ImmuLisa Enhanced AMA IgG Antibody ELISA; ImmuLisa Enhanced AMA IgA/IgG/IgM Antibody ELISA
Device Facts
| Record ID | K163133 |
|---|---|
| Device Name | ImmuLisa Enhanced AMA IgG Antibody ELISA; ImmuLisa Enhanced AMA IgA/IgG/IgM Antibody ELISA |
| Applicant | Immco Diagnostics, Inc. |
| Product Code | DBM · Immunology |
| Decision Date | Aug 8, 2017 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5090 |
| Device Class | Class 2 |
Intended Use
ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgG ELISA: An enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of anti-mitochondria IgG antibodies in human serum to aid in the diagnosis of primary biliary cirrhosis (PBC) in conjunction with other laboratory tests and clinical findings. ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA: An enzyme linked immunosorbent assay (ELISA) for the qualitative or semi-quantitative detection of anti-mitochondria antibodies (AMA) in human serum to aid in the diagnosis of primary biliary cirrhosis (PBC) in conjunction with other laboratory tests and clinical findings.
Device Story
The ImmuLisa Enhanced AMA ELISA is a solid-phase immunoassay used in clinical laboratories to detect anti-mitochondrial antibodies (AMA) in human serum. Microwells are coated with recombinant mitochondrial antigens (PDC-E2, OGDC-E2, BCOADC-E2). Patient serum, calibrators, and controls are incubated in the wells; specific AMA antibodies bind to the antigens. Bound antibodies are detected using an enzyme-labeled anti-human IgG or IgA/IgG/IgM conjugate, followed by TMB substrate addition. A spectrophotometer reads the resulting color change at 450 nm. Results are expressed in ELISA units per milliliter (EU/ml) and reported as positive or negative. The device aids clinicians in diagnosing primary biliary cirrhosis (PBC) by identifying specific autoantibody markers. It provides semi-quantitative results via a 5-point calibrator curve, assisting in the differentiation of AMA reactions that are otherwise difficult to distinguish via traditional indirect immunofluorescence.
Clinical Evidence
Clinical validation included 1,091 serum samples (193 PBC, 898 non-target disease). For AMA IgG: sensitivity 85.5%, specificity 99.0% (equivocal as positive). For AMA IgA/IgG/IgM: sensitivity 87.0%, specificity 98.5% (equivocal as positive). Method comparison against predicates showed high agreement (Overall % Agreement 94.0% for IgG; 93.8% for IgA/IgG/IgM).
Technological Characteristics
Solid-phase ELISA; 96-well microplate coated with recombinant mitochondrial antigens (PDC-E2, OGDC-E2, BCOADC-E2); enzyme-labeled anti-human IgG or IgA/IgG/IgM conjugate; TMB substrate; spectrophotometric detection at 450 nm; semi-quantitative output in EU/ml; linear range 3.3 to 160 EU/ml.
Indications for Use
Indicated for the qualitative or semi-quantitative detection of anti-mitochondria antibodies (IgG or IgA/IgG/IgM) in human serum to aid in the diagnosis of primary biliary cirrhosis (PBC) in patients, in conjunction with other laboratory tests and clinical findings.
Regulatory Classification
Identification
An antimitochondrial antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the antimitochondrial antibodies in human serum. The measurements aid in the diagnosis of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's own tissue), such as primary biliary cirrhosis (degeneration of liver tissue) and chronic active hepatitis (inflammation of the liver).
Predicate Devices
- INOVA QUANTA Lite™ M2 EP (MIT3) ELISA (K052262)
- Trinity Captia™ Mitochondria IgA/IgG/IgM Screen (K982121)
Related Devices
- K092736 — EUROIMMUN ANTI-M2-3E ELISA (IGG) · Euroimmun Us, Inc. · Sep 2, 2010
- K971909 — VIRGO AMA ELISA KIT · Hemagen Diagnostics, Inc. · Jun 20, 1997
- K030238 — BINDAZYME ANTI-MITOCHONDRIA M2 IGGAM EIA KIT · The Binding Site, Ltd. · Mar 24, 2003
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number: K163133
B. Purpose for Submission:
New device
C. Measurand:
IgG autoantibodies specific for mitochondrial proteins
IgA/IgG/IgM autoantibodies specific for mitochondrial proteins
D. Type of Test:
Immunoassay, qualitative and semi-quantitative
E. Applicant:
IMMCO Diagnostics Inc.
F. Proprietary and Established Names:
ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgG ELISA
ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA
G. Regulatory Information:
1. Regulation section:
21 CFR §866.5090, Antimitochondrial antibody immunological test system
2. Classification:
Class II
3. Product code:
DBM – Antimitochondrial antibody, indirect immunofluorescent, antigen, control
4. Panel:
Immunology (82)
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H. Intended Use:
1. Intended uses:
ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgG ELISA:
An enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of anti-mitochondria IgG antibodies in human serum to aid in the diagnosis of primary biliary cirrhosis (PBC) in conjunction with other laboratory tests and clinical findings.
ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA:
An enzyme linked immunosorbent assay (ELISA) for the qualitative or semi-quantitative detection of anti-mitochondria antibodies (AMA) in human serum to aid in the diagnosis of primary biliary cirrhosis (PBC) in conjunction with other laboratory tests and clinical findings.
2. Indications for use:
Same as Intended Use
3. Special conditions for use statements:
For prescription use only
4. Special instrument requirements:
An ELISA 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 12 x 8-antigen coated microwell strips, negative control (1x 1.75 mL), positive control (1x 1.75 mL), five assay calibrators (5x 1.75 mL), horseradish peroxidase anti-human IgA/IgG/IgM or anti-human IgG conjugate (1x 15 mL), TMB enzyme substrate (1x 15 mL), stop solution (1x 15 mL), wash buffer (2 vials) and diluent (1x 60 mL). The results are read by a spectrophotometer at 450 nm. Results are expressed in ELISA units per milliliter (EU/mL) and reported as positive or negative.
J. Substantial Equivalence Information:
1. Predicate device name (Predicate 510(k) number):
INOVA QUANTA Lite™ M2 EP (MIT3) ELISA (K052262)
Trinity Captia™ Mitochondria IgA/IgG/IgM Screen (K982121)
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2. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | New Device ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA | Predicate INOVA QUANTA Lite™ M2 EP (MIT3) ELISA |
| Intended Use | An enzyme linked immunoassay (ELISA) for the qualitative or semi-quantitative detection of anti-mitochondria IgG antibodies in human serum to aid in the diagnosis of primary biliary cirrhosis (PBC) in conjunction with other laboratory tests and clinical findings. | The QUANTA LITE™ M2 EP (MIT3) ELISA is an enzyme-linked immunosorbent assay (ELISA) for the semi-quantitative detection of mitochondria antibodies in human serum. The presence of mitochondria antibodies can be used in conjunction with clinical findings and other laboratory tests to aid in the diagnosis of primary biliary cirrhosis. |
| Assay Type | ELISA | Same |
| Capture Antigen | Recombinant BCOADC, PDC, and OGDC | Same |
| Sample Type | Serum | Same |
| Assay Format | Semi-quantitative and qualitative | Same |
| Labeled Detection Antibody (Conjugate) | Horseradish peroxidase conjugated to goat anti-human IgG | Same |
| Enzyme Substrate | Tetramethylbenzidene (TMB) | Same |
| Traceability | International Reference Preparation is not available. Results are traceable to in-house standards. | Same |
| Signal/Wavelength | Optical density/ 450nm | Same |
| Instrumentation | Microwell plate reader | Same |
| Storage | 2–8°C | Same |
| Differences | | |
| --- | --- | --- |
| Item | New Device ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA | Predicate INOVA QUANTA Lite™ M2 EP (MIT3) ELISA |
| Calibrators | Set of five: Values in EU/mL: 160, 80 40, 20, 1 | One calibrator Value in U/mL |
| Linear Range | 3.3 EU/mL–160 EU/mL | Not specified |
| Limit of Detection | 3.3 EU/mL | Not specified |
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| Differences | | |
| --- | --- | --- |
| Item | New Device ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA | Predicate INOVA QUANTA Lite™ M2 EP (MIT3) ELISA |
| Sample dilution | 1:101 | 1:51 |
| Cutoff | 20 EU/mL | 20 Units |
| Results Interpretation | Negative: < 20 EU/mL Indeterminate: 20–25 EU/mL Positive: > 25 UE/mL | Negative: ≤ 20 EU/mL Equivocal: 20.1–24.9 EU/mL Positive: ≥ 25 UE/mL |
| Similarities | | |
| --- | --- | --- |
| Item | New Device ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA | Predicate Trinity Captia Mitochondria IgA/IgG/IgM Screen |
| Intended Use | An enzyme linked immunosorbent assay (ELISA) for the qualitative or semi-quantitative detection of anti-mitochondria antibodies (AMA) in human serum to aid in the diagnosis of primary biliary cirrhosis (PBC) in conjunction with other laboratory tests and clinical findings. | On the IFU form: Intended for the in vitro determination of mitochondrial (M2) antibodies. Anti-M2 assays are used as an aid in the diagnosis of Primary Biliary Cirrhosis. In the Package Insert: The Trinity Biotech Captia™ Mitochondria Enzyme-Linked Immunosorbent Assay (ELISA) is intended for the detection and semi-quantitation of antibodies to mitochondria in human sera. The assay is to be used to detect antibodies in a single serum specimen. The results of the assay are to be used as an aid to the diagnosis of primary biliary cirrhosis. For in vitro diagnostic use. High complexity test. |
| Assay Type | ELISA | Same |
| Sample Type | Serum | Same |
| Assay Format | Semi-quantitative and qualitative | Same |
| Labeled Detection Antibody (Conjugate) | Horseradish peroxidase conjugated to goat anti-human IgA/IgG/IgM | Same |
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| Enzyme Substrate | Tetramethylbenzidene | Same |
| --- | --- | --- |
| Instrumentation | Microwell plate reader | Same |
| Storage | 2–8°C | Same |
| Differences | | |
| --- | --- | --- |
| Item | New Device
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA | Predicate
Trinity Captia Mitochondria IgA/IgG/IgM Screen |
| Calibrators | Set of five:
Values in EU/mL: 160, 80 40, 20, 1 | One |
| Capture Antigen | Recombinant BCOADC, PDC, and OGDC | Native immunoreactive domain of E2-PDC |
| Linear Range | 3.3 EU/mL–160 EU/mL | Not specified |
| Limit of Detection | 3.3 EU/mL | Not specified |
| Sample dilution | 1:101 | 1:21 |
| Results Interpretation | Negative: < 20 EU/mL
Indeterminate: 20–25 EU/mL
Positive: > 25 UE/mL | Index Value:
≤ 0.90 Negative
0.91–1.09 Equivocal
≥ 1.10 Positive |
K. Standard/Guidance Document Referenced:
CLSI guideline EP05-A2, "Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline Second Edition"
CLSI guideline EP06-A, "Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline"
CLSI guideline EP07-A2, "Interference Testing in Clinical Chemistry; Approved Guideline–Second Edition"
CLSI guideline EP09-A2, "Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline."
CLSI guideline EP12-A2, "User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline–Second Edition"
CLSI guideline EP17-A2, "Evaluation of Detection Capability for Clinical. Laboratory Measurement Procedures; Approved Guideline–Second Edition"
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L. Test Principle:
Mitochondrial antigen mixture composing of recombinant the E2 subunit of the branched-chain 2-oxoacid dehydrogenase complex (BCOADC-E2), the E2 component of pyruvate dehydrogenase complex (PDC-E2) and the E2 subunit of the 2-oxoglutarate dehydrogenase complex (OGDC-E2) is bound to the wells of a polystyrene microwell plate followed by blocking of the unreacted sites to reduce non-specific binding. Controls, calibrators and diluted patient sera are added to separate wells, allowing any anti-mitochondria antibodies present to bind to the immobilized antigen. Unbound sample is washed away and an enzyme labeled anti-human IgG 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 nm. Results are expressed in ELISA units per milliliter (EU/mL).
Semi-quantitative results are determined from a series of five calibrators (160 EU/mL, 80 EU/mL, 40 EU/mL, 20 EU/mL, and 1 EU/mL). Values less than 20 EU/mL are considered negative results while values greater than 25 EU/mL are considered positive; results between 20 EU/mL and 25 EU/mL are considered 'indeterminate/borderline'. The sponsor states the following recommendations in the Package Insert: "Indeterminate/borderline results should be retested and evaluated along with other laboratory methods".
Qualitative results are determined using a ratio of the absorbance of the sample to the absorbance of the cut-off calibrator (20 EU/mL). The ratio is multiplied by the concentration of the cut-off calibrator to give a numerical value. Values greater than or equal to 20 EU/mL are considered positive and <20 EU/mL values are considered negative.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Semi-Quantitative Precision:
Precision performance was evaluated in accordance with CLSI guideline EP05-A2 "Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline". A panel consisting of seven patient sera with levels of AMA antibodies that cover the analytical measuring range was assayed in duplicate, twice a day, for 20 days with one reagent lot (a total of 80 replicates per sample). Testing was performed by two operators using different equipment sets. The first operator performed the assay with a multi-channel pipettor, microplate washer and microplate reader. The second operator used different instruments with the same type of equipment. All %CV values were within the manufacturer's pre-determined acceptance limit. The results are summarized in the table below for each assay:
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ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA:
| Specimen | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Run | | Between-Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 13.8 | 0.8 | 5.8 | 1.1 | 7.9 | 0.6 | 4.2 | 1.4 | 9.8 |
| 2 | 16.0 | 0.7 | 4.1 | 1.0 | 6.0 | 1.2 | 7.7 | 1.6 | 10.0 |
| 3 | 19.7 | 0.7 | 3.7 | 1.3 | 6.7 | 0.4 | 2.0 | 1.5 | 7.6 |
| 4 | 23.6 | 0.7 | 3.1 | 1.1 | 4.5 | 0.6 | 2.3 | 1.3 | 5.5 |
| 5 | 78.1 | 3.2 | 4.1 | 6.4 | 8.2 | 1.9 | 2.5 | 7.2 | 9.2 |
| 6 | 111.2 | 4.5 | 4.0 | 7.8 | 7.0 | 1.1 | 6.9 | 9.0 | 8.1 |
| 7 | 159.9 | 4.9 | 3.0 | 8.8 | 5.5 | 2.7 | 1.7 | 10.0 | 6.3 |
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA:
| Specimen | Mean (EU/mL) | Within-Run (Repeatability) | | Between Run | | Between-Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 11.2 | 0.3 | 2.2 | 0.7 | 5.8 | 0.3 | 2.2 | 0.7 | 6.6 |
| 2 | 15.5 | 0.2 | 1.2 | 0.5 | 3.2 | 0.3 | 2.0 | 0.6 | 4.0 |
| 3 | 20.3 | 0.3 | 1.4 | 0.7 | 3.2 | 0.9 | 4.4 | 1.1 | 5.6 |
| 4 | 24.5 | 0.5 | 1.8 | 1.2 | 5.0 | 0.7 | 3.0 | 1.5 | 6.1 |
| 5 | 51.6 | 0.5 | 1.1 | 2.0 | 3.8 | 0.2 | 0.3 | 2.0 | 4.0 |
| 6 | 104.7 | 1.4 | 1.4 | 4.2 | 4.0 | 2.3 | 2.2 | 4.4 | 4.2 |
| 7 | 145.3 | 1.4 | 1.0 | 4.8 | 3.3 | 0.1 | 0.1 | 5.0 | 3.4 |
## Qualitative Reproducibility:
Studies were performed under the guidance of CLSI EP12-A2 "User Protocol for Evaluation of Qualitative Test Performance." Eighty replicates of each patient serum in the negative range, $\sim 20\%$ below cut-off, at cut-off, $\sim 20\%$ above cut-off and in the moderate positive range of the assays were performed to determine intra-assay qualitative reproducibility. Results were calculated using single-point (qualitative) analysis as indicated in the product insert. All values for qualitative agreement were within the manufacturer's pre-determined acceptance limit. Results are summarized below.
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA:
| Specimen | Mean (EU/mL) | Total replicates | Qualitative Agreement | |
| --- | --- | --- | --- | --- |
| | | | Positive/negative | % |
| Negative | 13.5 | 80 | 0/80 | 100 |
| Cut-off -20% | 15.8 | 80 | 2/78 | 98 |
| Cut-off | 19.6 | 80 | 30/50 | 63 |
| Cut-off +20% | 23.5 | 80 | 80/0 | 100 |
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| Specimen | Mean (EU/mL) | Total replicates | Qualitative Agreement | |
| --- | --- | --- | --- | --- |
| | | | Positive/negative | % |
| Moderate Positive | 83.6 | 80 | 80/0 | 100 |
| Moderate to High Positive | 101.6 | 80 | 80/0 | 100 |
| High Positive | 142.1 | 80 | 80/0 | 100 |
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA:
| Specimen | Mean (EU/mL) | Total replicates | Qualitative Agreement | |
| --- | --- | --- | --- | --- |
| | | | Positive/negative | % |
| Negative | 10.5 | 80 | 0/80 | 100 |
| Cut-off –20% | 15.2 | 80 | 0/80 | 100 |
| Cut-off | 20.2 | 80 | 34/46 | 58 |
| Cut-off +20% | 24.2 | 80 | 79/1 | 99 |
| Moderate Positive | 56.0 | 80 | 80/0 | 100 |
| Moderate to High Positive | 89.1 | 80 | 80/0 | 100 |
| High Positive | 118.0 | 80 | 80/0 | 100 |
## Lot-to-lot Reproducibility:
To evaluate lot-to-lot reproducibility, a panel of seven samples with levels of AMA antibodies spanning the assay range was tested in duplicate on three different lots over the course of five days. All %CV values were within the manufacturer's predetermined acceptance limit. The results are summarized in the table below for each assay:
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA:
| Specimen | Mean (EU/mL) | Within-Run | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| 1 | 4.1 | 0.1 | 1.4% | 0.1 | 1.5% | 0.1 | 1.9% |
| 2 | 16.2 | 0.6 | 3.9% | 0.3 | 2.1% | 0.7 | 4.0% |
| 3 | 20.5 | 1.2 | 6.0% | 0.4 | 1.7% | 1.2 | 5.7% |
| 4 | 24.2 | 0.9 | 3.7% | 1.1 | 4.7% | 0.9 | 3.9% |
| 5 | 57.1 | 3.7 | 6.5% | 2.1 | 3.7% | 3.4 | 5.9% |
| 6 | 67.2 | 5.7 | 8.4% | 3.4 | 5.1% | 5.8 | 8.6% |
| 7 | 149.0 | 5.7 | 3.9% | 6.2 | 4.2% | 7.5 | 5.0% |
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA:
| Specimen | Mean (EU/mL) | Within-Run | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| 1 | 10.7 | 1.0 | 9.4% | 0.4 | 3.4% | 0.9 | 8.8% |
| 2 | 16.6 | 1.3 | 7.5% | 0.9 | 5.1% | 1.3 | 7.9% |
| 3 | 21.5 | 1.9 | 8.6% | 0.7 | 3.4% | 1.8 | 8.5% |
| 4 | 28.3 | 1.6 | 5.8% | 1.0 | 3.4% | 1.6 | 5.6% |
| 5 | 38.3 | 3.3 | 8.6% | 0.8 | 2.0% | 3.0 | 7.8% |
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| Specimen | Mean (EU/mL) | Within-Run | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| 6 | 60.7 | 1.5 | 2.5% | 1.2 | 2.1% | 1.4 | 2.3% |
| 7 | 125.3 | 2.8 | 2.2% | 2.1 | 1.6% | 2.9 | 2.3% |
# b. Linearity/assay reportable range:
Linearity and recovery were tested by diluting positive specimens across the measuring range in equidistant dilutions with negative patient sera. The observed values were graphed against the calculated values and a linear regression was performed. Results summarized in the table below were within the manufacturer's pre-determined acceptance criteria.
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA:
| Sample | Test Range (EU/ml) | Slope (95% CI) | Y-intercept (95% CI) | R2 | % Recovery |
| --- | --- | --- | --- | --- | --- |
| 1 | 3.4 to 57.4 | 0.99 (0.94 to 1.04) | 0.94 (-0.858 to 2.74) | 0.997 | 93% to 102% |
| 2 | 6.0 to 81.0 | 1.00 (0.95 to 1.05) | -0.89 (-3.28 to 1.49) | 0.9978 | 100% to 109% |
| 3 | 54.9 to 162.4 | 0.96 (0.89 to 1.03) | 3.15 (-5.04 to 11.34) | 0.995 | 95% to 106% |
ImmuLisa™ Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA:
| Sample | Test Range (EU/ml) | Slope (95% CI) | Y-intercept (95% CI) | R2 | % Recovery |
| --- | --- | --- | --- | --- | --- |
| 1 | 5.9 to 31.9 | 1.01 (0.95 to 1.07) | 0.39 (-0.89 to 1.67) | 0.997 | 95% to 98% |
| 2 | 28.9 to 83.4 | 1.08 (0.92 to 1.23) | -5.94 (-15.48 to 3.59) | 0.979 | 96% to 113% |
| 3 | 46.6 to 166.5 | 0.93 (0.83 to 1.03) | 5.68 (-5.48 to 16.84) | 0.989 | 95% to 108% |
The linear range was determined to be 3.3-160 EU/mL for the AMA IgG assay and 3.3-160 EU/mL for the AMA IgA/IgG/IgM assay. For test results above 160 EU/mL, the package insert recommends dilution and retesting of the samples.
High dose hook effect: Four high concentrations specimens were serially diluted to assess the presence of any artifactual decrease in assay signal associated with AMA antibody excess (hook effect). No hook effect was demonstrated in sample levels up to 15,356.8 EU/mL in the AMA IgG assay and 17,915.7 EU/mL in the AMA IgA/IgG/IgM assay.
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c. Traceability, Stability, Expected values (controls, calibrators, or methods):
i. Traceability:
There are currently no recognized international standards for the measurement of AMA antibodies. Calibrator and Control values for both assays are directly traceable to in-house standards.
ii. Value Assignment:
Calibrators and positive controls are dilutions of pooled AMA antibody positive sera obtained from various commercial plasma centers. The calibrators and controls are taken from different pooled sera. In order to have traceability with newly made calibrators, each new lot of calibrators is assayed on an already cleared kit and compared to the existing calibrators as reference. Each lot of calibrator is also tested in comparison with normal human sera, clinical samples and internal standards. Five calibrator levels (Cal A-E) with assigned values from 0–160 EU/mL are included to provide semi-quantitation and must be used with each run. The cut-off calibrator for the qualitative analysis is derived from Cal D. Test results greater than or equal to Cal D are considered positive.
For semi-quantitative determinations the positive controls must give values in the range stated on the vial. The negative control contains negative human serum in buffer. The negative control must be <10 EU/mL.
iii. Stability:
Shelf-life Stability:
Accelerated and real-time stability studies for each ImmuLisa Enhanced AMA Antibody ELISAs were conducted on three lots of components/reagents. The accelerated stability study was conducted with materials incubated at 37°C. Data from the accelerated stability study support a shelf-life stability of 18 months. A real-time stability study is on-going and currently supports an 18-month shelf-life stability claim.
Open Kit Stability:
For the open kit stability study, materials in each ImmuLisa Enhanced AMA Antibody ELISA are opened and stored in a dark environment as required for bench-top usage, then assayed at 15, 45 and 90 day intervals. Data from the open kit stability study demonstrate that opened reagents are stable at 45 days, but the sponsor chose a more restrictive one month open kit stability claim.
Sample Stability and Storage:
The package insert recommends that sera be assayed soon after separation or stored in aliquots at 2–8°C for no longer than one week. For longer storage, serum specimens should be frozen. Avoid repeated freezing and thawing of samples. It is recommended that frozen specimens be tested within one year.
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# d. Detection limit:
The analytical sensitivity was determined in accordance with CLSI EP17-A2. The Limit of Blank (LoB) was determined by assaying a blank sample in 60 replicates. The limit of detection (LoD) was determined by assaying six low negative sera in 10 replicates over three days using two reagent lots of each ImmuLisa Enhanced AMA Antibody ELISAs. The LoB and LoD for the AMA IgG assay was 1.6 and 3.3 EU/mL, respectively. The LoB and LoD for the AMA IgA/IgG/IgM assay was 1.5 and 3.3 EU/mL, respectively.
# e. Analytical specificity:
# i. Endogenous Interference:
Interference studies were performed according to CLSI EP07-A2 by testing five serum samples with AMA antibody levels corresponding to the negative range, near the assay cut-off, and in the low, moderate and high positive range. Each sample was mixed with known quantities of potentially interfering substances and analyzed in one assay run with two replicates on one kit lot of each assay. The recovery was calculated by comparing to control samples spiked with the same volume of diluents. No significant interference was detected within the manufacturer's pre-determined acceptance criteria for the following substances up to the concentrations listed in the table below:
| Potential Interfering Compound | Test Concentration |
| --- | --- |
| Hemoglobin | 2 g/L |
| Bilirubin | 342 μmol/L |
| Triglycerides | 37 mmol/L |
| Rheumatoid Factor | 100 EU/mL |
| Total cholesterol | 13 mmol/L |
The 'Limitations of Procedure' section of the Package Insert states "This test should not be performed on grossly hemolyzed, microbially contaminated or lipemic samples".
# ii. Cross-reactivity:
Numerous potentially cross-reactive autoimmune and infectious disease sera were tested for AMA antibody levels. Refer to test results for 898 serum samples from patients in the Non-Target Disease Group in the table presented in the section below on Clinical studies. The number of serum samples tested positive is 12 $(0.1\%)$ for AMA IgG and 15 $(0.2\%)$ for AMA IgA/IgG/IgM.
# f. Assay cut-off:
The assay cut-offs for the ImmuLisa Enhanced AMA Antibody ELISAs were determined by testing specimens from 128 healthy blood donors. Using the $97.5^{\text{th}}$
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percentile value of the results obtained, an arbitrary value of 20 EU/mL was assigned for the cut-off to establish the following result interpretations:
| AMA antibody value | Result interpretation |
| --- | --- |
| <20 EU/mL | Negative |
| 20–25 EU/mL | Indeterminate (Borderline) |
| >25 EU/mL | Positive |
The cut-off using the 20 EU/mL was validated in a separate study using samples from healthy blood donors and samples from patients with autoimmune, infectious, and other conditions expected to be found in the differential diagnosis of PBC; $2.3\%$ (3/130) and $3.8\%$ (5/130) of the samples from were positive for AMA IgG and AMA IgA/IgG/IgM, respectively.
# 2. Comparison studies:
# a. Method comparison with predicate device:
For analysis of agreement with the predicate device, clinically defined serum samples from the target disease (PBC) and non-target disease group (defined in Section 3.a, below) were tested with both the predicate and the new device. A total of 434 samples were tested by the AMA IgG assay; 154 PBC samples and 289 non-target disease samples were included. A total of 453 samples were tested by the AMA IgA/IgG/IgM assay; 163 PBC samples and 290 non-target disease samples were included. Only specimens in the linear range of both the predicate device and the new device were included in the method comparison. The semi-quantitative results for each assay are summarized below:
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA:
| | Predicate AMA IgG ELISA | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Pos | Equiv | Neg | Total |
| ImmuLisa Enhanced AMA IgG | Pos | 98 | 11 | 14 | 123 |
| | Equiv | 3 | 0 | 1 | 4 |
| | Neg | 6 | 5 | 296 | 307 |
| | Total | 107 | 16 | 311 | 434 |
Both assays equivocal samples considered positive:
Positive % Agreement 91.1% 112/123 (95%CI: 84.2 - 95.2)
Negative % Agreement 95.2% 296/311 (95%CI: 92.0 - 97.2)
Overall % Agreement 94.0% 408/434 (95%CI: 91.2 - 96.0)
Both assays equivocal samples considered negative:
Positive % Agreement 91.6% 98/107 (95%CI: 84.2 - 95.8)
Negative % Agreement 92.4% 302/327 (95%CI: 88.8 - 94.9)
Overall % Agreement 92.2% 400/434 (95%CI: 89.1 - 94.4)
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ImmuLisa Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA:
| | Predicate AMA IgA/IgG/IgM ELISA | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Pos | Equiv | Neg | Total |
| ImmuLisa Enhanced AMA IgA/IgG/IgM | Pos | 91 | 22 | 18 | 131 |
| | Equiv | 4 | 5 | 5 | 14 |
| | Neg | 3 | 2 | 303 | 308 |
| | Total | 98 | 29 | 326 | 453 |
Both assays equivocal samples considered positive:
Positive % Agreement 96.1% 122/127 (95%CI: 90.6 - 98.5)
Negative % Agreement 92.9% 303/326 (95%CI: 89.5 - 95.4)
Overall % Agreement 93.8% 425/453 (95%CI: 91.1 - 95.8)
Both assays equivocal samples considered negative:
Positive % Agreement 92.9% 91/98 (95%CI: 85.3 - 96.8)
Negative % Agreement 88.7% 315/355 (95%CI: 84.9 - 91.7)
Overall % Agreement 89.6% 406/453 (95%CI: 86.4 - 92.2)
# b. Matrix comparison:
Serum is the only matrix indicated for these assays.
# 3. Clinical studies:
# a. Clinical Sensitivity and Specificity:
A total of 1091 serum samples were included in the clinical validation for the ImmuLisa™ Enhanced AMA Antibody ELISAs. The validation set of samples includes the target disease group consisting of 193 PBC subjects, and a non-target disease group consisting of 898 samples from subjects with autoimmune, infectious, and other conditions expected to be found in the differential diagnosis of PBC. Test results for each individual disease and both disease groups are shown below:
| | AMA IgG | | | AMA IgA/IgG/IgM | |
| --- | --- | --- | --- | --- | --- |
| Condition | n | n Pos* | % Pos | n Pos* | % Pos |
| Target Disease Group | | | | | |
| Primary biliary cirrhosis (PBC) | 193 | 165 | 85.5 | 168 | 87.0 |
| Non-Target Disease Group | | | | | |
| Type 1 Autoimmune hepatitis (AIH) | 31 | 1 | 3.2 | 4 | 12.9 |
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| | AMA IgG | | | AMA IgA/IgG/IgM | |
| --- | --- | --- | --- | --- | --- |
| Condition | n | n Pos* | % Pos | n Pos* | % Pos |
| Type 2 Autoimmune hepatitis (AIH) | 7 | 0 | 0 | 0 | 0 |
| Primary sclerosing cholangitis (PSC) | 20 | 0 | 0 | 0 | 0 |
| Antiphospholipid syndrome | 48 | 0 | 0 | 0 | 0 |
| Celiac disease | 99 | 0 | 0 | 0 | 0 |
| Crohn's disease | 25 | 0 | 0 | 0 | 0 |
| Mixed connective tissue disorder | 16 | 0 | 0 | 0 | 0 |
| Myositis | 26 | 0 | 0 | 0 | 0 |
| Rheumatoid arthritis | 106 | 1 | 0.9 | 0 | 0 |
| Sjogren's syndrome | 54 | 0 | 0 | 0 | 0 |
| Systemic lupus erythematosus | 154 | 0 | 0 | 0 | 0 |
| Systemic sclerosis | 26 | 0 | 0 | 1 | 3.8 |
| Ulcerative colitis | 25 | 0 | 0 | 0 | 0 |
| CMV | 20 | 1 | 5.0 | 1 | 5.0 |
| HepC | 74 | 0 | 0 | 0 | 0 |
| HSV 1 | 20 | 0 | 0 | 3 | 15.0 |
| HSV 2 | 20 | 1 | 5.0 | 0 | 0 |
| Lyme's disease | 16 | 0 | 0 | 0 | 0 |
| Mononucleosus | 20 | 0 | 0 | 3 | 15.0 |
| Rubella | 26 | 2 | 7.7 | 0 | 0 |
| Syphilis | 23 | 2 | 8.7 | 2 | 8.7 |
| Toxoplasmosis | 20 | 1 | 5.0 | 0 | 0 |
| Alcoholic liver disease | 12 | 0 | 0 | 1 | 8.3 |
| Hepatocellular carcinoma | 10 | 0 | 0 | | 0 |
| Non-Target Disease Group Total | 898 | 12 | 0.1 | 15 | 0.2 |
| | | | | | |
| Total Samples: | 1091 | | | | |
| | | | | | |
| *Borderline samples considered positive. | | | | | |
The performance of each ImmuLisa Enhanced AMA Antibody ELISAs was evaluated against the clinical diagnosis of PBC. The clinical sensitivity and specificity were calculated by grouping the assays' indeterminate results with its test negative results, and then sensitivity and specificity were calculated again by grouping the assays' equivocal results with its test positive results:
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ImmuLisa Enhanced Mitochondria Antibody (AMA) IgG ELISA:
| | | Primary Biliary Cirrhosis Diagnosis | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced AMA IgG | Positive | 162 | 5 | 167 |
| | Equivocal | 3 | 4 | 7 |
| | Negative | 28 | 889 | 917 |
| | Total | 193 | 898 | 1091 |
Equivocal samples considered positive:
Clinical Sensitivity: $85.5\%$ 165/193 $(95\% \mathrm{CI}:79.5 - 90.0)$
Clinical Specificity: $99.0\%$ 889/898 $(95\% \mathrm{CI}: 98.0 - 99.5)$
Equivocal samples considered negative:
Clinical Sensitivity $83.9\%$ 162/193 $(95\% \mathrm{CI}:77.8 - 88.7)$
Clinical Specificity $99.4\%$ 893/898 $(95\% \mathrm{CI}: 98.6 - 99.8)$
ImmuLisa Enhanced Mitochondria Antibody (AMA) IgA/IgG/IgM ELISA:
| | | Primary Biliary Cirrhosis Diagnosis | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| ImmuLisa Enhanced AMA IgA/IgG/IgM | Positive | 162 | 8 | 170 |
| | Equivocal | 6 | 7 | 13 |
| | Negative | 25 | 883 | 908 |
| | Total | 193 | 898 | 1091 |
Equivocal samples considered positive:
Clinical Sensitivity: $87.0\%$ 168/193 $(95\% \mathrm{CI}:81.3 - 91.3)$
Clinical Specificity: $98.5\%$ 883/193 $(95\% \mathrm{CI}: 97.2 - 99.0)$
Equivocal samples considered negative:
Clinical Sensitivity $83.9\%$ 162/193 $(95\% \mathrm{CI}:77.8 - 88.7)$
Clinical Specificity $99.1\%$ 890/898 $(95\% \mathrm{CI}: 98.2 - 99.6)$
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
A study of 190 normal, apparently disease-free samples tested with the ImmuLisa Enhanced AMA Antibody ELISAs yielded three (1.6%) borderline or positive results for the AMA IgG assay and three (1.6%) borderline results for the AMA IgA/IgG/IgM assay.
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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.
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