EU/ml | Single; value in units 25 EU/ml |
| Limit of detection | 3.3 EU/ml | Not specified |
K. Standard/Guidance Document Referenced (if applicable):
CLSI EP5-A2, EP7-A2, EP9-A2, EP12-A2, and EP17-A
L. Test Principle:
The test is performed as a solid phase immunoassay. Microwells are coated with purified MPO and PR3 antigen followed by a blocking step to reduce non-specific binding during the assay run. Controls, calibrators and patient sera are incubated in the antigen coated wells to allow specific antibodies present in the serum to bind to the MPO and PR3 antigen. Unbound antibodies and other serum proteins are removed by washing the microwells. Bound antibodies are detected by adding an enzyme labeled anti-human IgG conjugate to the microwells. Unbound conjugate is removed by washing. Specific enzyme substrate (TMB) is then added to the wells and the presence of antibodies is detected by a color change produced by the conversion of TMB substrate to a colored reaction product. The reaction is stopped and the intensity of the color change, which is proportional to the concentration of antibody, is ready by a spectrophotometer at 450 nm. Results are expressed in ELISA Units per milliliter (EU/ml) and reported as positive or negative.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Per CLSI EP5-A2, precision of the assay was tested with five specimens ranging in values from 10.43 to 87.21 EU/ml. The percent total imprecision ranged from 5.9-12.2%. Averaged results are shown in table below. Multiple studies were conducted. Assay runs of three replicates of five specimens were conducted using three different lots (n=45). Separately, assay runs of three replicates of five specimens were conducted over five days, twice a day (n=135). Lastly, assay runs of six replicates of five specimens were conducted. Repeatability was determined with 28 replicates of each of the five specimens. Results are also shown in the table below (last column). Percent CVs ranged from 4.1-9.5%. Sponsor's pre-defined acceptance criteria for precision was 20%.
| | | Total Imprecision | | Between days | | Within run
(Repeatability) | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Mean | SD | | SD | | SD | |
| Sample | (IU/ml) | (IU/ml) | CV% | (IU/ml) | CV% | (IU/ml) | CV% |
| 1 | 10.43 | 1.274 | 12.2% | 1.442 | 13.8% | 0.988 | 9.5% |
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| 2 | 16.03 | 1.211 | 7.6% | 1.335 | 8.5% | 0.868 | 5.3% |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 3 | 22.58 | 1.540 | 6.8% | 1.669 | 7.4% | 1.346 | 6.0% |
| 4 | 25.29 | 1.502 | 5.9% | 1.735 | 6.8% | 1.039 | 4.1% |
| 5 | 87.21 | 5.163 | 5.9% | 5.806 | 6.6% | 4.103 | 4.7% |
b. Linearity/assay reportable range:
Not applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
International reference materials for anti-MPO or anti-PR3 antibodies are not available. The assay is calibrated in relative arbitrary units (EU/ml).
Stability
Accelerated, real-time and open kit/reagent stability studies were conducted as part of design control to assign expiration dating to components and as part of ongoing quality control/quality assurance analysis. Accelerated and open kit studies were performed on three lots of components/reagents. They included materials incubated at 37°C where one day is considered to equivalent to one month stored at 2-8°C. Materials are removed from the incubator for testing at three day intervals for a minimum of 21 days. For open kit stability studies, materials are opened as required for bench-top usage, then assayed at 15, 45, and 90 day intervals. Based on these studies, the expiration date for this assay is 18 months. Real time stability studies are ongoing.
Positive control and calibrator were derived from the sera of subjects with autoimmune vasculitides obtained from a commercial source. For assignment of values, the samples were tested at various dilutions on at least two different lots of MPO and PR3 antigen coated plates.
d. Detection limit:
Per CLSI EP17-A, the limit of detection (LoD) for this assay was determined to be 3.3 EU/ml based on 60 replicates of the blank and 10 replicates of each of the six low-level samples. The limit of blank is 2.9 EU/ml.
e. Analytical specificity:
Interfering Substances:
Per CLSI EP7-A2, interference was studied by mixing sera with known ANCA levels with potentially interfering serum samples and analyzing deviation from expected results. Interference was calculated as follows: 1 - (obtained/expected)%. Results are presented in table below. No significant interference was demonstrated for the following substances at the levels indicated: hemoglobin range of interference -5.2-7.3% (2 g/L), bilirubin range of interference -13.9-2% (342 umol/L), rheumatoid factor range of interference 1.5-10.2% (100 EU/ml), and triglycerides range of interference -9.1-7.7% (37 mmol/L). Interference study with triglycerides was conducted separately. The sponsor's acceptance criteria for interference was set at less than 20% for negative samples and less than 15% for positive samples. Testing of grossly hemolyzed or lypemic samples is not recommended as
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stated in the package insert.
| Sample | | Hemoglobin | | Bilirubin | | RF | | Sample | | Triglycerides | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | EU/ml | EU/ml | % Int | EU/ml | % Int | EU/ml | % Int | | EU/ml | EU/ml | % Int |
| 1 | 13.7 | 14.7 | 7.3 | 11.8 | -13.9 | 15.1 | 10.2 | 1 | 11 | 12 | -9.1 |
| 2 | 19.4 | 18.4 | -5.2 | 16.8 | -13.4 | 19.7 | 1.5 | 2 | 18.1 | 16.8 | 7.7 |
| 3 | 24.8 | 25.1 | 1.2 | 22.7 | -8.5 | 27.2 | 9.7 | 3 | 21.4 | 22.8 | -6.1 |
| 4 | 62.1 | 63.8 | 2.7 | 58.3 | -6.1 | 67.4 | 8.5 | 4 | 46.2 | 48.3 | -4.3 |
| 5 | 92.1 | 92.6 | 0.5 | 89.4 | -2.9 | 95.6 | 3.8 | 5 | 63.8 | 64.3 | -0.8 |
| 6 | 145.2 | 151.7 | 4.5 | 148.1 | 2 | 158.2 | 9 | 6 | 117.7 | 113.5 | 3.7 |
# Cross-Reactivity:
A total of 96 potentially cross-reactive specimens from individuals with other autoimmune disorders or positive for other autoantibodies were tested for ANCA using the ImmuLisa ANCA ELISA for PR3 and MPO. ANCA may occur in patients with various connective tissue disorders. Inflammatory bowel disease samples were also included. The results are presented in the table below. Overall cross-reactivity studies are within acceptance criteria (set by sponsor at $10\%$ ).
| Condition | n | n Positive |
| --- | --- | --- |
| Non-ANCA associated vasculitis | 24 | 1 |
| Celiac disease | 8 | 0 |
| Crohn's disease | 10 | 0 |
| Ulcerative colitis | 6 | 0 |
| Hashimoto's thyroiditis | 8 | 0 |
| Rheumatoid arthritis | 8 | 1 |
| ENA* positive | | |
| La Ab | 8 | 1 |
| Ro Ab | 8 | 1 |
| Sm Ab | 8 | 1 |
| RNP Ab | 8 | 0 |
| Total | 96 | 5 (5.2%) |
* Antibodies to Extractable Nuclear Antigens
# Hook effect:
Not applicable
# f. Assay cut-off:
A normal range study was conducted using 64 normal human sera and 16 diseased controls on the assay. These samples were obtained from commercial sources. Based on ROC analysis, the mean plus 2.5 standard deviation of these values was established as the cut-off between normal and abnormal results at 0.367 OD. This value was assigned to $20\mathrm{EU / ml}$
# 2. Comparison studies:
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a. Method comparison with predicate device:
Per CLSI EP9-A2, the ImmuLisa ANCA ELISA for PR3 and MPO was tested in comparison with Inova Quanta Lite MPO ELISA and Inova Quanta Lite PR3 ELISA separately, using well-characterized sera of ANCA antibody positive subjects (88 ANCA positive), disease controls (56) and healthy individuals (54). Disease controls included Ceiliac Disease, ENA positive collagen vascular autoimmunity, Hashimoto's, and RA. The results are shown in the following 3 tables. 1) Table of IMMCO ANCA ELISA compared to Inova PR3 ELISA alone; 2) IMMCO ANCA ELISA compared to Inova MPO ELISA alone; and 3) Combined data from above two method comparison studies. These are results when the indeterminate range of the predicate assays were considered positive.
| Indeterminate as positive | | | | |
| --- | --- | --- | --- | --- |
| | | | Inova PR3 Ab | |
| | | Pos | Neg | Total |
| IMMCO | Pos | 37 | 41 | 78 |
| ANCA | Neg | 1 | 119 | 120 |
| ELISA | Total | 38 | 160 | 198 |
| | | | | |
| Positive. % Agreement | | 97.4% | (95% CI 84.6% to 99.9%) | |
| Negative. % Agreement | | 74.4% | (95% CI 66.8% to 80.8%) | |
| Overall % Agreement | | 78.8% | (95% CI 72.3% to 84.1%) | |
| Indeterminate as positive | | | | |
| --- | --- | --- | --- | --- |
| | | | Inova MPO Ab | |
| | | Pos | Neg | Total |
| IMMCO | Pos | 35 | 43 | 78 |
| ANCA | Neg | 0 | 120 | 120 |
| ELISA | Total | 35 | 163 | 198 |
| | | | | |
| Positive. % Agreement | | 100.0% | (95% CI 87.7% to 100%) | |
| Negative. % Agreement | | 73.6% | (95% CI 66.0% to 80.1%) | |
| Overall % Agreement | | 78.3% | (95% CI 71.8% to 83.7%) | |
These values are calculated by combining data from above two tables
| Indeterminate as positive | | | | |
| --- | --- | --- | --- | --- |
| | | Inova MPO or | Inova MPO and | |
| | | PR3 Ab Pos | PR3 Ab Neg | Total |
| IMMCO | Pos | 72 | 6 | 78 |
| ANCA | Neg | 1 | 119 | 120 |
| ELISA | Total | 73 | 125 | 198 |
| | | | | |
| Positive. % Agreement | | 98.6% | (95% CI 91.6% to 99.9%) | |
| Negative. % Agreement | | 95.2% | (95% CI 89.4% to 98.5%) | |
| Overall % Agreement | | 96.5% | (95% CI 92.6% to 98.4%) | |
These are results for the combined predicate assays (MPO and PR3) when the
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indeterminate range of the predicate assays were considered negative.
| Borderline as negative | | | | |
| --- | --- | --- | --- | --- |
| | | Inova MPO or | Inova MPO and | |
| | | PR3 Ab Pos | PR3 Ab Neg | Total |
| IMMCO | Pos | 67 | 1 | 68 |
| ANCA | Neg | 6 | 124 | 130 |
| ELISA | Total | 73 | 125 | 198 |
| | | | | |
| Positive. % Agreement | | 91.8% | (95% CI 82.4% to 96.6%) | |
| Negative. % Agreement | | 99.2% | (95% CI 95.0% to 99.9%) | |
| Overall % Agreement | | 96.5% | (95% CI 92.6% to 98.4%) | |
b. Matrix comparison:
Not applicable
3. Clinical studies:
a. Clinical sensitivity and specificity:
A set of 272 clinical samples from various disease groups (see table below for breakdown of actual disease groups) confirmed IFA+ were tested with the ImmunoLisa ANCA ELISA. Other autoimmune disease samples include Celiac Disease and Hashimoto's Thyroiditis. Results are presented in table below. The calculated clinical sensitivity of the assay is 98.7% (95% CI 92.2-99.9%). The calculated clinical specificity of the assay is 96.4% (95% CI 92.4-98.4%).
| ANCA ELISA | Positive > Cutoff 20 EU/ml | | | |
| --- | --- | --- | --- | --- |
| | | | Disease Status | |
| | | Pos | Neg | Total |
| | Pos | 78 | 7 | 85 |
| IMMCO | Neg | 1 | 186 | 187 |
| | Total | 79 | 193 | 272 |
| | | | | |
| Sensitivity | | 98.7% | (95% CI 92.2% to 99.9%) | |
| Specificity | | 96.4% | (95% CI 92.4% to 98.4%) | |
| Agreement | | 97.1% | (95% CI 94.1% to 98.6%) | |
| Patient Group | n | n Pos | % Pos |
| --- | --- | --- | --- |
| Disease Associated | | | |
| Glomerulonephritis | 32 | 32 | 100.0% |
| Wegener's granulomatosis | 47 | 46 | 97.9% |
| Undifferentiated ANCA positive | 17 | 1 | 5.9% |
| Disease Control | | | |
| Non-ANCA associated vasculitis | 24 | 1 | 4.2% |
| Crohn's disease | 10 | 0 | 0.0% |
| Ulcerative colitis | 6 | 0 | 0.0% |
| Systemic lupus erythematosus | 32 | 3 | 9.4% |
| Rheumatoid arthritis | 8 | 1 | 12.5% |
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b. Other clinical supportive data (when a. is not applicable):
Not applicable
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
Expected values in a normal population are negative. However, 2-4% of apparently healthy, asymptomatic individuals may test positive for ANCA antibodies. The following table depicts the frequency of MPO and PR3 specific ANCA in sera from 112 ANCA associated vasculitides patients.
Incidence of anti-PR3 and anti-MPO in ANCA associated vasculitides
| Antibody | Wegener's | Microscopic | Churg-Strauss |
| --- | --- | --- | --- |
| association | granulomatosis | polyangiitis | syndrome |
| ANCA positive by IFA | 78% | 59% | 67% |
| anti-PR3 positive | 90% | 0% | 10% |
| anti-MPO positive | 0% | 62% | 17% |
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
