ImmuLisa Enhanced RF IgA Antibody ELISA, ImmuLisa Enhanced RF IgG Antibody ELISA, ImmuLisa Enhanced RF IgM Antibody ELISA, ImmuLisa Enhanced RF Antibody Screen ELISA
Device Facts
| Record ID | K143736 |
|---|---|
| Device Name | ImmuLisa Enhanced RF IgA Antibody ELISA, ImmuLisa Enhanced RF IgG Antibody ELISA, ImmuLisa Enhanced RF IgM Antibody ELISA, ImmuLisa Enhanced RF Antibody Screen ELISA |
| Applicant | Immco Diagnostics, Inc. |
| Product Code | DHR · Immunology |
| Decision Date | Sep 23, 2015 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5775 |
| Device Class | Class 2 |
Indications for Use
ImmuLisa Enhanced™ RF IgA Antibody ELISA: Enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Rheumatoid Factor IgA antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings. ImmuLisa Enhanced™ RF IgG Antibody ELISA: Enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Rheumatoid Factor IgG antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings. ImmuLisa Enhanced™ RF IgM Antibody ELISA: Enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Rheumatoid Factor IgM antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings. ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA: Enzyme linked immunoassay (ELISA) for the qualitative detection of Rheumatoid Factor IgA, IgG and IgM antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings.
Device Story
ImmuLisa Enhanced™ RF Antibody ELISAs are sandwich-type enzyme immunoassays for detecting Rheumatoid Factor (RF) antibodies in human serum. The device uses microplates coated with purified rabbit IgG antigen. Patient serum samples are incubated in the wells; if RF antibodies are present, they bind to the antigen. After washing, HRP-conjugated anti-human immunoglobulins (IgA, IgG, IgM, or a mixture) are added. A TMB substrate is introduced, producing a color change proportional to the antibody concentration, measured by a spectrophotometer at 450 nm. The device is used in clinical laboratory settings by trained personnel. Results are reported as qualitative or semi-quantitative (EU/mL or IU/mL) to assist clinicians in diagnosing rheumatoid arthritis. The test provides diagnostic support when used alongside other clinical findings and laboratory tests.
Clinical Evidence
Clinical performance was evaluated using 179–249 clinically characterized RA samples and 310 disease controls. Sensitivity ranged from 39.4% to 84.9% and specificity from 85.5% to 94.8% depending on the specific isotype and cut-off interpretation. Method comparison against predicate devices showed overall percent agreement ranging from 79.7% to 96.3%. Cross-reactivity was assessed across 310 samples from various autoimmune and infectious diseases.
Technological Characteristics
Sandwich-type ELISA; rabbit IgG capture antigen; HRP-conjugated anti-human IgA, IgG, or IgM detection reagents; TMB substrate; sulfuric acid stop solution. Requires microplate reader (450 nm) and automatic washer. Qualitative and semi-quantitative modes. Traceable to in-house standards (IgA/IgG) or WHO NIBSC 64/2 (IgM).
Indications for Use
Indicated for the qualitative or semi-quantitative detection of Rheumatoid Factor (RF) IgA, IgG, or IgM antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings. For prescription use only.
Regulatory Classification
Identification
A rheumatoid factor immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the rheumatoid factor (antibodies to immunoglobulins) in serum, other body fluids, and tissues. Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis.
Related Devices
- K983083 — QUANTA LITE RF IGG ELISA · Inova Diagnostics, Inc. · Nov 4, 1998
- K983084 — QUANTA LITE RF IGA ELISA · Inova Diagnostics, Inc. · Nov 4, 1998
- K993557 — AUTOSTAT II RHEUMATOID FACTOR IGA ELISA · Cogent Diagnotics , Ltd. · Mar 3, 2000
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM
A. 510(k) Number:
K143736
B. Purpose for Submission:
New device
C. Measurand:
Rheumatoid Factors (RF) IgA, IgG, IgM, and Rheumatoid Factor Total
D. Type of Test:
Immunoassay, qualitative and semi-quantitative
E. Applicant:
IMMCO Diagnostics, Inc.
F. Proprietary and Established Names:
ImmuLisa Enhanced™ RF IgA Antibody ELISA
ImmuLisa Enhanced™ RF IgG Antibody ELISA
ImmuLisa Enhanced™ RF IgM Antibody ELISA
ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA
G. Regulatory Information:
1. Regulation section:
21 CFR §866.5775: Rheumatoid factor immunological test system
2. Classification:
Class II
3. Product code:
DHR: System, Test, Rheumatoid Factor
4. Panel:
Immunology (82)
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H. Intended Use:
1. Intended use(s):
ImmuLisa Enhanced™ RF IgA Antibody ELISA:
Enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Rheumatoid Factor IgA antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings.
ImmuLisa Enhanced™ RF IgG Antibody ELISA:
Enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Rheumatoid Factor IgG antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings.
ImmuLisa Enhanced™ RF IgM Antibody ELISA:
Enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Rheumatoid Factor IgM antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings.
ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA:
Enzyme linked immunoassay (ELISA) for the qualitative detection of Rheumatoid Factor IgA, IgG and IgM antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests 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:
A microplate reader capable of reading absorbance values at 450 nm. If a dual wavelength microplate reader is available, the reference filter should be set at 600-650 nm. An automatic microplate washer capable of accurately dispensing 200 µL of fluid is also required.
I. Device Description:
Each kit consists of one 12 x8 microplate with individual breakaway microwells coated with purified rabbit IgG antigen, a five-level calibrator set for semi-quantitative analysis, a cut-off calibrator for qualitative analysis, a negative control, a positive control, Tetramethylbenzidine (TMB) chromogenic substrate, stop solution, wash buffer, and diluent.
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The type of conjugate is specific for each kit: goat anti-human IgA, HRP (horseradish peroxidase) conjugate for the RF IgA Antibody ELISA; goat anti-human IgG HRP conjugate for the RF IgG Antibody ELISA; goat anti-human IgM HRP conjugate for the RF IgM Antibody ELISA; and a mixture of goat anti-human IgA, IgG, and IgM HRP conjugates for the RF IgA/IgG/IgM ELISA.
## J. Substantial Equivalence Information:
1. Predicate device name(s) and numbers:
Inova QuantaLite® RF IgA ELISA, K983084
Inova QuantaLite® RF IgG ELISA, K983083
Inova QuantaLite® RF IgM ELISA, K971614
2. Comparison with predicates:
| Similarities | | |
| --- | --- | --- |
| Item | New Devices: ImmuLisa Enhanced™ RF IgA/IgG/IgM Antibody ELISAs and RF IgA/IgG/IgM ELISA | Predicates: Inova QuantaLite® RF IgA/ IgG/IgM ELISA |
| Intended Use/Indication for Use | Enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Rheumatoid Factor IgA, IgG, or IgM antibodies in human serum to aid in the diagnosis of rheumatoid arthritis (RA) in conjunction with other laboratory tests and clinical findings. | Same |
| Test Principle | Enzyme-Linked Immunoassay (ELISA) | Same |
| Instrumentation | Spectrophotometer reading at 450 nm | Same |
| Analyte | RF IgA, IgG, or IgM antibodies | Same |
| Sample Type | Serum | Same |
| Sample Dilution | 1:101 | Same |
| Kit Components | Includes positive controls, negative controls, calibrators, conjugates, substrate, diluent, wash buffer, stop solution, microplates | Same |
| Capture Antigen | Rabbit IgG | Same |
| Detection Reagents | HRP conjugated to goat anti-human IgA, IgG, or IgM | Same |
| Substrate | TMB | Same |
| Incubation Times | Positive and negative controls and diluted patient samples: 30 min | Same |
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| Similarities | | |
| --- | --- | --- |
| Item | New Devices: ImmuLisa Enhanced™ RF IgA/IgG/IgM Antibody ELISAs and RF IgA/IgG/IgM ELISA | Predicates: Inova QuantaLite® RF IgA/ IgG/IgM ELISA |
| | Conjugate: 30 min Substrate: 30 min (in dark) | |
| Stop Solution | Sulfuric acid (H2SO4) | Same |
| Controls | Positive Controls: human sera positive for RF IgA, IgG, IgM, or total RF Negative Controls: human sera negative for RF IgA, IgG, IgM, or total RF | Same |
| Differences | | |
| --- | --- | --- |
| Item | New Devices: ImmuLisa Enhanced™ RF IgA/IgG/IgM Antibody ELISAs and RF IgA/IgG/IgM ELISA | Predicates: Inova QuantaLite® RF IgA/IgG/IgM ELISA |
| Measurement Type | RF IgA, IgG, and IgM Antibody ELISAs: Qualitative and semi-quantitative RF IgA/IgG/IgM ELISA: Qualitative only | Semi-Quantitative |
| Wash buffer | Powdered or optional liquid concentrate | Liquid concentrate |
| Calibrators | RF IgA and IgG Antibody ELISAs: Set of five vials with values in EU/ml: 1, 20, 40, 80, 160 RF IgM Antibody ELISA: Set of five vials with values in IU/mL: 1, 10, 20, 40, 80 RF IgA/IgG/IgM ELISA: 30 EU/mL (1-point calibration) | Set of five vials with value in units: 5, 12.5, 25, 50, 100 |
| Reagent Stability | Reagents: until the expiration date at 2–8°C Reconstituted wash buffer: until the kit expiration date at 2–8°C | Reagents: until the expiration date at 2–8°C Diluted wash buffer: 1 week at 2–8°C |
| Cut-offs | RF IgA/IgG and RF IgA/IgG/IgM ELISAs: 20 EU/mL RF IgM Antibody ELISA: 10 IU/mL | IgA, IgG, and IgM ELISAs: 6 Units |
| Linear Ranges | RF IgA Antibody ELISA: 3.7–160 EU/mL RF IgG Antibody ELISA: 2.2–160 EU/mL RF IgM Antibody ELISA: 1.3–80 IU/mL | Not Specified |
| Limits of Blank | RF IgA Antibody ELISA: 3.2 EU/mL RF IgG Antibody ELISA: 1.5 EU/mL RF IgM Antibody ELISA: 1.2 IU/mL RF IgA/IgG/IgM ELISA: 2.1 EU/mL | Not Specified |
| Limits of Detection | RF IgA Antibody ELISA: 3.7 EU/mL RF IgG Antibody ELISA: 2.2 EU/mL | Not Specified |
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| Differences | | |
| --- | --- | --- |
| Item | New Devices: ImmuLisa Enhanced™ RF IgA/IgG/IgM Antibody ELISAs and RF IgA/IgG/IgM ELISA | Predicates: Inova QuantaLite® RF IgA/IgG/IgM ELISA |
| | RF IgM Antibody ELISA: 1.3 IU/mL RF IgA/IgG/IgM ELISA: 2.7 EU/mL | |
| Traceability | RF IgA and IgG Antibody ELISAs: Reference standard or method is not available. Results are traceable to in-house standards. RF IgM Antibody ELISA: traceable to the WHO RF IgM International Standard (NIBSC 64/2) | Not Specified |
## K. Standard/Guidance Document Referenced (if applicable):
1. CLSI EP05-A2: Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline - Second Edition
2. CLSI EP06-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline
3. CLSI EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition
4. CLSI EP09-A3: Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline - Third Edition
5. CLSI EP12-A2: User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline- Second Edition
6. CLSI EP17-A2: Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline - Second Edition
## L. Test Principle:
All four ImmuLisa Enhanced RF Antibody tests are sandwich-type enzyme immunoassays. Serum samples diluted 1:101 are incubated in the microplates coated with purified rabbit IgG as the antigen. Patient antibodies, if present in the samples, bind to the antigen-coated wells. Unbound antibodies and other serum proteins are removed by washing the microwells. Bound antibodies are detected by adding anti-human immunoglobulins conjugated to horseradish peroxidase (conjugate) to the microwells. After incubation, excess conjugate is removed by washing. Specific enzyme substrate is then added to the microwells and the presence of antibodies to RF is detected by a color change produced by the conversion of the 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 read by a spectrophotometer at 450 nm.
Semi-quantitative results are determined from a standard curve generated from the results of each assay's five calibrators. Semi-quantitative results are reported as EU/mL for the IgA
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and IgG assays and as IU/mL for the IgM assay. Results are reported as positive, negative, or indeterminate according to the following table. The manufacturer recommends that indeterminate results should be retested and evaluated along with other laboratory methods.
| RF IgA or IgG Antibody ELISA Value (EU/mL) | RF IgM Antibody ELISA Value (IU/mL) | Interpretation |
| --- | --- | --- |
| <20 | <10 | Negative |
| 20–25 | 10–12.5 | Indeterminate |
| >25 | >12.5 | Positive |
Qualitative results for the RF IgA, IgG, and IgM Antibody ELISAs are determined using a ratio of the absorbance of the sample to the absorbance of the cut-off calibrator (20 EU/mL or 10 IU/mL). The ratio is multiplied by the concentration of the cut-off calibrator to give a numerical value. It is recommended that qualitative results be reported as positive or negative. Values greater than or equal to the cut-off calibrator are considered positive.
Qualitative results for the RF IgA/IgG/IgM ELISA are determined using a ratio of the absorbance of the sample to the absorbance of the single calibrator at 30 EU/mL. The ratio is multiplied by the concentration of the cut-off calibrator to give a numerical value. Values <20 EU/mL are reported as negative and values ≥20 EU/mL are reported as positive.
## M. Performance Characteristics:
### 1. Analytical performance:
#### a. Precision/Reproducibility:
**Semi-quantitative precision:**
The precision performance of each RF Antibody ELISA was evaluated in accordance with CLSI guideline EP05-A2. Seven serum samples from the intended use population that cover the measuring range of each ELISA were tested with six replicates in a single run per day over a period of 13 days. An additional 12 replicates were included on the final day (n = 90 replicates total). Assays were performed by two operators using one reagent lot and different instrumentation setups. The first instrumentation setup includes a multi-channel pipettor, microplate washer and microplate reader. The second instrumentation set was a liquid handling system that includes a microplate reader. The precision parameters were calculated and all values met the manufacturer’s pre-determined acceptance criteria. The data are presented in the tables below:
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| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Day | | Total | |
| | | SD | CV | SD | CV | SD | CV |
| 1 | 9.2 | 0.39 | 4.2% | 0.36 | 3.9% | 0.54 | 5.8% |
| 2 | 14.9 | 0.57 | 3.8% | 0.58 | 3.9% | 0.82 | 5.5% |
| 3 | 21.2 | 0.97 | 4.6% | 0.71 | 3.3% | 1.20 | 5.7% |
| 4 | 26.4 | 0.73 | 2.8% | 1.21 | 4.6% | 1.42 | 5.4% |
| 5 | 74.6 | 2.21 | 3.0% | 4.51 | 6.0% | 5.02 | 6.7% |
| 6 | 115.6 | 2.99 | 2.6% | 4.34 | 3.8% | 5.27 | 4.6% |
| 7 | 158.1 | 4.99 | 3.2% | 3.93 | 2.5% | 6.35 | 4.0% |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Day | | Total | |
| | | SD | CV | SD | CV | SD | CV |
| 1 | 8.8 | 0.60 | 6.8% | 0.47 | 5.3% | 0.76 | 8.7% |
| 2 | 16.4 | 0.72 | 4.4% | 0.74 | 4.5% | 1.03 | 6.3% |
| 3 | 19.4 | 0.88 | 4.5% | 0.60 | 3.1% | 1.06 | 5.5% |
| 4 | 22.7 | 0.94 | 4.1% | 0.54 | 2.4% | 1.08 | 4.8% |
| 5 | 45.9 | 1.86 | 4.0% | 1.12 | 2.4% | 2.17 | 4.7% |
| 6 | 79.7 | 2.64 | 3.3% | 4.16 | 5.2% | 4.93 | 6.2% |
| 7 | 133.0 | 4.83 | 3.6% | 4.15 | 3.1% | 6.37 | 4.8% |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (IU/mL) | Within-Run (Repeatability) | | Between-Day | | Total | |
| | | SD | CV | SD | CV | SD | CV |
| 1 | 4.8 | 0.12 | 2.5% | 0.13 | 2.7% | 0.18 | 3.7% |
| 2 | 8.5 | 0.28 | 3.3% | 0.18 | 2.1% | 0.34 | 3.9% |
| 3 | 10.3 | 0.63 | 6.1% | 0.48 | 4.6% | 0.79 | 7.7% |
| 4 | 11.9 | 0.45 | 3.8% | 0.54 | 4.5% | 0.70 | 5.9% |
| 5 | 25.6 | 0.70 | 2.7% | 0.82 | 3.2% | 1.08 | 4.2% |
| 6 | 45.6 | 1.16 | 2.6% | 1.46 | 3.2% | 1.87 | 4.1% |
| 7 | 80.2 | 1.88 | 2.3% | 3.12 | 3.9% | 3.64 | 4.5% |
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Inter-Operator Precision Tables:
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Day | | Between-Operator | | Total | |
| | | SD | CV | SD | CV | SD | CV | SD | CV |
| 1 | 9.2 | 0.39 | 4.2% | 0.37 | 4.0% | 0.00 | 0.0% | 0.54 | 5.8% |
| 2 | 14.9 | 0.57 | 3.8% | 0.52 | 3.5% | 0.39 | 2.6% | 0.86 | 5.8% |
| 3 | 21.2 | 0.97 | 4.6% | 0.60 | 2.8% | 0.55 | 2.6% | 1.26 | 6.0% |
| 4 | 26.4 | 0.73 | 2.8% | 1.08 | 4.1% | 0.81 | 3.1% | 1.53 | 5.8% |
| 5 | 74.6 | 2.21 | 3.0% | 3.88 | 5.2% | 3.33 | 4.5% | 5.57 | 7.5% |
| 6 | 115.6 | 2.99 | 2.6% | 3.43 | 3.0% | 3.85 | 3.3% | 5.96 | 5.2% |
| 7 | 158.1 | 4.99 | 3.2% | 3.94 | 2.5% | 0.00 | 0.0% | 6.35 | 4.0% |
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Operator/ Equipment 1 | | Operator/ Equipment 2 | | Total | | |
| | | SD | CV | SD | CV | SD | CV | |
| 1 | 9.2 | 0.59 | 6.3% | 0.48 | 5.2% | 0.54 | 5.8% | |
| 2 | 14.9 | 0.75 | 5.0% | 0.74 | 5.0% | 0.86 | 5.8% | |
| 3 | 21.2 | 0.94 | 4.4% | 1.18 | 5.6% | 1.26 | 6.0% | |
| 4 | 26.4 | 1.11 | 4.2% | 1.29 | 4.9% | 1.53 | 5.8% | |
| 5 | 74.6 | 3.32 | 4.5% | 4.60 | 6.2% | 5.57 | 7.5% | |
| 6 | 115.6 | 3.24 | 2.8% | 4.81 | 4.2% | 5.96 | 5.2% | |
| 7 | 158.1 | 3.87 | 2.4% | 7.01 | 4.4% | 6.35 | 4.0% | |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Day | | Between-Operator | | Total | |
| | | SD | CV | SD | CV | SD | CV | SD | CV |
| 1 | 8.8 | 0.60 | 6.8% | 0.33 | 3.7% | 0.48 | 5.5% | 0.83 | 9.5% |
| 2 | 16.4 | 0.72 | 4.4% | 0.72 | 4.4% | 0.24 | 1.5% | 1.04 | 6.4% |
| 3 | 19.4 | 0.86 | 4.4% | 0.58 | 3.0% | 0.32 | 1.6% | 1.08 | 5.6% |
| 4 | 22.7 | 0.89 | 3.9% | 0.57 | 2.5% | 0.33 | 1.4% | 1.11 | 4.9% |
| 5 | 45.9 | 1.70 | 3.7% | 1.40 | 3.1% | 0.00 | 0.0% | 2.20 | 4.8% |
| 6 | 79.7 | 2.66 | 3.3% | 3.94 | 4.9% | 1.44 | 1.8% | 4.97 | 6.2% |
| 7 | 133.0 | 4.86 | 3.7% | 3.18 | 2.4% | 3.59 | 2.7% | 6.83 | 5.1% |
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| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Operator/ Equipment 1 | | Operator/ Equipment 2 | | Total | |
| | | SD | CV | SD | CV | SD | CV |
| 1 | 8.8 | 0.74 | 8.4% | 0.62 | 7.1% | 0.83 | 9.5% |
| 2 | 16.4 | 0.93 | 5.7% | 1.00 | 6.1% | 1.04 | 6.4% |
| 3 | 19.4 | 0.92 | 4.7% | 1.05 | 5.4% | 1.08 | 5.6% |
| 4 | 22.7 | 1.20 | 5.3% | 0.93 | 4.1% | 1.11 | 4.9% |
| 5 | 45.9 | 2.11 | 4.6% | 2.14 | 4.7% | 2.20 | 4.8% |
| 6 | 79.7 | 3.76 | 4.7% | 4.85 | 6.1% | 4.97 | 6.2% |
| 7 | 133.0 | 6.34 | 4.8% | 5.26 | 4.0% | 6.83 | 5.1% |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (IU/mL) | Within-Run (Repeatability) | | Between-Day | | Between-Operator | | Total | |
| | | SD | CV | SD | CV | SD | CV | SD | CV |
| 1 | 4.8 | 0.12 | 2.5% | 0.13 | 2.7% | 0.00 | 0.0% | 0.18 | 3.7% |
| 2 | 8.5 | 0.28 | 3.3% | 0.16 | 1.9% | 0.11 | 1.3% | 0.34 | 4.0% |
| 3 | 10.3 | 0.63 | 6.1% | 0.50 | 4.8% | 0.00 | 0.0% | 0.81 | 7.8% |
| 4 | 11.9 | 0.45 | 3.8% | 0.55 | 4.6% | 0.00 | 0.0% | 0.71 | 6.0% |
| 5 | 25.6 | 0.70 | 2.7% | 0.85 | 3.3% | 0.00 | 0.0% | 1.10 | 4.3% |
| 6 | 45.6 | 1.16 | 2.6% | 1.44 | 3.2% | 0.38 | 0.8% | 1.89 | 4.1% |
| 7 | 80.2 | 1.88 | 2.3% | 2.56 | 3.2% | 2.57 | 3.2% | 4.09 | 5.1% |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (IU/mL) | Operator/ Equipment 1 | | Operator/ Equipment 2 | | Total | |
| | | SD | CV | SD | CV | SD | CV |
| 1 | 4.8 | 0.15 | 3.2% | 0.18 | 3.7% | 0.18 | 3.7% |
| 2 | 8.5 | 0.24 | 2.8% | 0.35 | 4.1% | 0.34 | 4.0% |
| 3 | 10.3 | 0.73 | 7.1% | 0.80 | 7.8% | 0.81 | 7.8% |
| 4 | 11.9 | 0.59 | 4.9% | 0.72 | 6.0% | 0.71 | 6.0% |
| 5 | 25.6 | 1.11 | 4.3% | 1.02 | 4.0% | 1.10 | 4.3% |
| 6 | 45.6 | 2.08 | 4.6% | 1.59 | 3.5% | 1.89 | 4.1% |
| 7 | 80.2 | 2.93 | 3.7% | 3.07 | 3.8% | 4.09 | 5.1% |
Lot-to-lot reproducibility:
Seven serum samples from the intended use population that cover the measuring range of each ELISA were measured with five replicates and one run per day for five
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days using three lots of reagents on one instrument and one operator at one site ( $n = 75$ replicates total). For Lot 1, the $\% \mathrm{CV}$ for total precision for the seven samples across all four ELISAs ranged from $2.4\% - 8.2\%$ . For Lot 2, the $\% \mathrm{CV}$ for total precision for the seven samples across all four ELISAs ranged from $2.2\% - 11.6\%$ . For Lot 3, the $\% \mathrm{CV}$ for total precision for the seven samples across all four ELISAs ranged from $2.8\% - 9.4\%$ . The combined lot-to-lot reproducibility parameters for each ELISA type are detailed in the tables below. All values met the manufacturer's acceptance criteria.
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Day | | Between-Lot | | Total | |
| | | SD | CV | SD | CV | SD | CV | SD | CV |
| 1 | 8.48 | 0.33 | 3.9% | 0.00 | 0.0% | 0.10 | 1.2% | 0.34 | 4.1% |
| 2 | 13.67 | 1.07 | 7.8% | 0.00 | 0.0% | 0.00 | 0.0% | 1.07 | 7.8% |
| 3 | 20.52 | 1.36 | 6.6% | 0.00 | 0.0% | 0.00 | 0.0% | 1.36 | 6.6% |
| 4 | 24.86 | 1.01 | 4.1% | 0.00 | 0.0% | 0.06 | 0.2% | 1.02 | 4.1% |
| 5 | 72.09 | 4.99 | 6.9% | 0.00 | 0.0% | 0.00 | 0.0% | 4.99 | 6.9% |
| 6 | 109.74 | 3.95 | 3.6% | 0.00 | 0.0% | 0.28 | 0.3% | 3.96 | 3.6% |
| 7 | 148.24 | 5.93 | 4.0% | 0.00 | 0.0% | 1.91 | 1.3% | 6.23 | 4.2% |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Day | | Between-Lot | | Total | |
| | | SD | CV | SD | CV | SD | CV | SD | CV |
| 1 | 8.36 | 0.82 | 9.8% | 0.06 | 0.8% | 0.00 | 0.0% | 0.82 | 9.8% |
| 2 | 15.80 | 0.79 | 5.0% | 0.00 | 0.0% | 0.30 | 1.9% | 0.84 | 5.3% |
| 3 | 19.74 | 1.27 | 6.4% | 0.00 | 0.0% | 0.34 | 1.7% | 1.31 | 6.7% |
| 4 | 22.11 | 0.83 | 3.8% | 0.00 | 0.0% | 0.22 | 1.0% | 0.86 | 3.9% |
| 5 | 44.11 | 1.24 | 2.8% | 0.00 | 0.0% | 0.30 | 0.7% | 1.28 | 2.9% |
| 6 | 77.03 | 4.71 | 6.1% | 0.00 | 0.0% | 0.00 | 0.0% | 4.71 | 6.1% |
| 7 | 127.59 | 5.72 | 4.5% | 1.10 | 0.9% | 0.00 | 0.0% | 5.82 | 4.6% |
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| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (IU/mL) | Within-Run (Repeatability) | | Between-Day | | Between-Lot | | Total | |
| | | SD | CV | SD | CV | SD | CV | SD | CV |
| 1 | 4.76 | 0.39 | 8.2% | 0.00 | 0.0% | 0.10 | 2.1% | 0.40 | 8.5% |
| 2 | 8.39 | 0.38 | 4.6% | 0.15 | 1.8% | 0.11 | 1.4% | 0.43 | 5.1% |
| 3 | 9.87 | 0.82 | 8.3% | 0.00 | 0.0% | 0.00 | 0.0% | 0.82 | 8.3% |
| 4 | 0.00 | 0.00 | 0.0% | 0.00 | 0.0% | 0.00 | 0.0% | 0.70 | 5.8% |
| 5 | 25.81 | 0.72 | 2.8% | 0.22 | 0.9% | 0.00 | 0.0% | 0.75 | 2.9% |
| 6 | 47.64 | 1.68 | 3.5% | 0.00 | 0.0% | 0.87 | 1.8% | 1.89 | 4.0% |
| 7 | 82.68 | 2.94 | 3.6% | 1.42 | 1.7% | 0.00 | 0.0% | 3.27 | 4.0% |
## Qualitative reproducibility:
Because the ImmuLisa Enhanced™ RF Antibody ELISAs can be used in either a semi-quantitative or a qualitative mode, a separate qualitative analysis using six human serum samples was performed according to CLSI EP12-A2. Ninety replicates were measured for sera in the negative range, ~20% below the cut-off, at ~cut-off, ~20% above the cut-off and in the moderate positive and high positive range of the assays. All values met the manufacturer's acceptance criteria and are summarized in the tables below.
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | |
| --- | --- | --- | --- |
| Sample | Mean (EU/mL) | %Negative | %Positive |
| Low Negative | 9.4 | 100% | 0% |
| Cut-off -20% | 14.9 | 100% | 0% |
| Cut-off | 21.0 | 13.3% | 86.7% |
| Cut-off +20% | 25.4 | 0% | 100% |
| Moderate Positive | 76.8 | 0% | 100% |
| High Positive | 117.9 | 0% | 100% |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | |
| --- | --- | --- | --- |
| Sample | Mean (EU/mL) | %Negative | %Positive |
| Low Negative | 7.7 | 100% | 0% |
| Cut-off -20% | 16.1 | 100% | 0% |
| Cut-off | 19.4 | 77.8% | 22.2% |
| Cut-off +20% | 23.2 | 0% | 100% |
| Moderate Positive | 48.8 | 0% | 100% |
| High Positive | 76.6 | 0% | 100% |
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| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | |
| --- | --- | --- | --- |
| Sample | Mean (IU/mL) | %Negative | %Positive |
| Low Negative | 4.8 | 100% | 0% |
| Cut-off -20% | 8.5 | 100% | 0% |
| Cut-off | 10.2 | 30% | 70% |
| Cut-off +20% | 11.5 | 0% | 100% |
| Moderate Positive | 19.1 | 0% | 100% |
| High Positive | 24.8 | 0% | 100% |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA | | | |
| --- | --- | --- | --- |
| Sample | Mean (EU/mL) | %Negative | %Positive |
| Low Negative | 12.5 | 100% | 0% |
| Cut-off -20% | 17.8 | 100% | 0% |
| Cut-off | 19.8 | 59% | 41% |
| Cut-off +20% | 24.4 | 0% | 100% |
| Moderate Positive | 55.4 | 0% | 100% |
| High Positive | 97.2 | 0% | 100% |
b. Linearity/assay reportable range:
i. Linearity:
The linear ranges for the ImmuLisa Enhanced™ RF Antibody ELISAs were determined in accordance with CLSI EP06-A. Three human serum samples were diluted with the assay kit sample diluent to cover the entire calibration range and tested in duplicate. The measured values were graphed against the expected values and a linear regression was performed. Results are summarized below and met the manufacturer's pre-determined acceptance criteria.
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample | Dilution range (EU/mL) | Slope (95% CI) | Intercept (95% CI) | R² | % Recovery Range |
| 1 | 4.8–13.7 | 0.96 (0.86–1.06) | 0.83 (-0.12–1.77) | 1.00 | 91.2%–100% |
| 2 | 10.5–64.9 | 0.96 (0.84–1.09) | 3.66 (-1.16–8.48) | 0.98 | 83.4%–101.3% |
| 3 | 17.3–155.0 | 1.00 (0.88–1.14) | 2.55 (-9.4–14.5) | 0.99 | 88%–104.1% |
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| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample | Dilution range (EU/mL) | Slope (95% CI) | Intercept (95% CI) | R² | % Recovery Range |
| 1 | 3.6–13.8 | 1.00 (0.92–1.08) | 0.33 (-0.40–1.07) | 1.00 | 92.2%–100.9% |
| 2 | 7.0–68.7 | 1.01 (0.89–1.12) | 1.81 (-2.85–6.47) | 0.99 | 87.5%–100.1% |
| 3 | 16.1–154.7 | 1.07 (0.95–1.20) | -1.59 (-13.3–10.2) | 1.00 | 88.2%–105.6% |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | | | |
| --- | --- | --- | --- | --- | --- |
| Sample | Dilution range (IU/mL) | Slope (95% CI) | Intercept (95% CI) | R² | % Recovery Range |
| 1 | 2.0–8.1 | 0.97 (0.82–1.26) | -0.50 (-2.60–1.25) | 1.00 | 88.2%–105.6% |
| 2 | 8.1–61.4 | 0.96 (0.91–1.01) | 1.43 (-0.56–3.43) | 1.00 | 87.6%–103.1% |
| 3 | 13.0–73.3 | 1.00 (0.84–1.15) | 2.24 (-4.88–9.36) | 1.00 | 89.4%–100.0% |
The claimed reportable ranges for the RF Antibody ELISAs are:
IgA: 3.7–160 EU/mL
IgG: 2.2–160 EU/mL
IgM: 1.3–80 IU/mL
ii. **High dose hook effect:**
High antibody concentrations specimens were serially diluted to assess the presence of decreases in assay signal associated with antigen excess. No hook effect was seen above the analytical measuring range.
c. **Traceability, Stability, Expected values (controls, calibrators, or methods):**
i. **Traceability:**
There are no reference standards or methods for RF IgA and IgG. The calibrators and controls for the ImmuLisa Enhanced™ RF IgA and RF IgG Antibody ELISAs are traceable to in-house standards. The ImmuLisa Enhanced™ RF IgM Antibody ELISA is traceable directly to the International Rheumatoid Arthritis Standard, NIBSC 64/2.
ii. **Value Assignment:**
Calibrators and Positive Controls are dilutions of pooled RF antibody positive sera. The manufacturer formulates new calibrator and control lots from an array of antibody positive sera obtained from various commercial plasma centers stored frozen at $-70^{\circ}\mathrm{C}$. The calibrators and controls are taken from different pooled
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sera. Manufactured calibrator sets are stored in aliquots frozen at $-70^{\circ}\mathrm{C}$. As new lots of calibrators are developed, comparison studies are performed to calibrate values against original calibrators. Each lot of calibrator is also tested in comparison with normal human sera, clinical samples and internal standards.
iii. **Kit Stability:**
**Shelf-life stability:** Accelerated and real-time stability studies were performed on three reagent lots of each ImmuLisa Enhanced™ RF IgA, IgG, and IgM Antibody ELISAs and the RF IgA/IgG/IgM ELISA. Data from the accelerated stability study support a shelf-life of 18 months. Currently available data from the ongoing real-time stability study support a stability claim of 9 months for ImmuLisa Enhanced™ RF IgA and RF IgG Antibody ELISAs and 12 months for ImmuLisa Enhanced™ RF IgM Antibody and RF IgA/IgG/IgM ELISAs.
**Open-kit stability:** Each ImmuLisa Enhanced™ RF Antibody and RF IgA/IgG/IgM ELISA kit was tested after first opening and determined to be stable at 45 days, but the sponsor chose a more restrictive 30-day open-kit stability claim.
**Sample Stability:** The manufacturer recommends storing specimens at $2^{\circ} - 8^{\circ}\mathrm{C}$ for no longer than one week. For longer storage, serum specimens should be frozen. It is further recommended that frozen specimens be tested within one year. Avoid repeated freezing and thawing of samples.
d. **Detection limit:**
The Limit of Blank (LoB) and Limit of Detection (LoD) were determined in accordance with CLSI EP17-A2. For LoB determinations, 60 samples of kit diluent were measured with one reagent lot of each ImmuLisa Enhanced™ RF Antibody kit. For LoD determination, ten replicates of each of six low-level serum samples were tested with one reagent lot of ImmuLisa Enhanced™ RF Antibody kit. The results are presented in the table below:
| ImmuLisa Enhanced™ RF Antibody | LoB | LoD |
| --- | --- | --- |
| IgA | 3.2 EU/mL | 3.7 EU/mL |
| IgG | 1.5 EU/mL | 2.2 EU/mL |
| IgM | 1.2 IU/mL | 1.3 IU/mL |
e. **Analytical specificity:**
i. **Endogenous interference:**
Interference studies were performed according to CLSI EP07-A2 by testing five samples: one negative, two with RF concentrations near the assay cutoff, and two
14
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moderate to high positive samples. Each sample was mixed with potentially interfering substances; hemoglobin (2 g/L), bilirubin (342 μmol/L), triglycerides (37 mmol/L) and cholesterol (13 mmol/L). The deviation from the expected result was calculated for each sample tested. Triglycerides and bilirubin interfered (≥15% in at least two of five samples) with the RF IgM assay at the concentrations tested. The sponsor has added a caution that icteric, lipemic and hemolysed samples should be avoided for all assays.
f. Assay Cut-Off:
The assay cutoff is >20 EU/mL for the ImmuLisa Enhanced™ RF IgA and IgG Antibody and RF IgA/IgG/IgM ELISAs. The assay cut-off is >10 IU/mL for the ImmuLisa Enhanced™ RF IgM Antibody ELISA.
2. Comparison studies:
a. Method comparison with predicate device:
The ImmuLisa Enhanced™ RF Antibody and RF IgA/IgG/IgM ELISAs were tested in comparison to the Inova QuantaLite® ELISA kits using sera from a total of 377–427 patients with rheumatoid arthritis or infectious diseases and other autoimmune conditions (disease controls). The number of specimens tested varies and is indicated in the respective tables. A direct comparison was made between the device and the predicate with the same isotype specificity: IgA vs. IgA, IgG vs. IgG, IgM vs. IgM. The ImmuLisa Enhanced™ IgA/IgG/IgM ELISA was compared to all predicate devices individually and in aggregate, where a sample was declared positive for the predicate if a single isotype was positive, and negative if all predicate isotype-specific devices were negative.
Positive, negative, and overall percent agreements were calculated by grouping ImmuLisa Enhanced™ RF IgA, IgG, and IgM Antibody semi-quantitative ELISA indeterminate results with both the positive and the negative results separately. The data presented in the tables below represent results from the semi-quantitative ImmuLisa Enhanced™ RF Antibody IgA, IgG, and IgM ELISAs. Results were also calculated using the qualitative IgA, IgG, and IgM ELISAs and found to be identical to the results from the semi-quantitative ELISAs.
15
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| ImmuLisa Enhanced™ RF IgA Antibody ELISA vs QuantaLite® Inova RF IgA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Positive | QuantaLite® Inova RF IgA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | Positive: >20 EU/mL | 113 | 9 | 122 |
| | Negative: ≤20 EU/mL | 5 | 250 | 255 |
| | Total | 118 | 259 | 377 |
| | | | | |
| Positive Percent Agreement | 95.8% | 95% CI: 90.5%-98.2% | | |
| Negative Percent Agreement | 96.5% | 95% CI: 93.5%-98.2% | | |
| Overall Percent Agreement | 96.3% | 95% CI: 93.9%-97.8% | | |
| n = 147 Rheumatoid Arthritis Samples, n = 230 Disease Control Samples | | | | |
| ImmuLisa Enhanced™ RF IgA Antibody ELISA vs QuantaLite® Inova RF IgA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Negative | QuantaLite® RF IgA ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | Positive: ≥25 EU/mL | 99 | 5 | 104 |
| | Negative: <25 EU/mL | 19 | 254 | 273 |
| | Total | 118 | 259 | 377 |
| | | | | |
| Positive Percent Agreement | 83.9% | 95% CI: 76.2%-89.4% | | |
| Negative Percent Agreement | 98.1% | 95% CI: 95.6%-99.2% | | |
| Overall Percent Agreement | 93.6% | 95% CI: 90.7%-95.7% | | |
| n = 147 Rheumatoid Arthritis Samples, n = 230 Disease Control Samples | | | | |
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| ImmuLisa Enhanced™ RF IgG Antibody ELISA vs QuantaLite® Inova RF IgG | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Positive | QuantaLite® RF IgG ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | Positive: >20 EU/mL | 88 | 24 | 112 |
| | Negative: ≤20 EU/mL | 7 | 271 | 278 |
| | Total | 95 | 295 | 390 |
| | | | | |
| Positive Percent Agreement | 92.6% | 95% CI, 85.6%-96.4% | | |
| Negative Percent Agreement | 91.9% | 95% CI, 88.2%-94.5% | | |
| Overall Percent Agreement | 92.1% | 95% CI, 88.9%-94.3% | | |
| n = 154 Rheumatoid Arthritis Samples, n = 236 Disease Control Samples | | | | |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA vs QuantaLite® Inova RF IgG | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Negative | QuantaLite® RF IgG ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | Positive: ≥25 EU/mL | 78 | 16 | 94 |
| | Negative: <25 EU/mL | 17 | 279 | 296 |
| | Total | 95 | 295 | 390 |
| | | | | |
| Positive Percent Agreement | 82.1% | 95% CI, 73.2%-88.5% | | |
| Negative Percent Agreement | 94.6% | 95% CI, 91.4%-96.6% | | |
| Overall Percent Agreement | 91.5% | 95% CI, 88.4%-93.9% | | |
| n = 154 Rheumatoid Arthritis Samples, n = 236 Disease Control Samples | | | | |
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| ImmuLisa Enhanced™ RF IgM Antibody ELISA vs QuantaLite® Inova RF IgM | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Positive | QuantaLite® RF IgM ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | Positive: >20 EU/mL | 124 | 16 | 140 |
| | Negative: ≤20 EU/mL | 4 | 235 | 239 |
| | Total | 128 | 251 | 379 |
| | | | | |
| Positive Percent Agreement | 96.9% | 95% CI: 92.2%–98.8% | | |
| Negative Percent Agreement | 93.6% | 95% CI: 89.9%–96.0% | | |
| Overall Percent Agreement | 94.7% | 95% CI: 92.0%–96.6% | | |
| n = 139 Rheumatoid Arthritis Samples, n = 240 Disease Control Samples | | | | |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA vs QuantaLite® Inova RF IgM | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Negative | QuantaLite® RF IgM ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | Positive: ≥25 EU/mL | 120 | 12 | 132 |
| | Negative: <25 EU/mL | 8 | 239 | 247 |
| | Total | 128 | 251 | 379 |
| | | | | |
| Positive Percent Agreement | 93.8% | 95% CI: 88.2%–96.8% | | |
| Negative Percent Agreement | 95.2% | 95% CI: 91.8%–97.2% | | |
| Overall Percent Agreement | 94.7% | 95% CI: 92.0%–96.6% | | |
| n = 139 Rheumatoid Arthritis Samples, n = 240 Disease Control Samples | | | | |
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| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA vs QuantaLite® Inova RF IgA | | | | |
| --- | --- | --- | --- | --- |
| | QuantaLite® RF IgA ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA | Positive | 115 | 53 | 168 |
| | Negative | 10 | 216 | 226 |
| | Total | 125 | 269 | 394 |
| | | | | |
| Positive Percent Agreement | 92.0% | 95% CI: 85.9%-95.6% | | |
| Negative Percent Agreement | 80.3% | 95% CI: 75.1%-84.6% | | |
| Overall Percent Agreement | 84.0% | 95% CI: 80.1%-87.3% | | |
| n = 152 Rheumatoid Arthritis Samples, n = 242 Disease Control Samples | | | | |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA vs QuantaLite® Inova RF IgG | | | | |
| --- | --- | --- | --- | --- |
| | QuantaLite® RF IgG ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA | Positive | 95 | 78 | 173 |
| | Negative | 3 | 223 | 226 |
| | Total | 98 | 301 | 399 |
| | | | | |
| Positive Percent Agreement | 96.9% | 95% CI: 91.4%-99.0% | | |
| Negative Percent Agreement | 74.1% | 95% CI: 68.9%-78.7% | | |
| Overall Percent Agreement | 79.7% | 95% CI: 75.5%-83.4% | | |
| n = 157 Rheumatoid Arthritis Samples, n = 242 Disease Control Samples | | | | |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA vs QuantaLite® Inova RF IgM | | | | |
| --- | --- | --- | --- | --- |
| | QuantaLite® RF IgM ELISA | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA | Positive | 161 | 35 | 196 |
| | Negative | 11 | 215 | 226 |
| | Total | 172 | 250 | 422 |
| | | | | |
| Positive Percent Agreement | 93.6% | 95% CI: 88.9%-96.4% | | |
| Negative Percent Agreement | 86.0% | 95% CI: 81.2%-89.8% | | |
| Overall Percent Agreement | 89.1% | 95% CI: 85.8%-91.7% | | |
| n = 180 Rheumatoid Arthritis Samples, n = 242 Disease Control Samples | | | | |
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| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA vs QuantaLite® Inova RF IgA/IgG/IgM | | | | |
| --- | --- | --- | --- | --- |
| | QuantaLite®RF IgA/IgG/IgM ELISA Aggregate Data | | | |
| | | At Least One Positive | All Negative | Total |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA | Positive | 183 | 18 | 201 |
| | Negative | 21 | 205 | 226 |
| | Total | 204 | 223 | 427 |
| | | | | |
| Positive Percent Agreement | | 89.7% | 95% CI: 84.8%–93.2% | |
| Negative Percent Agreement | | 91.9% | 95% CI: 87.6%–94.8% | |
| Overall Percent Agreement | | 90.9% | 95% CI: 87.8%–93.2% | |
| n = 184 Rheumatoid Arthritis Samples, n = 243 Disease Control Samples | | | | |
b. Matrix comparison:
Not applicable since human serum is the only claimed specimen matrix.
3. Clinical studies:
a. Clinical Sensitivity and Specificity:
The performances of the ImmuLisa Enhanced™ RF Antibody and RF IgA/IgG/IgM ELISAs were compared to a clinical diagnosis of RA. The validation set consisted of 179–249 clinically characterized sera from RA and 310 disease controls. Clinical sensitivity and specificity were calculated with 95% confidence intervals (95% CI).
Clinical sensitivity and specificity were calculated by grouping ImmuLisa Enhanced™ RF IgA, IgG, and IgM Antibody semi-quantitative ELISA indeterminate results with both the positive and the negative results separately. The data presented in the tables below represent results from the semi-quantitative ImmuLisa Enhanced™ RF Antibody IgA, IgG, and IgM ELISAs. Results were also calculated using the qualitative IgA, IgG, and IgM ELISAs and found to be identical to the results from the semi-quantitative ELISAs.
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| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Positive | RA Diagnosis | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | Positive: >20 EU/mL | 131 | 33 | 164 |
| | Negative: ≤20 EU/mL | 118 | 277 | 395 |
| | Total | 249 | 310 | 559 |
| | | | | |
| Sensitivity | 52.6% | 95% CI: 46.4%-58.7% | | |
| Specificity | 89.4% | 95% CI: 85.4%-92.3% | | |
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Negative | RA Diagnosis | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA Antibody ELISA | Positive: ≥25 EU/mL | 115 | 23 | 138 |
| | Negative: <25 EU/mL | 134 | 287 | 421 |
| | Total | 249 | 310 | 559 |
| | | | | |
| Sensitivity | 46.2% | 95% CI: 40.1%-52.4% | | |
| Specificity | 92.6% | 95% CI: 89.1%-95.0% | | |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Positive | RA Diagnosis | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | Positive: >20 EU/mL | 126 | 23 | 149 |
| | Negative: ≤20 EU/mL | 123 | 287 | 410 |
| | Total | 249 | 310 | 559 |
| | | | | |
| Sensitivity | 50.6% | 95% CI: 44.4%-56.8% | | |
| Specificity | 92.6% | 95% CI: 89.1%-95.0% | | |
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| ImmuLisa Enhanced™ RF IgG Antibody ELISA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Negative | RA Diagnosis | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgG Antibody ELISA | Positive: ≥25 EU/mL | 98 | 16 | 114 |
| | Negative: <25 EU/mL | 151 | 294 | 445 |
| | Total | 249 | 310 | 559 |
| | | | | |
| Sensitivity | 39.4% | 95% CI: 33.5%-45.5% | | |
| Specificity | 94.8% | 95% CI: 91.8%-96.8% | | |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Positive | RA Diagnosis | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | Positive: >20 EU/mL | 176 | 39 | 215 |
| | Negative: ≤20 EU/mL | 73 | 271 | 344 |
| | Total | 249 | 310 | 559 |
| | | | | |
| Sensitivity | 70.7% | 95% CI: 64.7%-76.0% | | |
| Specificity | 87.4% | 95% CI: 83.3%-90.7% | | |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | | | | |
| --- | --- | --- | --- | --- |
| Indeterminate Samples Called Negative | RA Diagnosis | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgM Antibody ELISA | Positive: ≥25 EU/mL | 159 | 36 | 195 |
| | Negative: <25 EU/mL | 90 | 274 | 364 |
| | Total | 249 | 310 | 559 |
| | | | | |
| Sensitivity | 63.9% | 95% CI: 57.7%-69.6% | | |
| Specificity | 88.4% | 95% CI: 84.3%-91.5% | | |
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23
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA | | | | |
| --- | --- | --- | --- | --- |
| | RA Diagnosis | | | |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced™ RF IgA/IgG/IgM ELISA | Positive: ≥25 EU/mL | 152 | 45 | 197 |
| | Negative: <25 EU/mL | 27 | 265 | 292 |
| | Total | 179 | 310 | 489 |
| | | | | |
| Sensitivity | 84.9% | 95% CI: 78.9%–89.4% | | |
| Specificity | 85.5% | 95% CI: 81.1%–89.0% | | |
b. Incidence of cross-reactivity in related autoimmune and infectious diseases
A total of 310 samples from related autoimmune and infectious diseases were tested with each ImmuLisa Enhanced™ RF Antibody ELISA and the ImmuLisa Enhanced™ IgA/IgG/IgM ELISA to determine incidence of positive results. Results are presented in the table below:
| Clinical Diagnosis | # Samples | % Positive | | | |
| --- | --- | --- | --- | --- | --- |
| | | RF IgA | RF IgG | RF IgM | RF IgA/IgG/IgM |
| Juvenile Arthritis | 10 | 0% | 10% | 10% | 10% |
| Osteoarthritis | 30 | 0% | 0% | 3% | 0% |
| Psoriatic Arthritis | 33 | 6% | 9% | 6% | 6% |
| Spondyloarthritis | 33 | 3% | 0% | 3% | 3% |
| Mixed Connective Tissue Disease | 10 | 10% | 20% | 40% | 30% |
| Churg-Strauss | 10 | 10% | 10% | 10% | 20% |
| Sjögren's Syndrome | 20 | 45% | 10% | 55% | 45% |
| Systemic Lupus Erythematosus | 30 | 37% | 20% | 17% | 40% |
| Systemic Sclerosis | 20 | 15% | 20% | 25% | 25% |
| Wegener's | 8 | 13% | 13% | 13% | 13% |
| Celiac Disease | 8 | 0% | 0% | 0% | 0% |
| Graves' Disease | 10 | 0% | 10% | 10% | 10% |
| Hashimoto's Disease | 8 | 0% | 0% | 0% | 0% |
| Ulcerative Colitis | 10 | 0% | 10% | 0% | 10% |
| Syphilis | 10 | 0% | 0% | 0% | 0% |
| Rubella | 10 | 0% | 0% | 10% | 10% |
| Mononucleosis | 5 | 20% | 0% | 0% | 0% |
{23}
| Clinical Diagnosis | # Samples | % Positive | | | |
| --- | --- | --- | --- | --- | --- |
| | | RF IgA | RF IgG | RF IgM | RF IgA/IgG/ IgM |
| Lyme | 5 | 0% | 0% | 0% | 0% |
| HCV | 10 | 10% | 10% | 20% | 30% |
| CMV | 10 | 0% | 0% | 0% | 10% |
| HSV | 10 | 10% | 0% | 20% | 10% |
| Toxoplasmosis | 10 | 10% | 0% | 10% | 10% |
# 4. Clinical cut-off:
See Assay Cut-Off
# 5. Expected values/Reference range:
A reference range study was performed with 62-80 normal human serum samples, and a verification study with 121-137 samples that were a combination of normal human sera and sera from patients with infectious diseases and other autoimmune conditions that were not RA (disease controls). The upper limit of the optical density (OD) results from the normal samples was calculated using the average plus 2 standard deviations for ImmuLisa Enhanced™ RF IgA and IgG ELISAs, and the average plus 2.5 standard deviations for ImmuLisa Enhanced™ RF IgM and IgA/IgG/IgM ELISAs. This OD was set to $20\mathrm{EU / mL}$ for the IgA, IgG, and IgA/IgG/IgM ELISA, and $10\mathrm{IU / mL}$ for the IgM ELISA. The data from the verification study are summarized in the table below:
| ImmuLisa Enhanced™ RF Antibody | # of Samples | % Samples Negative | % Samples Indeterminate | % Samples Positive |
| --- | --- | --- | --- | --- |
| IgA | 127 | 94.5% | 3.9% | 1.6% |
| IgG | 121 | 99.0% | 1.0% | 0.0% |
| IgM | 137 | 99.0% | 1.0% | 0.0% |
| IgA/IgG/IgM | 137 | 100.0% | 0.0% | 0.0% |
# 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.
