IMMULISA ENHANCED (TM) CARDIOLIPIN IGA, IGG, IGM AND IGA/IGG/IGM ANTIBODY (ACA) ELISAS

{0} # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: k113020 B. Purpose for Submission: New Device C. Measurand: IgG, IgM, and IgA antibodies to Cardiolipin (aCL) D. Type of Test: Qualitative and semi-quantitative microwell-based ELISA assay E. Applicant: IMMCO Diagnostics, Inc. F. Proprietary and Established Names: ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA G. Regulatory Information: 1. Regulation section: 21 CFR §866.5660 – Multiple autoantibodies immunological test system 2. Classification: Class II 3. Product code: MID – System, Test, Anti-Cardiolipin Immunological {1} 4. Panel: Immunology (82) H. Intended Use: 1. Intended use(s): ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA: Enzyme linked immunoassays (ELISAs) for the qualitative or semi-quantitative detection of Cardiolipin IgA antibodies in human serum to aid in the diagnosis of antiphospholipid syndrome (APS) and APS associated with systemic lupus erythematosus (SLE) in conjunction with other laboratory tests and clinical findings. ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA: Enzyme linked immunoassays (ELISAs) for the qualitative or semi-quantitative detection of Cardiolipin IgG antibodies in human serum to aid in the diagnosis of antiphospholipid syndrome (APS) and APS associated with systemic lupus erythematosus (SLE) in conjunction with other laboratory tests and clinical findings. ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA: Enzyme linked immunoassays (ELISAs) for the qualitative or semi-quantitative detection of Cardiolipin IgM antibodies in human serum to aid in the diagnosis of antiphospholipid syndrome (APS) and APS associated with systemic lupus erythematosus (SLE) in conjunction with other laboratory tests and clinical findings. ImmuLisa Enhanced™ Cardiolipin Antibody IgA/IgG/IgM Antibody (ACA) ELISA: Enzyme linked immunoassay (ELISA) for the qualitative detection of Cardiolipin IgA, IgG and IgM antibodies in human serum to aid in the diagnosis of anti-phospholipid syndrome (APS) and APS associated with systemic lupus erythematosus (SLE) 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: {2} An ELISA microplate reader capable of reading absorbance values at 450nm. If a dual wavelength microplate reader is available, the reference filter should be set at 600-650 nm. I. Device Description: Each ImmuLisa Enhanced™ Cardiolipin Antibody IgA, IgG or IgM (ACA) ELISA kit contains: a microplate with individual breakaway wells coated with Cardiolipin antigen and bovine/human Beta-2 glycoprotein 1 (β2GP1); Five levels of calibrators (120, 60, 30, 15, and 1 APL or GPL or MPL); antibody specific positive and negative controls; horseradish peroxidase (HRP) conjugated goat anti-human IgG or IgA or IgM; serum diluent; wash buffer concentrate; 3,3',5,5', tetramethylbenzidine (TMB); and stop solution. Each ImmuLisa Enhanced™ Cardiolipin Antibody IgA/IgG/IgM Antibody (ACA) ELISA kit contains: a microplate with individual breakaway wells coated with Cardiolipin antigen and bovine/human Beta-2 glycoprotein 1 (β2GP1); One level of calibrators (30 EU/mL); positive and negative controls; horseradish peroxidase (HRP) conjugated goat anti-human IgG/IgA/IgM; serum diluent; wash buffer concentrate; 3,3',5,5', tetramethylbenzidine (TMB); and stop solution. J. Substantial Equivalence Information: 1. Predicate device name(s) and 510(k) number(s) QUANTA Lite™ ACA IgA ELISA, k953366 QUANTA Lite™ ACA IgG ELISA, k946034 QUANTA Lite™ ACA IgM ELISA, k946385 QUANTA Lite™ ACA Screen ELISA, k953291 2. Comparison with predicate: ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA/ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA/ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA {3} | Similarities | | | | --- | --- | --- | | Item | Device ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Predicate QUANTA Lite™ ACA IgA ELISA QUANTA Lite™ ACA IgA ELISA QUANTA Lite™ ACA IgM ELISA | | Intended Use | An enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of Cardiolipin antibodies in human serum to aid in the diagnosis of anti-phospholipid syndrome (APS) and APS associated with systemic lupus erythematosus (SLE) in conjunction with other laboratory tests and clinical findings. | An enzyme-linked immunosorbent assay (ELISA) for the qualitative or semi-quantitative detection of cardiolipin antibodies in human serum. The presence of cardiolipin antibodies can be used in conjunction with clinical findings and other laboratory tests to aid in assessing the risk of thrombosis in individuals with systemic lupus erythematosus (SLE) or lupus-like disorders. | | Methodology | ELISA | Same | | Assay Format | Semi-quantitative and qualitative | Same | | Sample Matrix | Serum | Same | | Coating Antigens | Purified Cardiolipin antigen (1,1',2,2' Tetraoleoyl Cardiolipin (sodium salt)) and β2GP1 from human/bovine serum) | Same | | Detection antibody | Goat anti-human IgA, IgG or IgM | Same | | Conjugate | HRP | Same | | Substrate/Chromogen | TMB | Same | | Controls | One Cardiolipin IgA or IgG or IgM Positive Control, one Negative Control | Same | | Calibration | 5-level Calibrators | Same | | Stop solution | H2SO4 | Same | | Dilution | 1:101 | Same | | Reading | 450 nm on spectrophotometer | Same | {4} | Similarities | | | | --- | --- | --- | | Item | Device ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Predicate QUANTA Lite™ ACA IgA ELISA QUANTA Lite™ ACA IgA ELISA QUANTA Lite™ ACA IgM ELISA | | Storage | 2 – 8°C | Same | | IgG Cutoff | 15 GPL | Same | | Differences | | | | --- | --- | --- | | Item | Device ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Predicate QUANTA Lite™ ACA IgA ELISA QUANTA Lite™ ACA IgA ELISA QUANTA Lite™ ACA IgM ELISA | | Wash Buffer | Powdered or optional liquid concentrate | Liquid concentrate | | IgA Cutoff | 15 APL | 12 APL | | IgM Cutoff | 15 MPL | 12.5 MPL | | Calibrators | Set of 5: 120, 60, 30, 15, 1 APL or GPL or MPL | Set of 5: 150, 75, 37.5, 18.75, 9.375 APL or GPL or MPL | | Reportable Range | IgA: 1.8 APL – 120 APL IgG: 1.0 GPL – 120 GPL IgM: 2.8 MPL – 120 MPL | Not specified | | Limit of Detection | IgA: 1.8 APL IgG: 1.0 GPL IgM: 2.8 MPL | Not specified | | Results Interpretation | IgA: < 15 APL – negative 15 – 22.5 APL – indeterm > 22.5 APL – positive IgG: < 15 GPL – negative 15 – 22.5 GPL – indeterm > 22.5 GPL – positive IgM: < 15 GPL – negative 15 – 22.5 MPL – indeterm > 22.5 MPL – positive | IgA: < 12 APL – negative 12 – 20 APL – indeterm > 20 APL – positive IgG: < 15 GPL – negative 15 – 20 GPL – indeterm > 20 GPL – positive IgM: < 15 GPL – negative 15 – 22.5 MPL – indeterm > 20 MPL – positive | {5} ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA | Similarities | | | | --- | --- | --- | | Item | Device:ImmuLisa Enhanced™Cardiolipin IgA/IgG/IgMAntibody (ACA) ELISA | Predicate:QUANTA Lite™ ACA ScreenELISA | | Intended Use | An enzyme linkedimmunoassay (ELISA) forthe qualitative detection ofCardiolipin IgA, IgG andIgM antibodies in humanserum to aid in the diagnosisof anti-phospholipidsyndrome (APS) and APSassociated with systemiclupus erythematosus (SLE)in conjunction with otherlaboratory tests and clinicalfindings. | An enzyme-linked immunosorbentassay (ELISA) for the qualitativedetection of cardiolipin antibodies inhuman serum. The presence ofcardiolipin antibodies can be used inconjunction with clinical findingsand other laboratory tests to aid inassessing the risk of thrombosis inindividuals with systemic lupuserythematosus (SLE) or lupus-likedisorders. | | Methodology | ELISA | Same | | Sample Matrix | Serum | Same | | CoatingAntigens | Purified Cardiolipin antigen(1,1',2,2' TetraoleoylCardiolipin (sodium salt))andβ2GP1 from human/bovineserum | Same | | DetectionAntibody | Goat anti-human IgA, IgG orIgM | Same | | Assay Format | Qualitative | Same | | Conjugate | HRP | Same | | Substrate/Chromogen | TMB | Same | | Controls | One Cardiolipin IgA, IgG,and IgM Positive Control,one Negative Control | Same | | Calibration | Single Calibrator | Same | | Stop Solution | H2SO4 | Same | | Dilution | 1:101 | Same | | Reading | 450nm on spectrophotometer | Same | | Storage | 2-8°C | Same | {6} | Differences | | | | --- | --- | --- | | Item | Device:ImmuLisa Enhanced™Cardiolipin IgA/IgG/IgMAntibody (ACA) ELISA | Predicate:QUANTA Lite™ Cardiolipin ScreenELISA | | Wash Buffer | Powdered or optional liquid concentrate | Liquid concentrate | | Calibrator | Single Calibrator. 30 EU/mL | Single decision point calibrator. No units assigned. | | Cut-off | 20 EU/mL | Not specified (≥ decision point calibrator) | | Limit of Detection | 5.2 EU/mL | Not specified | | Results Interpretation | < 20 EU/mL – negative≥ 20EU/mL – positive | All results with a mean absorbance greater than the decision point calibrator absorbance are considered positive< decision point calibrator – negative≥ decision point calibrator – positive | # K. Standard/Guidance Document Referenced (if applicable): EN 13640: 2002, Stability Testing of In Vitro Diagnostic Reagents CLSI EP06-A: Evaluation of Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline CLSI EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline – Second Edition CLSI EP09-A2: Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline - Second Edition CLSI EP12-A2: User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline - Second Edition CLSI EP17-A: Protocols for Determination of Limits of Detection and Limits of Quantification; Approved Guideline # L. Test Principle: Cardiolipin antigen and $\beta 2\mathrm{GP1}$ are bound to the wells of a polystyrene microwell plate followed by blocking the unreacted sites to reduce non-specific binding. {7} Controls, calibrators and diluted patient sera are added to separate wells, allowing any cardiolipin antibodies present to bind to the immobilized antigen. Unbound sample is washed away and an enzyme labeled anti-human IgG, IgA or IgM conjugate is added to each well. These enzyme conjugated antibodies bind specifically to the human immunoglobulin of the appropriate class. After washing away any unbound conjugate, specific enzyme substrate (TMB) is then added to the wells. After stopping the enzymatic reaction, the intensity of color change, which is proportional to the concentration of antibody, is read by a spectrophotometer at $450~\mathrm{nm}$ . Results are expressed in units per milliliter $(\mathrm{U} / \mathrm{mL})$ according to the Harris classification. # M. Performance Characteristics (if/when applicable): # 1. Analytical performance: # a. Precision/Reproducibility: # Semi-Quantitative Precision: Six samples from different parts of the claimed assay ranges were tested to establish between-day precision and within-run repeatability. Between-day imprecision was tested in replicates of six over thirteen assay runs $(n = 78)$ ; Within-run repeatability was tested by 12 replicates performed in a single run. The total number of replicates was 90. Results are summarized below. ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | Sample | Mean (APL) | Within-Run (Repeatability) | | Between-Day | | Total Imprecision | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | %CV | SD | %CV | SD | %CV | | 1 | 2.5 | 0.234 | 8.8% | 0.366 | 14.6% | 0.4 | 14.0% | | 2 | 12.4 | 0.915 | 7.4% | 1.014 | 8.2% | 1.0 | 8.0% | | 3 | 17.9 | 1.233 | 6.9% | 1.470 | 8.2% | 1.4 | 8.0% | | 4 | 29.1 | 1.466 | 5.2% | 2.499 | 8.5% | 2.4 | 8.2% | | 5 | 53.1 | 3.088 | 5.9% | 4.182 | 7.9% | 4.1 | 7.6% | | 6 | 103.7 | 5.847 | 5.5% | 6.922 | 6.7% | 6.8 | 6.5% | ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA | Sample | Mean (GPL) | Within-Run (Repeatability) | | Between-Day | | Total Imprecision | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | %CV | SD | %CV | SD | %CV | | 1 | 3.8 | 0.207 | 9.6% | 0.177 | 8.6% | 0.183 | 9.2% | | 2 | 12.0 | 1.050 | 9.1% | 1.118 | 9.3% | 1.117 | 9.3% | | 3 | 18.0 | 1.383 | 7.7% | 1.723 | 9.5% | 1.676 | 9.3% | | 4 | 31.3 | 1.651 | 5.5% | 2.805 | 8.9% | 2.738 | 8.7% | | 5 | 80.4 | 3.317 | 4.1% | 8.049 | 10.0% | 7.586 | 9.4% | | 6 | 99.0 | 4.560 | 4.7% | 7.011 | 7.0% | 6.806 | 6.9% | {8} ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Sample | Mean (MPL) | Within-Run (Repeatability) | | Between-Day | | Total Imprecision | | | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | %CV | SD | %CV | SD | %CV | | 1 | 11.6 | 0.407 | 3.5% | 0.930 | 8.0% | 0.930 | 8.0% | | 2 | 13.0 | 0.904 | 7.0% | 1.084 | 8.4% | 1.084 | 8.4% | | 3 | 17.0 | 0.598 | 3.5% | 1.257 | 7.4% | 1.257 | 7.4% | | 4 | 45.7 | 1.305 | 2.9% | 2.292 | 5.0% | 2.292 | 5.0% | | 5 | 79.9 | 3.609 | 4.5% | 5.580 | 7.0% | 5.580 | 7.0% | | 6 | 114.8 | 4.811 | 4.2% | 6.603 | 5.8% | 6.603 | 5.8% | # Lot-to-lot Reproducibility: To assess lot-to-lot reproducibility, samples were selected through the linear range for ImmuLisa Enhanced™ Cardiolipin IgA, IgG and IgM Antibody (ACA) ELISAs. Three runs of each lot were performed. Results are summarized below. ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA Lot-to-Lot Reproducibility | Sample | Mean (APL) | Lot 1 | | Lot 2 | | Lot 3 | | Lot-to-Lot Variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 8.2 | 0.9 | 11.6% | 1.3 | 14.7% | 0.8 | 9.8% | 1.0 | 12.2% | | 2 | 14.3 | 0.8 | 5.5% | 0.8 | 5.0% | 0.6 | 4.1% | 0.9 | 6.4% | | 3 | 19.0 | 1.7 | 9.2% | 1.7 | 8.7% | 1.1 | 5.9% | 1.6 | 8.2% | | 4 | 36.1 | 2.0 | 5.7% | 1.7 | 4.4% | 2.7 | 7.7% | 2.3 | 6.3% | | 5 | 56.7 | 1.8 | 3.3% | 2.2 | 3.7% | 2.1 | 3.9% | 3.3 | 5.8% | | 6 | 77.1 | 3.1 | 4.0% | 2.9 | 3.7% | 3.3 | 4.3% | 2.9 | 3.8% | | 7 | 104.2 | 4.0 | 3.8% | 2.4 | 2.2% | 2.2 | 2.2% | 4.4 | 4.3% | ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA Lot-to-Lot Reproducibility | Sample | Mean (GPL) | Lot 1 | | Lot 2 | | Lot 3 | | Lot-to-Lot Variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 3.7 | 0.6 | 16.4% | 0.6 | 13.5% | 1.2 | 33.7% | 0.8 | 21.2% | | 2 | 13.4 | 0.5 | 3.5% | 0.8 | 5.3% | 0.9 | 7.1% | 0.9 | 6.7% | | 3 | 20.1 | 1.6 | 8.0% | 1.8 | 8.6% | 1.2 | 6.3% | 1.9 | 9.6% | | 4 | 26.2 | 2.3 | 9.4% | 1.2 | 4.5% | 1.6 | 5.9% | 2.0 | 7.5% | | 5 | 47.5 | 2.8 | 6.1% | 3.0 | 6.0% | 2.0 | 4.3% | 3.4 | 7.2% | | 6 | 80.4 | 8.1 | 10.1% | 3.6 | 4.2% | 3.4 | 4.5% | 6.6 | 8.2% | | 7 | 111.4 | 4.6 | 4.2% | 5.1 | 4.4% | 5.0 | 4.6% | 5.1 | 4.6% | {9} ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA Lot-to-Lot Reproducibility | Sample | Mean (MPL) | Lot 1 | | Lot 2 | | Lot 3 | | Lot-to-Lot Variation | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | | 1 | 10.1 | 1.0 | 8.8% | 0.7 | 8.7% | 1.3 | 12.2% | 1.6 | 15.3% | | 2 | 15.0 | 0.5 | 3.4% | 0.9 | 6.2% | 1.0 | 6.6% | 0.9 | 5.9% | | 3 | 19.1 | 1.0 | 5.1% | 1.5 | 8.5% | 1.5 | 7.3% | 1.6 | 8.3% | | 4 | 28.4 | 1.6 | 5.3% | 2.8 | 10.4% | 2.3 | 7.9% | 2.3 | 7.9% | | 5 | 43.8 | 3.1 | 7.0% | 2.4 | 5.7% | 3.0 | 6.5% | 2.8 | 6.4% | | 6 | 82.1 | 8.1 | 9.9% | 3.9 | 5.1% | 5.4 | 6.1% | 7.2 | 8.8% | | 7 | 96.6 | 3.2 | 3.3% | 4.5 | 4.9% | 4.1 | 4.0% | 6.2 | 6.4% | ## Qualitative Reproducibility: Five samples were tested in 90 runs; a low negative (but above limit of detection), a sample ~ 20% below cut-off, a sample at the cut-off concentration (15-APL, -GPL, or -MBL for IgA, IgG, or IgM kits respectively; 20 EU/mL for Screen), a sample ~ 20% above cut-off, and a moderate positive. Results are summarized below: ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | Sample | Test Range (APL) | Expected Result | # Samples with expected result | % Correct | | --- | --- | --- | --- | --- | | Negative | 2.2 | Negative | 90/90 | 100% | | ~ 20% below Cutoff | 12.1 | Negative | 89/90 | 99% | | ~ Cutoff | 15.3 | Positive | 47/90 | 52% | | ~ 20% above Cutoff | 17.5 | Positive | 88/90 | 98% | | Moderate Positive | 27.7 | Positive | 90/90 | 100% | ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA | Sample | Test Range (GPL) | Expected Result | # Samples with expected result | % Correct | | --- | --- | --- | --- | --- | | Negative | 3.1 | Negative | 90/90 | 100% | | ~ 20% below Cutoff | 11.5 | Negative | 90/90 | 100% | | ~ Cutoff | 15.4 | Positive | 54/90 | 60% | | ~ 20% above Cutoff | 18.6 | Positive | 89/90 | 99% | | Moderate Positive | 30.4 | Positive | 90/90 | 100% | {10} ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Sample | Test Range (MPL) | Expected Result | # Samples with expected result | % Correct | | --- | --- | --- | --- | --- | | Negative | 11.5 | Negative | 90/90 | 100% | | ~ 20% below Cutoff | 13.0 | Negative | 87/90 | 97% | | ~ Cutoff | 15.1 | Positive | 54/90 | 60% | | ~ 20% above Cutoff | 17.0 | Positive | 88/90 | 98% | | Moderate Positive | 46.1 | Positive | 90/90 | 100% | ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA | Sample | Test Range (EU/mL) | Expected Result | # Samples with expected result | % Correct | | --- | --- | --- | --- | --- | | Negative | 7 | Negative | 90/90 | 100% | | ~ 20% below Cutoff | 17.4 | Negative | 87/90 | 97% | | ~ Cutoff | 18.3 | Negative | 54/90 | 60% | | ~ 20% above Cutoff | 24.6 | Positive | 90/90 | 100% | | Moderate Positive | 60 | Positive | 90/90 | 100% | b. Linearity/assay reportable range: Linearity studies were performed according to CLSI EP-6A. Three serum samples for each of the isotype with values to cover the range of the assays were selected. Each sample was proportionally diluted with a known negative serum sample and tested. The observed values were graphed against the calculated values and a linear regression was performed. In cases where the high positive sample was above the highest calibrator (120 U/mL), the dilution points were truncated from the curve and recovery calculations. The results are summarized in the tables below: ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | Sample | Test Range (APL) | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery (obtained/expected) | | --- | --- | --- | --- | --- | --- | | 1 | 2.6 – 47.6 | 0.999 (0.95 to 1.05) | -0.52 (-1.95 to 9.91) | 1.00 | 97.7% to 114.0% | | 2 | 18.4 – 70.4 | 0.997 (0.85 to 1.15) | 1.57 (-5.28 to 8.43) | 0.98 | 85.1% to 101.9% | | 3 | 20.0 –107.6 | 1.126 (0.995 to 1.257) | 0.066 (-9.488 to 9.620) | 0.99 | 84.4% to 100% | {11} ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA | Sample | Test Range (GPL) | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery (obtained/expected) | | --- | --- | --- | --- | --- | --- | | 1 | 2.4 to 98.1 | 0.989 (0.899 to 1.078) | -0.725 (-6.114 to 4.664) | 0.99 | 95.9% to 114.6% | | 2 | 4.5 to 27.8 | 0.995 (0.938 to 1.052) | 0.348 (-0.657 to 1.353) | 1.00 | 90.8% to 101.1% | | 3 | 3.1 to 107.5 | 1.168 (0.940 to 1.396) | -4.734 (-20.637 to 11.170) | 0.97 | 82.6% to 102.8% | ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Sample | Test Range (MPL) | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery (obtained/expected) | | --- | --- | --- | --- | --- | --- | | 1 | 5.6 to 28.3 | 0.925 (0.830 to 1.021) | 1.696 (-0.051 to 3.443) | 0.99 | 83.0% to 102.3% | | 2 | 5.8 to 86.8 | 0.97 (0.87 to 1.07) | 1.159 (-4.371 to 6.689) | 0.99 | 93.4% to 106.9% | | 3 | 5.5 to 103.6 | 0.939 (0.764 to 1.113) | 4.780 (-5.89 to 15.46) | 0.99 | 81.6% to 105.2% | Additional IgA samples were tested to cover the lower range of the assay. Results are summarized below: | Test Range (APL) | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery (obtained/expected) | | --- | --- | --- | --- | --- | | 9.2 – 36.1 | 0.953 (0.861 to 1.045) | 1.319 (-1.049 to 3.687) | 0.99 | 95.8% to 104.6% | | 3.6 – 34.2 | 1.041 (0.884 to 1.198) | -0.207 (-3.666 to 3.251) | 0.99 | 87.7% to 104.4% | | 8.1 –30.6 | 0.999 (0.941 –1.057) | -0.235 (-1.450 –0.980) | 1 | 98.6% to 105.4% | The claimed reportable ranges of the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISAs are as follows: ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA: 1.8 – 120 APL; ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA: 1.0 – 120 GPL; ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA: 2.8 – 120 MPL. Linearity was not assessed for the qualitative ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA. {12} c. Traceability, Stability, Expected values (controls, calibrators, or methods): ## Traceability: There is no FDA recognized international reference material available for anti-cardiolipin antibodies. The calibrators and controls are assigned relative arbitrary units (U/mL -APL, -GPL, -MPL). Calibrator material (high positive patient sera) dilutions are compared to the Harris Standards to evaluate assay reference range. ## Value Assignment: Positive control and calibrators were derived from different pooled sera of cardiolipin/phospholipid antibody positive SLE/APS patients obtained from commercial sources. Antibody positivity and concentrations were confirmed using commercially available assays for cardiolipin IgA, IgG and IgM antibodies. Serial dilutions of calibrator are made to establish the 120 U/mL (Calibrator A), 60 U/mL (Calibrator B), 30 U/mL (Calibrator C), 15 U/mL (Calibrator D), and 1 U/mL (Calibrator E) calibrators. The samples are tested at various dilutions on at least two different lots of cardiolipin-coated plates. 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 as well as the Harris standards. Positive control lots are developed in the same manner as calibrators, but are derived from different sets of positive sera. ## Device Stability: Accelerated shelf life, real time shelf life, and open kit/reagent stability studies were performed as part of design control to assign expiration dating to components and as part of ongoing quality control/quality assurance analysis. All studies were performed on three lots of components/reagents. Accelerated studies were conducted with materials incubated at 37°C. In these conditions, one day was considered equivalent to one month stored at 2°–8°C. Materials were removed from the incubator for testing at three day intervals for a minimum of 21 days. Based on these studies, an 18 month expiration date was established for this product. Real time stability studies are ongoing and currently support device stability up to 12 months. For open kit stability studies, materials were opened as required for bench-top usage and then assayed at designated intervals. The sponsor demonstrated {13} open kits were stable for 30 days when stored at the recommended conditions (2°–8°C). ## Sample Stability The sponsor presented a study that supports a claim that serum samples are stable at 2°–8°C for one week. ### d. Detection limit: The limit of blank (LoB) and limit of detection (LoD) studies were performed following the protocol in CLSI EP-17A. To determine the LoB, sixty replicates of the blank (kit diluent) were run; the LoB was determined by sorting the results from low to high by OD and averaging the value of the 57th and 58th positions. The Limit of Blank for the ImmuLisa Enhanced™ Cardiolipin IgA, IgG and IgM Antibody (ACA) ELISAs was determined to be 1.8 APL for IgA, 1.0 GPL for IgG, 2.3 MPL for IgM and 4.7 EU/mL for the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA. To determine the LoD, six low negative samples were tested ten times each for a total of 60 measurements. The LoD was calculated using the equation $$\mathrm{LoD} = \mathrm{LoB} + \mathrm{c}_{\beta}\mathrm{SD}_{\mathrm{s}}$$. EU/mL determined by plotting OD value vs. standard curve. The Limit of Detection for the ImmuLisa Enhanced™ Cardiolipin IgA, IgG and IgM Antibody (ACA) ELISAs was determined to be 1.8 APL for IgA, 1.0 GPL for IgG, 2.8 MPL for IgM and 5.2 EU/mL for the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA. ### e. Analytical specificity: #### Interfering Substances: Interference was studied by mixing sera with known cardiolipin antibody levels with potentially interfering serum samples and studying deviation from expected results. Five samples for IgA, IgG and IgM were evaluated for interference spanning the measuring range. There was no significant trend of interference observed. Results were within the expected CV (±15%) of the assay. The greatest impact is in lower negative samples. The instructions for use caution against the use of grossly hemolyzed or lipemic samples. The substances and the maximum levels tested are shown in the table below: {14} | Substance | Final Concentration | | --- | --- | | Hemoglobin | 2 g/L | | Bilirubin | 342 μmol/L | | Cholesterol | 13 mmol/L | | Triglycerides | 37 mmol/L | | Rheumatoid Factor | 100 EU/mL | ## Cross-Reactivity: A set of potentially cross-reactive specimens from individuals with other connective tissue/autoimmune disorders were tested for cardiolipin antibodies using the ImmuLisa Enhanced™ Cardiolipin IgA, IgG, and IgM Antibody (ACA) ELISAs and ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA. The table below shows the number (n) of samples containing potential cross reactants as disease state evaluated by the ImmuLisa Enhanced™ Cardiolipin IgA, IgG, and IgM Antibody (ACA) ELISAs and the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA. The cross reactivity of ImmuLisa™ Cardiolipin assays was obtained as the positivity rate (%pos) among the total number (n) of cross reactant samples evaluated. | Cross Reactive Disease State | IgA | | IgG | | IgM | | IgA/IgG/IgM | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | n | % pos | n | % pos | n | % pos | n | % pos | | SLE | 77 | 16.5% | 79 | 24.1% | 86 | 9.3% | 69 | 32.4% | | Thrombocytopenia | 15 | 6.7% | 15 | 13.3% | 15 | 0.0% | 15 | 13.3% | | Pre-eclampsia | 15 | 6.7% | 15 | 20.0% | 15 | 0.0% | 15 | 20.0% | | Celiac Disease | 21 | 0.0% | 22 | 0.0% | 24 | 0.0% | 18 | 0.0% | | Mixed Connective Tissue Disease | 24 | 4.2% | 23 | 4.3% | 29 | 10.3% | 15 | 6.7% | | Myositis | 3 | 0.0% | 3 | 33.3% | 4 | 0.0% | 2 | 0.0% | | Rheumatoid Arthritis | 89 | 2.2% | 89 | 6.7% | 95 | 7.4% | 85 | 10.6% | | Sjögren's Syndrome | 25 | 8.0% | 25 | 8.0% | 27 | 11.1% | 20 | 10.0% | | Syphilis* | 40 | 15.0% | 40 | 82.5% | 40 | 35.0% | 40 | 97.5% | | Systemic Sclerosis | 44 | 2.3% | 44 | 0.0% | 45 | 4.4% | 36 | 5.6% | | Thyroiditis | 8 | 0.0% | 8 | 0.0% | 7 | 0.0% | 8 | 0.0% | | Vasculitis | 0 | not tested | 0 | not tested | 0 | not tested | 8 | 0.0% | * High percent of seropositive syphilis specimens were tested positive in {15} particular for IgG and the combined screening tests. A warning is included in the product insert which states that “Confirmed active or seropositive syphilis patients can have elevated anti-cardiolipin antibody (ACA) levels. To rule out syphilis, confirmatory tests should be performed”. **High dose hook effect:** To assess high dose hook effect, four to five high APLS specimens all above the highest calibrator for IgA, IgG and IgM Cardiolipin were tested in 1:100, 1:200, 1:400, 1:800 dilutions. These dilution series did not demonstrate hook effect. Results above the upper limit of the measuring range are reported as “over”. No recommendations are made for dilution of samples outside the measuring range in the package insert. **f. Assay cut-off:** The assay cut-off for each assay was established using the mean OD plus optimal standard deviations of ODs obtained by testing a set of normal samples and disease control sera. Based on internal comparison with the Harris cardiolipin IgA, IgG and IgM standards, the cut-offs for the ImmuLisa Enhanced™ Cardiolipin IgA, IgG and IgM Antibody (ACA) ELISA was assigned a value of 15 U/mL -APL, -GPL, and -MBL respectively. The indeterminate/borderline range was set within 50% of the cutoff. For ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA, cutoff was assigned a value of 20 EU/mL. There is no indeterminate/borderline range for the screen assay. **2. Comparison studies:** **a. Method comparison with predicate device:** ImmuLisa Enhanced™ Cardiolipin IgA, IgG, and IgM Antibody (ACA) ELISAs and the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA were compared to predicate ELISA kits using sera of APS and APS with SLE subjects and disease controls. Only samples in the linear range of the IMMCO assay are included in this method comparison. The number (n) and disease state of specimens tested is indicated in the table below: | Disease Category | IgA | IgG | IgM | IgA/IgG /IgM | | --- | --- | --- | --- | --- | | | n | n | N | n | | APS | 71 | 58 | 70 | 100 | | APS with SLE | 49 | 74 | 55 | 90 | | Suspected APS* | 167 | 180 | 149 | 150 | | Celiac Disease | 12 | 18 | 16 | 8 | {16} | Disease Category | IgA | IgG | IgM | IgA/IgG /IgM | | --- | --- | --- | --- | --- | | | n | n | N | n | | Mixed Connective Tissue Disease | 6 | 20 | 15 | 4 | | Polymyositis/Dermatomyositis | 1 | 1 | 1 | 0 | | Pre-Eclampsia | 15 | 13 | 12 | 10 | | Rheumatoid Arthritis | 81 | 80 | 76 | 57 | | Systemic Sclerosis (Scleroderma) | 34 | 40 | 28 | 20 | | Sjögren’s Syndrome | 25 | 23 | 22 | 16 | | SLE | 65 | 68 | 55 | 56 | | Submitted SLE | 80 | 84 | 79 | 73 | | Thrombocytopenia | 14 | 15 | 9 | 8 | | Hashimoto’s Thyroiditis (TPO) | 1 | 7 | 3 | 7 | | Vasculitis | 0 | 0 | 0 | 6 | | Total samples tested: | 621 | 681 | 590 | 605 | * Suspected APS were samples from patients suspected of having APS but did not have a definitive diagnosis available. ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | | Predicate ACA IgA ELISA | | | | | | --- | --- | --- | --- | --- | --- | | | | Positive | Borderline (12–20 APL) | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | Positive | 70 | 19 | 8 | 97 | | | Borderline (15 – 22.5 APL) | 6 | 10 | 7 | 23 | | | Negative | 17 | 22 | 462 | 501 | | | Total | 93 | 51 | 477 | 621 | | Borderline Samples Considered Positive | Predicate ACA IgA ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | Positive | 105 | 15 | 120 | | | Negative | 39 | 462 | 501 | | | Total | 144 | 477 | 621 | Positive Percent Agreement: 72.9% (95% CI: 64.8% –79.8%) Negative Percent Agreement: 96.9% (95% CI: 95.7% – 98.2%) Overall Percent Agreement: 91.3% (95% CI: 88.7% – 93.3%) | Borderline Samples Considered Negative | Predicate ACA IgA ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | Positive | 70 | 27 | 97 | | | Negative | 23 | 501 | 524 | | | Total | 93 | 528 | 621 | {17} Positive Percent Agreement: 75.3% (95% CI: 65.0% -83.4%) Negative Percent Agreement: 94.9% (95% CI: 92.6% - 96.5%) Overall Percent Agreement: 91.9% (95% CI: 89.5% - 93.9%) | Borderline Samples Excluded | Predicate ACA IgA ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgA Antibody (ACA) ELISA | Positive | 70 | 8 | 78 | | | Negative | 17 | 462 | 479 | | | Total | 87 | 470 | 557 | Positive Percent Agreement: 80.5% (95% CI: 70.3% -87.9%) Negative Percent Agreement: 98.3% (95% CI: 96.5% - 99.2%) Overall Percent Agreement: 95.5% (95% CI: 93.5% - 96.9%) ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA | | Predicate ACA IgG ELISA | | | | | | --- | --- | --- | --- | --- | --- | | | | Positive | Borderline (15 – 20 GPL) | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA | Positive | 140 | 5 | 19 | 164 | | | Borderline (15 – 22.5 GPL) | 5 | 5 | 13 | 23 | | | Negative | 9 | 16 | 469 | 494 | | | Total | 154 | 26 | 501 | 681 | | Borderline Samples Considered Positive | Predicate ACA IgG ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA | Positive | 155 | 32 | 187 | | | Negative | 25 | 469 | 494 | | | Total | 180 | 501 | 681 | Positive Percent Agreement: 86.1% (95% CI: 80.0% - 90.6%) Negative Percent Agreement: 93.6% (95% CI: 91.0% - 95.5%) Overall Percent Agreement: 91.6% (95% CI: 89.2% - 93.5%) | Borderline Samples Considered Negative | Predicate ACA IgG ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgG Antibody (ACA) ELISA | Positive | 140 | 24 | 164 | | | Negative | 14 | 503 | 517 | | | Total | 154 | 527 | 681 | Positive Percent Agreement: 90.9% (95% CI 84.9% - 94.8%) {18} Negative Percent Agreement: 95.4% (95% CI 93.2% - 97.0%) Overall Percent Agreement: 94.4% (95% CI 92.3% - 96.0%) | Borderline Samples Excluded | Predicate ACA IgG ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ | Positive | 140 | 19 | 159 | | Cardiolipin IgG | Negative | 9 | 469 | 478 | | Antibody (ACA) ELISA | Total | 149 | 488 | 637 | Positive Percent Agreement: 94.0% (95% CI 88.5% - 97.0%) Negative Percent Agreement: 96.1% (95% CI 93.9% - 97.6%) Overall Percent Agreement: 95.6% (95% CI 93.7% - 96.9%) ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | | Predicate ACA IgM ELISA | | | | | | --- | --- | --- | --- | --- | --- | | | | Positive | Borderline (12.5–20 MPL) | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Positive | 102 | 4 | 1 | 107 | | | Borderline (15 – 22.5 MPL) | 8 | 5 | 1 | 14 | | | Negative | 20 | 30 | 419 | 469 | | | Total | 130 | 39 | 421 | 590 | | Borderline Samples considered Positive | Predicate ACA IgM ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Positive | 119 | 2 | 121 | | | Negative | 50 | 419 | 469 | | | Total | 169 | 421 | 590 | Positive Percent Agreement: 70.4% (95% CI: 62.8% - 77.1%) Negative Percent Agreement: 99.5% (95% CI: 98.1% - 99.9%) Overall Percent Agreement: 91.2% (95% CI: 88.5% - 93.3%) | Borderline Samples Considered Negative | Predicate ACA IgM ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ Cardiolipin IgM Antibody (ACA) ELISA | Positive | 102 | 5 | 107 | | | Negative | 28 | 455 | 483 | | | Total | 130 | 460 | 590 | Positive Percent Agreement: 78.5% (95% CI: 70.2% - 85.0%) Negative Percent Agreement: 98.9% (95% CI: 97.3% - 99.6%) {19} Overall Percent Agreement: 94.4% (95% CI: 92.2% - 96.1%) | Borderline Samples Excluded | Predicate ACA IgM ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ | Positive | 102 | 1 | 103 | | Cardiolipin IgM | Negative | 20 | 419 | 439 | | Antibody (ACA) ELISA | Total | 122 | 420 | 542 | Positive Percent Agreement: 83.6% (95% CI: 75.6% - 89.5%) Negative Percent Agreement: 99.8% (95% CI: 98.5% - 100%) Overall Percent Agreement: 96.1% (95% CI: 94.2% - 97.5%) ImmuLisa Enhanced™ Cardiolipin IgA/IgG/IgM Antibody (ACA) ELISA | ≥ Cutoff (20 EU/mL) = Positive | Predicate ACA Antibody Screen ELISA | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | ImmuLisa Enhanced™ | Positive | 243 | 27 | 270 | | Cardiolipin IgA/IgG/ | Negative | 36 | 299 | 335 | | IgM Antibody (ACA) ELISA | Total | 279 | 326 | 605 | Positive Percent Agreement: 87.1% (95% CI: 80.5% - 90.7%) Negative Percent Agreement: 91.7% (95% CI: 88.0% - 94.4%) Overall Percent Agreement: 89.6% (95% CI: 86.8% - 91.9%) b. Matrix comparison: Not applicable. 3. Clinical studies: a. Clinical Sensitivity and Specificity: The study used a set of clinically characterized sera from APS, APS with SLE and non-APS diseased controls. The study was designed to be reflective of the intended use population. For that reason, the study did not include any samples from diagnosed APS patients with known anti-cardiolipin antibody status. The results of the ImmuLisa Enhanced™ Cardiolipin IgA, IgG, and IgM Antibody (ACA) ELISAs and the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/ IgM Antibody (ACA) ELISA in each of disease category are shown below: {20} | Disease Category | IgA | | | IgG | | | IgM | | | IgA/IgG/ IgM | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | n | pos | % | n | pos | % | n | pos | % | n | pos | % | | APS | 127 | 11 | 8.7% | 99 | 56 | 56.6% | 108 | 38 | 35.2% | 90 | 56 | 62.2% | | APSwSLE | 53 | 30 | 56.6% | 95 | 47 | 49.5% | 80 | 17 | 21.3% | 66 | 60 | 90.9% | | Total Diagnosed APS | 180 | 41 | 22.8% | 194 | 103 | 53.1% | 188 | 55 | 29.3% | 156 | 116 | 74.4% | | | | | | | | | | | | | | | | Other disease sera | (included in non-APS diagnosis population) | | | | | | | | | | | | | Submitted for APS testing/suspected APS | 188 | 14 | 7.4% | 184 | 45 | 24.5% | 188 | 13 | 6.9% | 185 | 38 | 20.5% | | Submitted SLE testing/suspected SLE | 90 | 5 | 5.6% | 88 | 13 | 14.8% | 90 | 5 | 5.6% | 88 | 16 | 18.2% | | Thrombocytopenia | 15 | 1 | 6.7% | 15 | 2 | 13.3% | 15 | 0 | 0.0% | 15 | 2 | 13.3% | | Pre-eclampsia | 15 | 1 | 6.7% | 15 | 3 | 20.0% | 15 | 0 | 0.0% | 15 | 3 | 20.0% | | Celiac Disease | 21 | 0 | 0.0% | 22 | 0 | 0.0% | 24 | 0 | 0.0% | 18 | 0 | 0.0% | | Mixed Connective Tissue Disease | 24 | 1 | 4.2% | 23 | 1 | 4.3% | 29 | 3 | 10.3% | 15 | 1 | 6.7% | | Myositis | 3 | 0 | 0.0% | 3 | 1 | 33.3% | 4 | 0 | 0.0% | 2 | 0 | 0.0% | | Rheumatoid Arthritis | 89 | 2 | 2.2% | 89 | 6 | 6.7% | 95 | 7 | 7.4% | 85 | 9 | 10.6% | | Sjögren's Syndrome | 25 | 2 | 8.0% | 25 | 2 | 8.0% | 27 | 3 | 11.1% | 20 | 2 | 10.0% | | Systemic Lupus Erythematosus | 77 | 13 | 16.5% | 79 | 20 | 24.1% | 86 | 8 | 9.3% | 69 | 24 | 32.4% | | Systemic Sclerosis | 44 | 1 | 2.3% | 44 | 0 | 0.0% | 45 | 2 | 4.4% | 36 | 2 | 5.6% | | Thyroiditis | 8 | 0 | 0.0% | 8 | 0 | 0.0% | 7 | 0 | 0.0% | 8 | 0 | 0.0% | | Vasculitis | 0 | 0 | not tested | 0 | 0 | not tested | 0 | 0 | not tested | 8 | 0 | 0.0% | | Total Diseased Samples: | 779 | | | 789 | | | 813 | | | 719 | | | The following table summarizes the clinical sensitivity and specificity of the ImmuLisa Enhanced™ Cardiolipin IgA, IgG, and IgM Antibody (ACA) ELISAs and the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/ IgM Antibody (ACA) ELISA: {21} | Disease | IgA | | IgG | | IgM | | IgA/IgG/IgM | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | Clin Sen (95% CI) | Clin Spec (95%CI) | Clin Sen (95% CI) | Clin Spec (95%CI) | Clin Sen (95% CI) | Clin Spec (95%CI) | Clin Sen (95% CI) | Clin Spec (95%CI) | | APS | 8.7% (4.6%, 15.3%) | 93.7% (91.3%, 95.4%) | 56.6% (46.2%, 66.4%) | 85.0% (81.9%, 87.8%) | 35.2% (26.4%, 45.0%) | 93.4% (91.1%, 95.2%) | 62.2% (50.7%, 71.4%) | 83.8% (80.4%, 86.7%) | | APS with SLE | 56.6% (42.4%, 69.9%) | 93.7% (91.3%, 95.4%) | 49.5% (39.1%, 59.9%) | 85.0% (81.9%, 87.8%) | 21.3% (13.2%, 32.1%) | 93.4% (91.1%, 95.2%) | 90.9% (80.4%, 86.7%) | 83.8% (80.4%, 86.7%) | b. Other clinical supportive data (when a. is not applicable): Not applicable. 4. Clinical cut-off: See Assay Cutoff 5. Expected values/Reference range: The expected value in the general population is negative. The normal range was established by testing a population of normal blood donor specimens on the assays. Sets of normal human sera were tested on the ImmuLisa Enhanced™ Cardiolipin IgA, IgG, and IgM Antibody (ACA) ELISAs and the ImmuLisa Enhanced™ Cardiolipin IgA/IgG/ IgM Antibody (ACA) ELISA. Results demonstrating incidence in the apparently-disease free samples for this study are provided below: | ACA IgA | | ACA IgG | | ACA IgM | | ACA IgA/IgG/IgM | | | --- | --- | --- | --- | --- | --- | --- | --- | | n | % pos | n | % pos | n | % pos | n | % pos | | 167 | 1.2% | 164 | 3.0% | 154 | 2.6% | 158 | 1.9% | 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.