IMMULISA PR3 ANTIBODY ELISA

K092600 · Immco Diagnostics, Inc. · MOB · Oct 7, 2010 · Immunology

Device Facts

• Record ID: K092600
• Device Name: IMMULISA PR3 ANTIBODY 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 detection and semi-quantitation of antibodies to proteinase 3 (PR3) in human serum to aid in the diagnosis of Wegener’s granulomatosis in conjunction with other laboratory and clinical findings.

Device Story

ImmuLisa™ PR3 Antibody ELISA is a solid-phase immunoassay for detecting human IgG antibodies to proteinase 3 (PR3) in serum. The device uses microwell strips coated with purified PR3 antigen. Patient serum, controls, and calibrators are incubated in the wells; specific antibodies bind to the antigen. After washing, an HRP-labeled anti-human IgG conjugate is added, followed by a TMB substrate. The resulting color change, proportional to antibody concentration, is measured via spectrophotometer at 450 nm. The test is performed in a clinical laboratory setting by trained personnel. Results are expressed in U/ml and reported as positive or negative. The assay aids clinicians in diagnosing Wegener’s granulomatosis by identifying specific autoantibodies, supporting clinical decision-making alongside other laboratory and clinical findings.

Clinical Evidence

Clinical performance was evaluated using 275 samples, including 59 confirmed Wegener’s granulomatosis cases and various disease/healthy controls. Clinical sensitivity was 96.6% (95% CI 87.3-99.4%) and clinical specificity was 98.1% (95% CI 95-99.4%). Method comparison against the predicate device showed 94.3% overall agreement (cut-off 10 U/ml). Analytical performance included precision (CV% 3.4-12.8%), linearity (2.2-156 U/ml), and LoD (2.2 U/ml). No significant interference was observed for hemoglobin, bilirubin, rheumatoid factor, or triglycerides.

Technological Characteristics

Solid-phase ELISA; microwells coated with purified PR3 antigen. Uses HRP-conjugated anti-human IgG and TMB substrate. Requires microplate reader (450 nm/600-650 nm reference). Calibrated in arbitrary U/ml. Standards referenced: CLSI EP5-A2, EP6-A, EP7-A2, EP9-A2, EP12-A2, and EP17-A.

Indications for Use

Indicated for the detection and semi-quantitation of anti-PR3 antibodies in human serum to aid in the diagnosis of Wegener’s granulomatosis. 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 PR3 Antibody ELISA (k981328)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k092600

B. Purpose for Submission:
New Device

C. Measurand:
Anti-PR3 (proteinase 3)

D. Type of Test:
ELISA (Semi-quantitative)

E. Applicant:
Immco Diagnostics, Inc.

F. Proprietary and Established Names:
ImmuLisa™ PR3 Antibody ELISA

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 detection and semi-quantitation of antibodies to proteinase 3 (PR3) in human serum to aid in the diagnosis of Wegener’s granulomatosis 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 PR3, Calibrators A-E (156, 62.5, 25, 10, 1 U/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 PR3 Antibody ELISA

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2. Predicate K number(s):
k981328

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | ImmuLisa PR3 Antibody ELISA | Inova Quanta Lite PR3 ELISA |
| Intended use | An enzyme linked immunosorbent assay (ELISA) for the detection and semi-quantitation of antibodies to proteinase 3 (PR3) in human serum, as an aid in the diagnosis of Wegener's granulomatosis in conjunction with other laboratory and clinical findings. | Same |
| Methodology | ELISA | Same |
| Capture antigen | PR3 | Same |
| Analyte detected | Human IgG antibodies to PR3 | Same |
| Assay type | Semi-quantitative | 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 | PR3 IgG antibody | Same |
| Stop solution | H2SO4 | Same |
| Screening dilution | 1:101 | Same |
| Signal detection | 450nm on spectrophotometer | Same |
| Storage | 2-8°C | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | ImmuLisa PR3 Antibody ELISA | Inova Quanta Lite PR3 ELISA |
| Cut-off | 10 U/ml | 20 EU/ml |
| Wash buffer | Powdered or liquid | Liquid concentrate |

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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | concentrate | |
| Positive control | Acceptance range printed on vial | No value/range assigned |
| Calibrators | Set of 5; values in U/ml
E: 1
D: 10
C: 25
B: 62.5
A: 156 | Single; value in units 25 EU/ml |
| Linear range | 2.2-156 U/ml | Not specified |
| Indeterminate range | 10-12.5 U/ml | None |
| Limit of detection | 2.2 U/ml | Not specified |

K. Standard/Guidance Document Referenced (if applicable):
CLSI EP5-A2, EP6-A, 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 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 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 arbitrary Units per milliliter (U/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 seven specimens with values spanning the range of the assay. The linear range of the assay is 2.2-156 U/ml. The seven samples spanned concentrations 6.34 to 141.59. The percent total imprecision ranged from 5.0-12.8%. Averaged results are shown in table below. Multiple studies were conducted. Assay runs of three replicates of seven specimens were conducted using three different lots (n=63). Separately, assay runs of three replicates of seven specimens were conducted over five days, twice a day (n=189). Lastly, assay runs of six replicates of seven specimens were conducted. Repeatability was determined with 28 replicates of each of the seven specimens. Results are also shown in the table below (last column). Percent CVs ranged from 3.4-8.1%. Sponsor's pre-defined acceptance criteria for precision was 20%.

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| | | | | | | Within run | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Total Imprecision | | Between days | | (Repeatability) | |
| | Mean | SD | | SD | | SD | |
| Sample | (IU/ml) | (IU/ml) | CV% | (IU/ml) | CV% | (IU/ml) | CV% |
| 1 | 6.34 | 0.808 | 12.8% | 0.964 | 15.2% | 0.517 | 8.1% |
| 2 | 8.50 | 0.750 | 8.8% | 0.818 | 9.7% | 0.643 | 7.5% |
| 3 | 12.26 | 1.215 | 9.9% | 1.220 | 9.8% | 1.167 | 9.8% |
| 4 | 27.27 | 2.627 | 9.6% | 3.112 | 11.4% | 1.711 | 6.3% |
| 5 | 56.94 | 3.088 | 5.4% | 3.485 | 6.1% | 2.390 | 4.2% |
| 6 | 111.06 | 5.518 | 5.0% | 6.406 | 5.8% | 3.789 | 3.4% |
| 7 | 141.59 | 7.763 | 5.5% | 8.205 | 5.8% | 6.850 | 4.8% |

b. Linearity/assay reportable range:

To determine the linear range of the assay, studies were performed per CLSI EP6-A using equidistant dilution series of positive samples with values throughout the calibrator range. Up to nine serial dilutions in sample diluent (low positive or negative samples) were made and run in duplicate. The linear range of the assay is 2.2-156 U/ml. Linear regressions are summarized in the table below. Samples producing results greater than the top calibrator are recommended to be diluted and retested.

| Sample | Test Range | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery |
| --- | --- | --- | --- | --- | --- |
| 1 | 3.7 to 77.2 | 1.07 (0.94 to 1.20) | -0.031 (-0.134 to 0.071) | 0.986 | 89.8 to 111.9 |
| 2 | 3.8 to 158.2 | 1.012 (0.934 to 1.090) | -0.053 (-0.153 to 0.047) | 0.994 | 88.9 to 102.2 |
| 3 | 3.4 to 43.4 | 1.023 (0.929 to 1.118) | 0.009 (-0.045 to 0.063) | 0.991 | 100 to 112.3 |

c. Traceability, Stability, Expected values (controls, calibrators, or methods): An international reference material for anti-PR3 antibodies is not available. The assay is calibrated in relative arbitrary units (U/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 $37^{\circ}\mathrm{C}$ where one day is considered to equivalent to one month stored at $2 - 8^{\circ}\mathrm{C}$ . 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 calibrators 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 PR3 antigen coated plates.

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d. Detection limit:

Per CLSI EP17-A, the limit of detection (LoD) for PR3 antibody using this assay was determined to be $2.2\mathrm{U / ml}$ based on 60 replicates of the blank and 10 replicates of each of the six low-level samples. The limit of blank is 2.1 U/ml.

e. Analytical specificity:

Interfering Substances:

Per CLSI EP7-A2, interference was studied by mixing sera with known PR3 antibody 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 -9.6-10.6% (2 g/L), bilirubin range of interference -5.7-1.7% (342 umol/L), rheumatoid factor range of interference -13.4-4.2% (100 EU/ml), and triglycerides range of interference -4.4-8.5% (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 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 | 4 | 4.4 | -9.6 | 4.1 | -1.7 | 4.5 | -13.4 | 1 | 3.8 | 3.7 | 2.7 |
| 2 | 9.8 | 9.6 | 1.4 | 9.7 | 0.8 | 10 | -2 | 2 | 8.9 | 8.2 | 8.5 |
| 3 | 9.8 | 8.7 | 10.6 | 9.8 | 0 | 9.6 | 2 | 3 | 7.9 | 8.3 | -4.4 |
| 4 | 36 | 35.4 | 1.7 | 38 | -5.7 | 37 | -3 | 4 | 18.8 | 17.8 | 5.6 |
| 5 | 72 | 75.1 | -4.3 | 75.9 | -5.5 | 73.9 | -2.6 | 5 | 42.1 | 41.4 | 1.7 |
| 6 | 192.9 | 176.9 | 8.3 | 189.7 | 1.7 | 184.7 | 4.2 | 6 | 134.4 | 138.1 | -2.7 |

Cross-Reactivity:

A total of 136 potentially cross-reactive specimens from individuals with other autoimmune disorders or positive for other autoantibodies were tested for PR3 antibodies using the ImmuLisa PR3 Antibody ELISA. MPO positive samples were tested MPO positive by ELISA and confirmed for a pANCA positive reaction using FDA cleared IFA. 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 | Positive n |
| --- | --- | --- |
| Glomerulonephritis | 29 | 2 |
| Undifferentiated pANCA positive | 11 | 0 |
| Non-ANCA associated vasculitis | 24 | 1 |

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| Crohn's disease | 10 | 0 |
| --- | --- | --- |
| Ulcerative Colitis | 6 | 0 |
| Systemic lupus erythematosus | 32 | 0 |
| Rheumatoid Arthritis | 8 | 0 |
| Other autoimmune disease | 16 | 0 |
| Total | 136 | 3 (2.2%) |

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 0.250 OD. This value was assigned to 10 U/ml. An indeterminate range has been established 20% above the cut-off at 12.5 U/ml. Samples in this indeterminate range should be retested or confirmed using a secondary method such as IFA.

2. Comparison studies:

a. Method comparison with predicate device:
Per CLSI EP9-A2, the ImmuLisa PR3 Antibody ELISA was tested in comparison with Inova Quanta Lite PR3 ELISA using well-characterized sera of ANCA antibody positive subjects (53 cANCA positive), disease controls (28) and healthy individuals (24). Included were 12 samples near the cut-off of 10 U/ml. Only samples within the measuring range of the assay were included for method comparison analysis. The disease controls included Celiac Disease, ENA positive collagen vascular autoimmunity, Hashimoto's, and RA. Of the four samples that tested negative on the Inova assay and positive on the IMMCO assay, three were IFA positive Wegnener's cases and one was a disease control. The two samples that tested negative on the IMMCO assay and positive on the Inova assay were IFA positive Wegener's cases. The results are as follows for a cut-off of 10 U/ml using a 5 point calibrator (indeterminate results are considered positive):

| 5 point calibrator, indeterminate as positive | | | | |
| --- | --- | --- | --- | --- |
| | | | Inova | |
| | | Pos | Neg | Total |
| | Pos | 41 | 4 | 45 |
| IMMCO | Neg | 2 | 58 | 60 |
| | Total | 43 | 62 | 105 |
| | | | | |
| Positive % Agreement | | 95.3% | (95% CI 82.9% to 99.2%) | |
| Negative % Agreement | | 93.5% | (95% CI 83.5% to 99.2%) | |
| Overall % Agreement | | 94.3% | (95% CI 87.5% to 97.7%) | |

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The following results are for a cut-off of 12.5 U/ml using a 5 point calibrator (indeterminate results are considered negative):

| 5 point calibrator, indeterminate as negative | | | | |
| --- | --- | --- | --- | --- |
| | | | Inova | |
| | | Pos | Neg | Total |
| | Pos | 38 | 0 | 38 |
| IMMCO | Neg | 5 | 62 | 67 |
| | Total | 43 | 62 | 105 |
| | | | | |
| Positive % Agreement | | 88.4% | (95% CI 74.1% to 95.6%) | |
| Negative % Agreement | | 100.0% | (95% CI 92.7% to 100%) | |
| Overall % Agreement | | 95.2% | (95% CI 88.7% to 98.2%) | |

b. Matrix comparison:

Not applicable

3. Clinical studies:

a. Clinical sensitivity and specificity:

A set of 75 clinical samples from various disease groups (see table below for breakdown of actual disease groups) confirmed IFA+ were tested with the ImmunoLisa PR3 Antibody ELISA and another commercially available PR3 antibody ELISA. Other autoimmune disease samples include Celiac Disease and Hashimoto's Thyroiditis. Results are presented in table below. When indeterminate results are considered positive, the calculated clinical sensitivity of the assay is 96.6% (95% CI 87.3-99.4%). The calculated clinical specificity of the assay is 98.1% (95% CI 95-99.4%).

| PR3 | Positive > Cutoff 10 IU/ml, 5 point Calibrator | | | |
| --- | --- | --- | --- | --- |
| | | | Disease Status | |
| | | Pos | Neg | Total |
| | Pos | 57 | 4 | 61 |
| IMMCO | Neg | 2 | 212 | 214 |
| | Total | 59 | 216 | 275 |
| | | | | |
| Sensitivity | | 96.6% | (95% CI 87.3% to 99.4%) | |
| Specificity | | 98.1% | (95% CI 95.0% to 99.4%) | |
| Agreement | | 97.8% | (95% CI 95.1% to 99.1%) | |
| | IMMCO | | | Inova PR3 Ab ELISA | | |
| --- | --- | --- | --- | --- | --- | --- |
| Patient Group | n | n Pos | % Pos | n | n Pos | % Pos |
| Disease Associated | | | | | | |
| Wegener's granulomatosis | 59 | 57 | 96.6% | 59 | 56 | 94.9% |
| Glomerulonephritis | 29 | 2 | 6.9% | 29 | 2 | 6.9% |
| Undifferentiated ANCA positive | 11 | 0 | 0.0% | 11 | 0 | 0.0% |
| Disease Control | | | | | | |
| Non-ANCA associated vasculitis | 24 | 1 | 4.2% | | | |
| Crohn's disease | 10 | 0 | 0.0% | | | |
| Ulcerative colitis | 6 | 0 | 0.0% | | | |

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| Systemic lupus erythematosus | 32 | 1 | 3.1% | 32 | 0 | 0.0% |
| --- | --- | --- | --- | --- | --- | --- |
| Rheumatoid arthritis | 8 | 0 | 0.0% | 8 | 0 | 0.0% |
| Other autoimmune disease | 16 | 0 | 0.0% | 16 | 0 | 0.0% |
| Healthy normals | 80 | 0 | 0.0% | 80 | 0 | 0.0% |

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 anti-PR3 antibodies. The following table depicts the frequency of PR3 and MPO 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.