EUROIMMUN ANTI-LKM-1 ELISA(LGG)
Device Facts
| Record ID | K112223 |
|---|---|
| Device Name | EUROIMMUN ANTI-LKM-1 ELISA(LGG) |
| Applicant | Euroimmun US |
| Product Code | NBS · Immunology |
| Decision Date | Sep 11, 2012 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5660 |
| Device Class | Class 2 |
Indications for Use
The EUROIMMUN Anti-LKM-1 ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against LKM-1 (cytochrome P450 IID6 of liver-kidney-microsomes) in human serum and plasma. It is used as an aid in the diagnosis of autoimmune hepatitis, type 2, in conjunction with other laboratory and clinical findings.
Device Story
ELISA-based in vitro diagnostic test; detects IgG autoantibodies against LKM-1 (cytochrome P450 IID6) in human serum or plasma. Kit includes microplate strips coated with recombinant cytochrome P450 IID6 antigen, calibrator, controls, HRP-conjugated rabbit anti-human IgG, and TMB substrate. Procedure: patient sample added to antigen-coated wells; autoantibodies bind; unbound proteins washed away; HRP-conjugate added; bound conjugate visualized via TMB substrate color development. Intensity measured by microplate reader at 450 nm (reference 620-650 nm). Results interpreted as ratio of sample OD to cut-off calibrator OD. Used in clinical laboratories by trained personnel to aid diagnosis of autoimmune hepatitis, type 2.
Clinical Evidence
Clinical study of 515 samples (68 AIH-2, 65 AIH-1, 15 AIH/PBC overlap, 367 controls). Sensitivity for AIH-2 was 95.6% (95% CI: 87.6–99.1%). Specificity was 100.0% (95% CI: 99.2–100.0%) across various liver diseases and autoimmune conditions. Method comparison with predicate (n=196) showed 96.9% overall agreement.
Technological Characteristics
ELISA-based immunoassay. Components: microwell plate coated with recombinant cytochrome P450 IID6 antigen, peroxidase-labeled anti-human IgG conjugate, TMB substrate. Requires microwell plate reader (450nm). Qualitative result reported as ratio. Manual or automated processing.
Indications for Use
Indicated for qualitative detection of IgG autoantibodies against LKM-1 in human serum/plasma as an aid in diagnosing autoimmune hepatitis type 2. For prescription use only.
Regulatory Classification
Identification
A multiple autoantibodies immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies (antibodies produced against the body's own tissues) in serum and other body fluids. Measurement of multiple autoantibodies aids in the diagnosis of autoimmune disorders (disease produced when the body's own tissues are injured by autoantibodies).
Predicate Devices
- Inova Quanta Lite LKM-1 ELISA (K000535)
Related Devices
- K112221 — EUROIMMUN ANTI-SLA/LP ELISA(LGG) · Euroimmun US · Sep 11, 2012
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k112223
B. Purpose for Submission:
New assay
C. Measurand:
Anti-liver/kidney microsome, type 1 (LKM-1) IgG antibodies
D. Type of Test:
Qualitative
E. Applicant:
EUROIMMUN US Inc.
F. Proprietary and Established Names:
EUROIMMUN Anti-LKM-1 ELISA (IgG)
G. Regulatory Information:
1. Regulation section:
21 CFR §866.5660, Multiple autoantibodies immunological test system
2. Classification:
Class II
3. Product code:
NBS, autoantibodies, LKM (liver/kidney microsome, type 1)
4. Panel:
Immunology (82)
H. Intended Use:
1. Intended use(s):
The EUROIMMUN Anti-LKM-1 ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against LKM-1 (cytochrome P450 IID6 of liver-kidney-microsomes) in human serum and plasma. It is used as an aid in the diagnosis of autoimmune hepatitis, type 2, in conjunction with other laboratory and clinical findings.
2. Indication(s) for use:
Same as intended use above.
3. Special conditions for use statement(s):
For prescription use only.
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4. Special instrument requirements:
Microplate reader capable of measuring OD at 450 nm and 620 to 650 nm.
I. Device Description:
The device is packaged as a kit containing: 12 microplate strips each containing 8 antigen coated wells and frame, one cut-off calibrator (20 RU/mL), human serum-based positive and negative controls, horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG, ready-to-use sample buffer, 10x wash buffer concentrate, 3,3',5,5' tetramethylbenzidine (TMB) substrate and sulfuric acid stop solution.
J. Substantial Equivalence Information:
1. Predicate device name(s) and k number(s):
INOVA QUANTA Lite LKM-1 ELISA, k000535
2. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Detection anti-LKM-1 IgG autoantibodies to “aid in the diagnosis of autoimmune hepatitis, type 2, in conjunction with other laboratory and clinical findings”. | Same |
| Assay | ELISA | Same |
| Antigen | Recombinant cytochrome P450 IID6 | Same |
| Substrate | TMB | Same |
| Controls | One Positive Control, One Negative Control | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Assay Type | Qualitative | Semi-quantitative |
| Conjugate | Rabbit anti-human IgG labeled with HRP | Goat anti-human IgG labeled with HRP |
| Calibrators and Controls | 1 cut-off calibrator2 controls: 1 positive, 1 negative | 3 controls:1 high positive, 1 low positive, 1 negative |
| Sample Matrix | Serum or plasma (EDTA, heparin, and citrate) | Serum only |
| Reported Units | Ratio | U/mL |
| Cut-Off Level | Ratio 1.0 | 25 U/mL |
K. Standard/Guidance Document Referenced (if applicable):
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CEN 13640:2002, Stability Testing of In Vitro Diagnostic Reagents.
## L. Test Principle:
The test wells are coated with recombinant cytochrome P450 IID6. The cut-off calibrator, controls, or diluted patient samples are added to the wells and autoantibodies recognizing the antigen bind during the first incubation. After washing the wells to remove all unbound proteins, conjugate is added. The conjugate binds to the captured human autoantibodies. Excess unbound conjugate is removed by another wash step. The bound conjugate is visualized TMB substrate. The intensity of color development is proportional to the concentration of autoantibody in the sample. Microtiter plates are read at 450 nm and a reference wavelength of 620 to 650 nm. The controls and patient results are interpreted by comparing them as a ratio of the cut-off calibrator.
## M. Performance Characteristics (if/when applicable):
### 1. Analytical performance:
#### a. Precision/Reproducibility:
The reproducibility of the assay was assessed using patient sera with a range of values. Intra-assay reproducibility was assessed by 20 determinations on one day while inter-assay reproducibility was based on 24 determinations performed over six different days. The samples were the same in both determinations. The expected result was determined from the sample mean. The results that correctly matched the expected result were tallied. The results are summarized in the tables below:
Intra-assay reproducibility:
| Sample # | Sample Mean Ratio | Ratio Range | Expected Result | # Correct Results |
| --- | --- | --- | --- | --- |
| 1 | 0.2 | 0.2 – 0.2 | Neg | 20/20 |
| 2 | 0.8 | 0.7 – 0.8 | Neg | 20/20 |
| 3 | 1.2 | 1.1 – 1.3 | Pos | 20/20 |
| 4 | 1.9 | 1.8 – 2.0 | Pos | 20/20 |
| 5 | 3.0 | 2.9 – 3.2 | Pos | 20/20 |
| 6 | 4.7 | 4.1 – 5.0 | Pos | 20/20 |
Inter-assay reproducibility:
| Sample # | Sample Mean Ratio | Ratio Range | Expected Result | # Correct Results |
| --- | --- | --- | --- | --- |
| 1 | 0.2 | 0.2 – 0.3 | Neg | 24/24 |
| 2 | 0.8 | 0.7 – 1.0 | Neg | 23/24 |
| 3 | 1.2 | 1.0 – 1.3 | Pos | 24/24 |
| 4 | 1.9 | 1.5 – 2.2 | Pos | 24/24 |
| 5 | 2.9 | 2.4 – 3.4 | Pos | 24/24 |
| 6 | 4.6 | 3.9 – 5.2 | Pos | 24/24 |
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Lot-to-lot reproducibility was assessed using patient sera with a range of values distributed over the measurement range. Each sample/lot combination was tested between six and 38 times. The expected result was determined from the sample mean. The numbers of results that correctly match the expected result were determined. The results are summarized in the table below:
| Sample | Sample Mean Ratio | Ratio Range | Lots Tested | Runs /Lot | Total Replicates | Expected Result | # Correct Results |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.9 | 0.9 – 0.9 | 3 | 2 | 6 | Neg | 6/6 |
| 2 | 1.2 | 1.2 – 1.4 | 3 | 2 | 6 | Pos | 6/6 |
| 3 | 1.7 | 1.6 – 1.8 | 3 | 2 | 6 | Pos | 6/6 |
| 4 | 2.4 | 2.3 – 2.5 | 3 | 2 | 6 | Pos | 6/6 |
| 5 | 2.9 | 2.8 – 3.0 | 3 | 2 | 6 | Pos | 6/6 |
| 6 | 4.8 | 4.5 – 5.1 | 3 | 2 | 6 | Pos | 6/6 |
| 7 | 5.8 | 5.7 – 6.0 | 3 | 2 | 6 | Pos | 6/6 |
| 8 | 7.3 | 5.4 – 8.3 | 32 | 1 | 32 | Pos | 32/32 |
| 9 | 8.0 | 7.2 – 8.7 | 7 | 1 | 7 | Pos | 7/7 |
| 10 | 0.1 | 0.1 – 0.1 | 14 | 1 | 14 | Neg | 10/10 |
| 11 | 0.1 | 0.1 – 0.2 | 8 | 1 | 8 | Neg | 8/8 |
b. Linearity/assay reportable range:
Not applicable.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
There are no international standards for LKM-1 antibodies. The results are expressed as ratios of the cut-off calibrator.
Calibrators and Controls:
The cut-off calibrator and controls are derived from human serum and match pre-specified performance criteria. Each lot of cut-off calibrator and controls are traceable to a master lot.
Stability studies:
Stability studies are conducted in accordance to CEN 13640:2002. Three production lots of all kit reagents were tested. Real-time stability studies (+2°C to +8°C) support a 12-month unopened stability claim. Other studies support that the re-constituted wash buffer is stable for up to 28 days, and opened reagents stored at +2°C to +8°C are stable for 12 months.
Sample stability:
The sponsor recommends following the guidelines in CLSI H18-A3 for sample storage.
d. Detection limit:
Not applicable.
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e. Analytical specificity:
Endogenous interferents:
Five samples containing various levels of anti-LKM-1 were spiked with varying concentrations of hemoglobin up to 1,000 mg/dL, triglycerides up to 2,000 mg/dL, or bilirubin up to 40 mg/dL. Comparison of all spiked sample/interferent combinations to the unspiked sample was within ±15% of the original sample concentration except one; a sample with a ratio of 7.2 spiked with 2,000 mg/dL triglycerides had 119% recovery. Lower concentrations of triglycerides in the same sample showed recovery ± 15% of the unspiked sample.
Cross-reactivity with anti-SLA/LP:
Twenty-eight (28) sera serologically positive for anti-SLA/LP antibodies were tested with the assay and found negative for anti-LKM-1 antibodies suggesting that antibodies specific for anti-SLA/LP do not bind to the LKM-1 antigen coating the assay wells. Sample ratios ranged from 0.1 – 0.9.
f. Assay cut-off:
The assay cut-off is 1.0. Results > 1.0 are positive.
2. Comparison studies:
a. Method comparison with predicate device:
One hundred and sixty-seven (167) clinically characterized samples were tested with the predicate device and the EUROIMMUN Anti-LKM-1 ELISA (IgG). These samples were from patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), AIH/PBC overlap syndrome, and viral hepatitis. Twenty-nine samples intended to challenge the assay cut-off were created by mixing positive and negative samples:
| | Predicate* | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| EUROIMMUNE
Anti-LKM-1 ELISA | Positive | 35 | 5 | 40 |
| | Negative | 1 | 155 | 156 |
| | Total | 36 | 160 | 196 |
*The predicate assay has an equivocal zone for result interpretation but none of the samples fell in this range.
Positive agreement 35/36 = 97.2% 95% CI: 85.5% – 99.9%
Negative agreement 155/160 = 96.6% 95% CI: 92.9% – 99.0%
b. Matrix comparison:
The purpose of this study was to demonstrate that the new devices give the same results for lithium heparin plasma, citrate plasma and EDTA plasma for serum samples collected from the same patient. Normal sample pairs were spiked with anti-LKM-1 positive sera to create 16 samples that spanned the assay range. Serum sample ratios ranged from 0.4 – 9.6. Bias of the heparin plasma, citrate plasma, and EDTA plasma samples ranged from 93% to 109%,
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87% to 106%, and 93% to 106%, respectively.
## 3. Clinical studies:
a. Clinical sensitivity and specificity:
Five hundred and fifteen (515) clinically characterized samples from several university, hospital, and private laboratories were analyzed with the EUROIMMUN Anti-LKM-1 ELISA (IgG) test to assess the clinical sensitivity and specificity. The samples consisted of 68 AIH-2, 65 AIH-1, 111 primary biliary cirrhosis (PBC), 15 from patients with AIH/PBC overlap syndrome, 47 hepatitis B, 71 hepatitis C, 19 primary sclerosing cholangitis (PSC), 24 steatohepatitis, 14 toxic liver damage, and 81 samples from other autoimmune diseases.
| | Autoimmune Hepatitis Type 2 | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| Anti-LKM-1 | Positive | 65 | 0 | 65 |
| | Negative | 3 | 447 | 450 |
| | Total | 68 | 447 | 515 |
Sensitivity: 95.6%
95% CI: 87.6% – 99.1%
Specificity: 100.0%
95% CI: 99.2% – 100.0%
The results for non-AIH-1 subjects are shown below:
| Results by Diagnosis | Anti-LKM-1 (IgG) | |
| --- | --- | --- |
| | n | Positive (%) |
| Autoimmune hepatitis type 1 (AIH-1) | 65 | 0 (0%) |
| AIH/PBC overlap syndrome | 15 | 0 (0%) |
| Viral hepatitis | 118 | 0 (0.0%) |
| Toxic liver damages | 14 | 0 (0.0%) |
| Steatohepatitis | 24 | 0 (0.0%) |
| Primary biliary liver cirrhosis (PBC) | 111 | 0 (0.0%) |
| Primary sclerosing cholangitis (PSC) | 19 | 0 (0.0%) |
| Other autoimmune diseases* | 81 | 0 (0.0%) |
* Other autoimmune diseases include: MCTD, celiac disease, Type 1 diabetes, Hashimoto thyroiditis, Graves' disease, and ulcerative colitis
b. Other clinical supportive data:
Not applicable.
4. Clinical cut-off:
Not applicable.
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5. Expected values/Reference range:
The levels of anti-LKM-1 antibodies in 150 apparently healthy blood donor samples (Caucasian, mixed age and sex) were analyzed with the EUROIMMUN Anti-LKM-1 ELISA (IgG). One sample was positive (ratio = 3.4). Negative sample ratios ranged from 0 to 0.9.
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.
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