EUROIMMUN ANTI-NRNP/SM ELISA (IGG)
Device Facts
| Record ID | K123261 |
|---|---|
| Device Name | EUROIMMUN ANTI-NRNP/SM ELISA (IGG) |
| Applicant | Euroimmun US |
| Product Code | LKO · Immunology |
| Decision Date | Jun 12, 2013 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5100 |
| Device Class | Class 2 |
Intended Use
The EUROIMMUN Anti-nRNP/Sm ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against nRNP/Sm in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of mixed connective tissue diseases and systemic lupus erythematosus, in conjunction with other laboratory and clinical findings. The EUROIMMUN Anti-Sm ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against Sm in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of systemic lupus erythematosus, in conjunction with other laboratory and clinical findings. The EUROIMMUN Anti-SS-A ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against SS-A in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of Sjögren’s syndrome and systemic lupus erythematosus, in conjunction with other laboratory and clinical findings. The EUROIMMUN Anti-SS-B ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against SS-B in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of Sjögren’s syndrome and systemic lupus erythematosus, in conjunction with other laboratory and clinical findings. The EUROIMMUN Anti-Scl-70 ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against Scl-70 in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of progressive systemic sclerosis, in conjunction with other laboratory and clinical findings. The EUROIMMUN Anti-Centromeres ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against Centromeres in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of limited form of progressive systemic sclerosis (CREST syndrome), in conjunction with other laboratory and clinical findings. The EUROIMMUN Anti-Jo-1 ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against Jo-1 in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of polymyositis and dermatomyositis, in conjunction with other laboratory and clinical findings. The EUROIMMUN Anti-ribosomal P-proteins ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against ribosomal P-proteins in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of systemic lupus erythematosus, in conjunction with other laboratory and clinical findings.
Device Story
The EUROIMMUN ELISA test kits are qualitative enzyme immunoassays for detecting specific IgG autoantibodies in human serum or plasma. The device consists of a microwell plate coated with specific antigens (nRNP/Sm, Sm, SS-A, SS-B, Scl-70, Centromeres, Jo-1, or Ribosomal P-proteins). Patient samples are diluted and incubated in the wells; unbound antibodies are washed away. Peroxidase-labeled anti-human IgG conjugate is added, followed by a second incubation and wash. TMB substrate is added, producing a color change proportional to the antibody concentration, which is stopped with sulfuric acid. The plate is read at 450nm using a microwell plate reader. Results are calculated as a ratio against a calibrator. The device is used in clinical laboratories by trained personnel to aid in the diagnosis of various autoimmune conditions. The two-step assay design minimizes matrix effects and interference from binding proteins. Healthcare providers use the qualitative results in conjunction with clinical findings to support diagnostic decisions, potentially improving patient management for connective tissue diseases.
Clinical Evidence
Clinical performance was evaluated using large cohorts of clinically characterized samples (e.g., 1046 for nRNP/Sm, 1036 for Sm, 1026 for SS-A/SS-B, 909 for Scl-70, 877 for Centromeres, 876 for Jo-1, 876 for Ribosomal P). Sensitivity and specificity were calculated against clinical diagnoses. Specificity ranged from 94.8% to 99.8% across the panels. Method comparison studies against predicate devices showed high overall agreement (95.9% to 100.0%).
Technological Characteristics
ELISA platform using 96-well microtiter plates. Antigen-coated wells; peroxidase-labeled anti-human IgG conjugate; TMB substrate; 0.5 M sulfuric acid stop solution. Manual or automated processing; requires 450nm plate reader. Qualitative result reporting via ratio. Sample dilution 1:201. Compatible with serum, EDTA, Li-heparin, and Citrate plasma.
Indications for Use
Indicated for qualitative detection of IgG autoantibodies in human serum/plasma to aid diagnosis of mixed connective tissue disease, systemic lupus erythematosus, Sjögren’s syndrome, progressive systemic sclerosis, CREST syndrome, polymyositis, and dermatomyositis.
Regulatory Classification
Identification
An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues).
Predicate Devices
- Inova Quanta Lite RNP ELISA (K922833)
- Inova Quanta Lite Sm ELISA (K922831)
- Inova Quanta Lite SS-A ELISA (K922830)
- Inova Quanta Lite SS-B ELISA (K922832)
- Inova Quanta Lite Scl-70 ELISA (K924898)
- Inova Quanta Lite Centromeres ELISA (K003488)
- Inova Quanta Lite Jo-1 ELISA (K922832)
- Inova Quanta Lite Ribosomal P ELISA (K981237)
Related Devices
- K112996 — EUROIMMUN ANTI-ENA POOL ELISA (IGG) · Euroimmun US · Apr 9, 2013
Submission Summary (Full Text)
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K 1.23261
JUN 1 2 2013
ATTACHMENT I
### PREMARKET NOTIFICATION 210(K) SAFETY AND EFFECTIVENESS SUMMARY (as required by 21 CFR § 807.92)
- A. 510(k)Number: K123261
- B. Purpose for Submission: New device
- C. Measurand:
D.
H.
- Anti-nRNP/Sm autoantibodies
- Type of Test: Qualitative enzyme immunoassay
- E. Applicant:
- EUROIMMUN US INC.
- Proprietary and Established Names: ಿ. EUROIMMUN Anti-nRNP/Sm ELISA (IgG)
- G. Requlatory Information:
- Regulation: 1.
- 21 CFR 866.5110 Antinuclear antibody immunological test system
- 2. Classification: Class II
- 3. Product code: LKO
- 4. Panel:
Immunology Intended Use:
- Intended use(s): 1 .
The EUROIMMUN Anti-nRNP/Sm ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against nRNP/Sm in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of mixed connective tissue diseases and systemic lupus erythematosus, in conjunction with other laboratory and clinical findings.
- 2. Indication(s) for use: Same as intended use.
- 3. Special conditions for the use statement(s):
For prescription use only.
- 4. Special instrument requirements:
Microwell plate reader capable of measuring OD at 450nm for dual wavelength readings.
#### l. Device Description:
The EUROIMMUN Anti-nRNP/Sm ELISA (IgG) consists of a microwell ELISA plate coated with nRNP/Sm antigen, calibrator, positive and negative control, peroxidase-labelled anti-human IgG conjugate, sample buffer, wash buffer concentrate, TMB chromogen/substrate solution and stop solution.
#### J. Substantial Equivalence Information:
- Predicate device name (s): 1 .
- Inova Quanta Lite RNP ELISA
- 2. Predicate 510(k) number(s): K922833
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#### 3. Comparison with predicate:
| Item | New Device | Predicate Device |
|-----------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------|
| Intended use | Detection of IgG antibodies to nRNP/Sm | Same |
| Technology | ELISA | Same |
| Assay platform | 96-well microtiter plates | Same |
| Calibration | Relative evaluation | Same |
| Antigen | Purified U1-nRNP complex; U1-nRNP contains RNP as<br>well as Sm reactive proteins | Same |
| Conjugate | Anti-human IgG labeled with horseradish peroxidase | Same |
| Substrate | TMB | Same |
| Reagent preparation | All reagents, calibrators and controls are ready to use,<br>except for the wash buffer. | Same |
| Procedure | Sample incubation with micro-well antigen coated plate,<br>followed by a wash step, incubation with an anti-human<br>IgG enzyme conjugate; wash step, incubation with<br>substrate; then the addition of a stop solution and<br>reading at 450nm. | Same |
| Item | New Device | Predicate Device |
| Assay format | Qualitative | "Semi-quantitative" (using low positive control) |
| Sample dilution | 1:201 | 1:101 |
| Calibrators and<br>controls | 1 calibrator<br>2 controls: 1 positive, 1 negative | 3 controls: 1 high positive, 1 low positive (used for<br>calculation of results), 1 negative |
| Wash buffer | 10x concentrate | 40x concentrate |
| Stop solution | 0.5 M sulphuric acid | 0.344 M sulphuric acid |
| Sample types | Serum or plasma (EDTA, Li-heparin, Citrate) | Serum |
| Reported results | Ratio | Units |
| Cut off level | Ratio 1.0 | 20 Units |
#### K. Standard/Guidance Document Referenced (if applicable):
Guidance for Industry and FDA Staff: Recommendations for Antibody (ANA) Test System Premarket (510(k)) Submissions (January 22, 2009)
#### L. Test Principle:
1.
Patient samples are diluted 1:201 in sample buffer, 100 µl of each diluted patient sample and pre-diluted controls and calibrators are added to the antigen coated microtiter wells and incubated for 30 minutes at room temperature. After incubation the microtiter well strips are washed with wash buffer to remove unbound antibodies and 100 µl of the anti-human IgG enzyme conjugate reagent is added to each microtiter well. After an additional 30-minutes incubation at room temperature, the microtiter wells are again washed 3 times with 300 pl of wash buffer to remove any unbound enzyme conjugate and 100 µl of the chromogen substrate is added. The strips are incubated for 15 minutes at room temperature and 100 µl stop solution is added. The microtiter plates are placed in an ELISA reader and read at a wavelength of 450 nm and a reference wavelength of between 620 nm and 650 nm within 30 minutes.
#### Performance Characteristics (where applicable): M.
- Analytical performance:
- Precision/Reproducibility: a.
The reproducibility of the test was investigated using sera with different concentrations. Intra-assay reproducibility is based on 20 determinations and inter-assay reproducibility on 40 determinations performed in 10 different runs on 5 days with 2 runs per day according to the package insert. The following results were obtained:
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### Intra-assay reproducibility
| n = 20 | Anti-nRNP/Sm ELISA (IgG) Ratio | | | | | |
|------------------|--------------------------------|-----------|-----------|-----------|-----------|-----------|
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| Mean value (x): | 5.1 | 3.3 | 1.9 | 1.2 | 0.8 | 0.4 |
| Range of values: | 5.0 - 5.1 | 3.2 - 3.4 | 1.8 - 2.0 | 1.1 - 1.2 | 0.7 - 0.9 | 0.3 - 0.4 |
| Expected result: | positive | positive | positive | positive | negative | negative |
| % positive: | 100% | 100% | 100% | 100% | 0% | 0% |
| % negative: | 0% | 0% | 0% | 0% | 100% | 100% |
Inter-assay reproducibility
| | Anti-nRNP/Sm ELISA (IgG) | | | | | |
|------------------|--------------------------|-----------|-----------|-----------|-----------|-----------|
| n = 10 x 4 | Ratio | | | | | |
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| Mean value (x): | 5.2 | 3.3 | 1.8 | 1.1 | 0.8 | 0.4 |
| Range of values: | 4.8 - 5.5 | 3.1 - 3.6 | 1.6 - 1.9 | 1.1 - 1.2 | 0.7 - 0.9 | 0.3 - 0.4 |
| Expected result: | positive | positive | positive | positive | negative | negative |
| % positive: | 100% | 100% | 100% | 100% | 0% | 0% |
| % negative: | 0% | 0% | 0% | 0% | 100% | 100% |
The lot to lot reproducibility was investigated during the validaton and quality control of the kit using different lots with QC samples distributed over the measurement range. The following results were obtained:
Lot to lot reproducibility
| | Anti-nRNP/Sm ELISA (IgG)<br>Ratio | | | | | | | Anti-nRNP/Sm ELISA (IgG)<br>Ratio | | | | |
|------------------|-----------------------------------|-----------|-----------|-----------|-----------|-----------|------------------|-----------------------------------|-----------|-----------|-----------|-----------|
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 |
| n | 6* | 6* | 6* | 6* | 6* | 6* | n | 11** | 11** | 11** | 11** | 11** |
| Mean value (x): | 3.6 | 2.5 | 5.4 | 0.4 | 0.9 | 1.1 | Mean value (x): | 0.2 | 3.4 | 5.2 | 6.6 | 8.5 |
| Range of values: | 3.4 - 3.9 | 2.3 – 2.8 | 4.8 - 5.7 | 0.3 - 0.4 | 0.9 - 0.9 | 1.1 - 1.1 | Range of values: | 0.1 - 0.3 | 3.1 - 3.8 | 4.7 - 5.9 | 5.7 - 7.2 | 8.0 - 9.7 |
| Expected result: | positive | positive | positive | negative | negative | positive | Expected result: | negative | positive | positive | positive | positive |
| % positive: | 100% | 100% | 100% | 0% | 0% | 100% | % positive: | 0% | 100% | 100% | 100% | 100% |
| % negative: | 0% | 0% | 0% | 100% | 100% | 0% | % negative: | 100% | 0% | 0% | 0% | 0% |
*3 lots x 2 runs ** n lots x 1 run
> b. Linearity/assay reportable range: Not applicable.
Not applicable.
High dose/leak-off.
C. High dose Hook effect
Potential for a high dose Hook effect is a phenomenon that is inherent with one step "sandwich" assay designs: Very high concentrations of antigen in the patient sample bind to all available sites saturating them - on both the antibody-solid phase and the antibody-labeled conjugate and thereby prevent the "sandwich" formation. Under these conditions, the measured level of analyte may be significantly lower than the actual level present in the sample. The two-step immunoassay design of the Anti-nRNP/Sm ELISA (IgG) eliminates the adverse contribution of binding proteins, endogenous interfering substances and general matrix effects due to the extra wash step.
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Image /page/3/Picture/1 description: The image shows a pattern of alternating black and white shapes. The black shapes are arranged in a row and are separated by white spaces. The black shapes are rounded and symmetrical, resembling beads or capsules. The white spaces between the black shapes are rectangular and uniform in size.
#### d. Traceability, Stability, Expected values (controls, calibrators or methods):
A recognized standard or reference material for anti-nuclear antibodies is not available. Results of this assay are given in ratios. The reactivity of the Anti-nRNP/Sm ELISA was verified using the ANA reference panel of the CDC Centers for Disease Control and Prevention, Atlanta, USA.
- Limit of detection: e.
- Not applicable.
- f Analytical specificity:
Cross-reactivity: The quality of the antigen coated on the plates ensures a high specificity of the ELISA. Cross reactivity was investigated using a panel of 30 sera serologically positive for antibodies against rib.P-P, SS-A, SS-B, Scl-70, Jo-1, centromeres, Rubella virus, Measles virus, Herpes simplex virus type 1 and Borrelia burgdorferi. All 30 sera were negative in the Anti-nRNP/Sm ELISA (IgG), so no cross reactivity is expected.
Interference: To investigate the influence from hemoglobin, triglycerides and bilirubin, 5 different sera at different anti-nRNP/Sm concentrations (ratio 0.9 - 5.6) were spiked with potential interfering substances and were incubated with the test system acording to the package insert. Interferences from rheumatoid factor were investigated similarly by spiking with a RF positive serum. The recovery in relation to the unspiked sample without interferent was calculated. The individual recovery was within the range of 85 - 109 %. No significant interference was observed for concentrations of up to 1000 mg/dl for hemoglobin, 2000 mg/dl for triglyceride, 40 mg/dl for billrubin and 500 IU/ml for rheumatoid factor.
- Assay cut-off: g.
Ratio 1.0
#### 2. Comparison studies:
- Method comparison with predicate device: a.
A comparison study was performed using 287 clinically characterized samples (52 MCTD, 69 SLE, 51 Sjögren's syndrome, 15 systemic sclerosis, 15 fibromyalgia, 35 RA, 30 borreliosis and 20 healthy), obtained from different sources from Europe and North America. The panel consisted of 49 men and 238 women. Age ranged from 14 to 85 years with an average age of 46 years. AntinRNP/Sm antibodies are expected in either MCTD or SLE. The other disease groups serve as control cohorts. The samples were tested with the EUROIMMUN Anti-nRNP/Sm ELISA (IgG) and with the Inova Quanta Lite RNP ELISA as the predicate device. Of the 8 discrepant samples, one was from a MCTD patient and the other 7 were from controls.
| All samples<br>n = 287 | | Predicate ELISA | |
|---------------------------------------|---------------|-----------------|--------------------------|
| | | positive | negative |
| EUROIMMUN<br>Anti-nRNP/Sm ELISA (IgG) | positive | 83 | 0 |
| | negative | 8 | 196 |
| Negative agreement | $196 / 196 =$ | 100.0% | 95% C.I.: 98.1% - 100.0% |
| Positive agreement | $83 / 91 =$ | 91.2% | 95% C.I.: 83.4% - 96.1% |
| Overall agreement | $279 / 287 =$ | 97.2% | 95% C.I.: 94.6% - 98.8% |
- b. Matrix comparison:
The usability of plasma was investigated using sample pairs each of serum and corresponding plasma (EDTA, Li-heparin, Citrate). Passing-Bablok regression was calculated for the comparison of serum to plasma. The regression equation is near the ideal correlation (intercept 0; slope 1.0) indicating equivalence of concentrations between serum and the corresponding plasma matrices. The comparison below satisfies this condition. Coefficients of determination were found to be above 0.99 and %recovery compared toserum was in the range of 91 to 120 % (serum = 100 %)
| | EDTA plasma | Li-heparin plasma | Citrate plasma |
|------------------------------------------------|---------------------|----------------------|-------------------|
| n | 15 | 15 | 15 |
| Regression equation<br>(y = plasma, x = serum) | $y = 0.46 + 0.98 x$ | $y = -0.25 + 1.05 x$ | $y = 0.37 + 1.04$ |
| 95% C.I. of intercept | -0.56 - 2.41 | -1.54 - 1.27 | -1.72 - 1.82 |
| 95% C.I. of slope | 0.95 - 1.01 | 0.97 - 1.09 | 0.97 - 1.09 |
| Coefficient of determination R² | 0.9988 | 0.9929 | 0.9915 |
| Mean %recovery | 101 % | 103 % | 105 % |
| Range of %recovery | 91 - 117 % | 92 - 114 % | 93 - 120 % |
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### Clinical studies:
Clinical studies were performed in cooperation with different sites. In total 1046 clinically characterized samples (65 from MCTD patients, 404 from SLE patients, 151 from myositis patients and 426 from control groups) were investigated for antibodies (IgG). With the EUROIMMUN Anti-nRNP/Sm ELISA (IgG) a prevalence of 100.0% (95% C.I.: 94.5 - 100.0%) was found in MCTD (clinical sensitivity) and a prevalence of 23.3% (95% C.I.: 19.2 - 27.7%) in SLE with a specificity of 99.3% (95% C.I.: 98.0 - 99.9%). The myositis panel was not considered for calculation of sensitivity and specificity, because anti-nRNP/Sm antibodies may occur in this disease (Tomer 1993). The results are shown in the table below. 95% C.I. are calculated by the exact method.
- Clinical sensitivity: a.
| No. | Panel | n | Anti-nRNP/Sm ELISA (IgG) | | |
|-----|----------------------------------|-----|--------------------------|--------|---------------|
| | | | positive | % | 95% C.I. |
| 1 | Mixed connective tissue diseases | 65 | 65 | 100.0% | 94.5 - 100.0% |
| 2 | Systemic lupus erythematosus | 404 | 94 | 23.3% | 19.2 - 27.7% |
- Clinical specificity: b.
| No. | Panel | n | Anti-nRNP/Sm ELISA (IgG) | | |
|-----|------------------------------|-----|--------------------------|--------|---------------|
| | | | negative | % | 95% C.I. |
| 3 | Polymyositis/dermatomyositis | 151 | 143 | 94.7% | 89.8 - 97.7% |
| 4 | Rheumatoid arthritis | 164 | 164 | 100.0% | 97.8 - 100.0% |
| 5 | Systemic sclerosis | 81 | 81 | 100.0% | 95.5 - 100.0% |
| 6 | Sjögren's syndrome | 88 | 86 | 97.7% | 92.0 - 99.7% |
| 7 | Other autoimmune diseases* | 63 | 62 | 98.4% | 91.5 - 100.0% |
| 8 | Borreliosis | 30 | 30 | 100.0% | 88.4 - 100.0% |
| | Total | 577 | 566 | 98.1% | 96.6 - 99.9% |
*from the following groups: AIH (n = 8), Grave's disease (n = 12), Hashimoto (n = 11), celiac disease (n = 11), Diabetes Type I (n = 12)
- Other clinical supportive data (when a. and b. are not applicable): C. Not applicable.
- 4. Clinical cut-off:
See Assay cut-off.
- ട. Expected values/Reference range:
The levels of anti-nRNP/Sm antibodies (IgG) were analyzed in a panel of 200 samples from apparently healthy blood donors (120 men and 80 women with an average age of 40 y; age range: 19 - 68 y). The results are shown in the table below.
| n | 200 |
|---------------|-------|
| Positives | 1 |
| Negatives | 199 |
| Prevalence | 0.5% |
| | Ratio |
| Lowest value | 0.1 |
| Highest value | 1.3 |
| Mean value | 0.1 |
| Std deviation | 0.09 |
#### 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.
Michael Locke
Signature
Dir. Regulatory Affairs Title
12 June 2013 Date
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ATTACHMENT 1
### PREMARKET NOTIFICATION રાજીન્દ) SAFETY AND EFFECTIVENESS SUMMARY (as required by 21 CFR § 807.92)
- A. 510(k)Number: K123261
- B. Purpose for Submission: New device
- C. Measurand:
- Anti-Sm autoantibodies
- D. Type of Test: Qualitative enzyme immunoassay
- E. Applicant:
- EUROIMMUN US INC.
- F. Proprietary and Established Names: EUROIMMUN Anti-Sm ELISA (IgG)
#### G. Regulatory Information:
- 1. Regulation:
- 21 CFR 866.5110 Antinuclear antibody immunological test system
- 2. Classification: Class II
- 3. Product code: LKP
- 4. Panel:
H.
- Immunology Intended Use:
- 1. Intended use(s):
The EUROIMMUN Anti-Sm ELISA (IgG) test kit is intended for the qualitative of IgG class autoantibodies against Sm in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of systemic lupus erythematosus, in conjunction with other laboratory and clinical findings.
- 2. Indication(s) for use:
- Same as intended use.
- 3. Special conditions for the use statement(s):
- For prescription use only.
- র্ম Special instrument requirements:
Microwell plate reader capable of measuring OD at 450nm for dual wavelength readings.
#### l. Device Description:
The EUROIMMUN Anti-Sm ELISA (IgG) consists of a microwell ELISA plate coated with Sm antigen, calibrator, positive and negative control, peroxidase-labelled anti-human IgG conjugate, sample buffer, wash buffer concentrate, TMB chromogen/substrate solution and stop solution.
#### J. Substantial Equivalence Information:
- Predicate device name (s): 1.
- Inova Quanta Lite Sm ELISA 2. Predicate 510(k) number(s):
- K922831
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Image /page/6/Picture/1 description: The image shows a black and white illustration of a ladder. The ladder has four rungs that are evenly spaced apart. The rungs are connected to two vertical rails on either side.
#### 3. Comparison with predicate:
| Similarities | | |
|---------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|
| Item | New Device | Predicate Device |
| Intended use | Detection of IgG antibodies to Sm | Same |
| Technology | ELISA | Same |
| Assay platform | 96-well microtiter plates | Same |
| Calibration | Relative units | Same |
| Antigen | Purified Sm antigen | Same |
| Conjugate | Anti-human IgG labeled with horseradish peroxidase | Same |
| Substrate | TMB | Same |
| Reagent preparation | All reagents, calibrator and controls are ready to use,<br>except for the wash buffer. | Same |
| Procedure | Sample incubation with micro-well antigen coated plate,<br>followed by a wash step, incubation with an anti-human<br>IgG enzyme conjugate; wash step, incubation with<br>substrate; then the addition of a stop solution and<br>reading at 450nm. | Same |
| Differences | | |
| Item | New Device | Predicate Device |
| Assay format | Qualitative | "Semi-quantitative" (using low positive control) |
| Sample dilution | 1:201 | 1:101 |
| Calibrators and | 1 calibrator | 3 controls: 1 high positive, 1 low positive (used for |
| controls | 2 controls: 1 positive, 1 negative | calculation of results), 1 negative |
| Wash buffer | 10x concentrate | 40x concentrate |
| Stop solution | 0.5 M sulphuric acid | 0.344 M sulphuric acid |
| Sample types | Serum or plasma (EDTA, Li-heparin, Citrate) | Serum |
| Reported results | Ratio | Units |
| Cut off level | Ratio 1.0 | 20 Units |
#### K. Standard/Guidance Document Referenced (if applicable):
Guidance for Industry and FDA Staff: Recommendations for Antibody (ANA) Test System Premarket (510(k)) Submissions (January 22, 2009)
#### L. Test Principle:
1.
Patient samples are diluted 1:201 in sample buffer, 100 pl of each diluted patient sample and pre-diluted controls and calibrators are added to the antigen coated microtiter wells and incubated for 30 minutes at room temperature. After incubation the microtier well strips are washed with wash buffer to remove unbound antibodies and 100 ul of the anti-human IgG enzyme coniugate reagent is added to each microtiter well. After an additional 30-minutes incubation at room temperature, the microtiter wells are again washed 3 times with 300 µl of wash buffer to remove any unbound enzyme conjugate and 100 µl of the chromogen substrate is added. The strips are incubated for 15 minutes at room temperature and 100 ul stop solution is added. The microtiter plates are placed in an ELISA reader and read at a wavelength of 450 nm and a reference wavelength of between 620 nm and 650 nm within 30 minutes.
#### Performance Characteristics (where applicable): M.
- Analytical performance:
- Precision/Reproducibility: a.
The reproducibility of the test was investigated using sera with different concentrations. Intra-assay reproducibility is based on 20 determinations and inter-assay reproducibility on 40 determinations performed in 10 different runs on 5 days with 2 runs per day according to the package insert. The following results were obtained:
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Image /page/7/Picture/0 description: The image shows a repeating pattern of black shapes against a white background. The black shapes are vertically oriented and have a rounded, almost pill-like form, with a narrower section in the middle. These shapes are evenly spaced and aligned in a row, creating a rhythmic visual sequence. The top and bottom of the image are bordered by solid black lines, which frame the repeating pattern.
#### Intra-assay reproducibility Anti-Sm ELISA (IgG) Ratio n = 20 Sample 2 Sample 5 Sample 6 Sample 1 Sample 3 Sample 4 Mean value (x): 5.7 3.7 2.0 1.2 0.8 0.4 5.4 - 5.8 3.5 - 3.9 1.9 – 2.0 1.2 - 1.3 0.7 - 0.9 0.4 - 0.4 Range of values: negative Expected result: positive positive positive positive negative % positive: 100% 100% 100% 100% 0% 0% 0% 100% 100% % negative: 0% 0% 0%
Inter-assay reproducibility
| n = 10 x 4 | Anti-Sm ELISA (IgG)<br>Ratio | | | | | |
|------------------|------------------------------|-----------|-----------|-----------|-----------|-----------|
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| Mean value (x): | 5.8 | 3.9 | 1.7 | 1.1 | 0.8 | 0.4 |
| Range of values: | 5.3 - 7.4 | 3.5 - 4.6 | 1.5 - 2.2 | 1.0 - 1.2 | 0.6 - 1.0 | 0.3 - 0.5 |
| Expected result: | positive | positive | positive | positive | negative | negative |
| % positive: | 100% | 100% | 100% | 100% | 2.5% | 0% |
| % negative: | 0% | 0% | 0% | 0% | 97.5% | 100% |
The lot to lot reproducibility was investigated during the validaton and quality control of the kit using different lots with QC samples distributed over the measurement range. The following results were obtained:
Lot to lot reproducibility
| | | Anti-Sm ELISA (IgG) | | | | | |
|------------------|-----------|---------------------|-----------|-----------|-----------|-----------|--|
| | | Ratio | | | | | |
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | |
| n | 6* | 6* | 6* | 6* | 6* | 6* | |
| Mean value (x): | 3.6 | 3.4 | 3.1 | 0.3 | 0.9 | 1.2 | |
| Range of values: | 3.3 - 3.9 | 3.2 - 3.9 | 2.9 - 3.4 | 0.3 - 0.3 | 0.8 - 0.9 | 1.1 - 1.3 | |
| Expected result: | positive | positive | positive | negative | negative | positive | |
| % positive: | 100% | 100% | 100% | 0% | 0% | 100% | |
| % negative: | 0% | 0% | 0% | 100% | 100% | 0% | |
| | Anti-Sm ELISA (IgG) Ratio | | | | |
|------------------|---------------------------|-----------|-----------|-----------|-----------|
| | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 |
| n | 9** | 10** | 10** | 9** | 10** |
| Mean value (x): | 0.1 | 1.9 | 3.4 | 5.7 | 8.3 |
| Range of values: | 0.0 - 0.2 | 1.5 - 2.2 | 2.6 - 3.8 | 4.9 - 6.7 | 7.5 - 9.6 |
| Expected result: | negative | positive | positive | positive | positive |
| % positive: | 0% | 100% | 100% | 100% | 100% |
| % negative: | 100% | 0% | 0% | 0% | 0% |
*3 lots x 2 runs ** n lots x 1 run
> Linearity/assay reportable range: b. Not applicable.
High dose Hook effect C.
> Potential for a high dose Hook effect is a phenomenon that is inherent with one step "sandwich" assay designs: Very high concentrations of antigen in the patient sample bind to all available sites saturating them - on both the antibody-solid phase and the antibody-labeled conjugate and thereby prevent the "sandwich" formation. Under these conditions, the measured level of analyte may besignificantly lower than the actual level present in the sample. The two-step immunoassay design of the Anti-Sm ELISA (IgG) eliminates the adverse contribution of binding proteins, endogenous interfering substances and general matrix effects due to the extra wash step.
{8}------------------------------------------------
Image /page/8/Picture/1 description: The image shows a black and white pattern. There are four black shapes that are evenly spaced. The shapes are connected to a black line at the bottom of the image. There is a white line at the top and bottom of the image.
d. Traceability, Stability, Expected values (controls, calibrators or methods):
A recognized standard or reference material for anti-nuclear antibodies is not available. Results of this assay are given in ratios. The reactivity of the Anti-Sm ELISA was verified using the ANA reference panel of the CDC Centers for Disease Control and Prevention, Atlanta, USA.
- Limit of detection: e.
- Not applicable
- f. Analvtical specificity:
Cross-reactivity: The quality of the antigen coated on the plates ensures a high specificity of the ELISA. Cross reactivity was investigated using a panel of 30 sera serologically positive for antibodies against rib.P-P, nRNP/Sm, SS-A, Scl-70, Jo-1, centromeres, Rubella virus, Measles virus, Herpes simplex virus type 1 and Borrelia burgdorferi. All 30 sera were negative in the Anti-Sm ELISA (IgG), so no cross reactivity is expected.
Interference: To investigate the influence from hemoglobin, triglycerides and bilirubin, 5 different sera at different anti-Sm concentrations (ratio 0.7 ~ 3.9) were spiked with potential interfering substances and were incubated with the test system according to the package insert. Interferences from rheumatoid factor were investigated similarly by spiking with a RF positive serum. The recovery in relation to the unspiked sample without interferent was calculated. The individual recovery was within the range of 90 - 111 %. No significant interference was observed for concentrations of up to 1000 ma/dl for hemoalobin, 2000 mg/dl for trialyceride, 40 mg/dl for billrubin and 500 lU/ml for rheumatoid factor.
- Assay cut-off: ਉਂ
- Ratio 1.0
- 2. Comparison studies:
- Method comparison with predicate device: a.
A comparison study was performed using 294 clinically characterized samples (128 SLE, 51 Sjögren's syndrome, 15 systemic sclerosis, 15 fibromyalgia, 35 RA, 30 borreliosis and 20 healthy), obtained from different sources from Europe and North America. The panel consisted of 60 men and 234 women. Age ranged from 14 to 85 years with an average age of 45 years. Anti-Sm antibodies are expected in SLE. The other disease groups serve as control cohorts. The samples were tested with the EUROMMUN Anti-Sm ELISA (IqG) and with the Inova Quanta Lite Sm ELISA as the predicate device. The results are shown in the table below. All of the 7 discrepant samples negative in the EUROIMMUN test were from controls. The 5 discrepant samples positive in the EUROIMMUN test were from SLE patients.
| All samples<br>n = 294 | | Predicate ELISA | |
|----------------------------------|---------------------|-----------------|----------|
| | | positive | negative |
| EUROIMMUN<br>Anti-Sm ELISA (IgG) | positive | 37 | 5 |
| | negative | 7 | 245 |
| Negative agreement | $245 / 250 = 98.0%$ | 95% C.I.: 95.4% | 99.3% |
| Positive agreement | $37 / 44 = 84.1%$ | 95% C.I.: 69.9% | 93.4% |
| Overall agreement | $282 / 294 = 95.9%$ | 95% C.I.: 93.0% | 97.9% |
- b. Matrix comparison:
The usability of plasma was investigated using sample pairs each of serum and corresponding plasma (EDTA, Li-heparin, Citrate). Passing-Bablok regression was calculated for the comparison of serum to plasma. The regression equation is near the ideal correlation (intercept 0) indicating equivalence of concentrations between serum and the corresponding plasma matrices. Coefficients of determination were found to be above 0.99 and %recovery compared to serum was in the range of 89 to 118 % (serum = 100 %).
| | EDTA plasma | Li-heparin plasma | Citrate plasma |
|------------------------------------------------|--------------------|--------------------|-------------------|
| n | 16 | 16 | 16 |
| Regression equation<br>(y = plasma, x = serum) | $y = 0.33 + 1.00x$ | $y = 0.19 + 0.97x$ | $y = 0.05 + 1.02$ |
| 95% C.I. of intercept | -0.32 - 1.24 | -0.92 - 1.95 | -0.69 - 3.27 |
| 95% C.I. of slope | 0.96 - 1.03 | 0.94 - 1.01 | 0.99 - 1.05 |
| Coefficient of determination R2 | 0.9953 | 0.9948 | 0.9931 |
| Mean %recovery | 102 % | 99 % | 103 % |
| Range of %recovery | 94 - 115 % | 90 - 111 % | 89 - 118 % |
{9}------------------------------------------------
#### 3. Clinical studies:
Clinical studies were performed in cooperation with different sites. In total 1036 clinically characterized samples (414 from SLE patients and 622 from control groups) were investigated for antibodies (IgG). With the EUROIMMUN Anti-Sm ELISA (IgG) a prevalence of 11.4% (95% C.I.: 8.5 - 14.8%) was found in SLE with a specificity of 99.0% (95% C.I.: 97.9 - 99.6%). The results are shown in the table below. 95% C.I. are calculated by the exact method.
- a. Clinical sensitivity:
| No. | Panel | n | Anti-Sm ELISA (IgG) | | |
|-----|----------------------------------|-----|---------------------|--------|---------------|
| | | | positive | % | 95% C.I. |
| 1 | Systemic lupus erythematosus | 414 | 47 | 11.4% | 8.5 - 14.8% |
| No. | Panel | n | negative | % | 95% C.I. |
| 2 | Rheumatoid arthritis | 164 | 164 | 100.0% | 97.8 - 100.0% |
| 3 | Systemic sclerosis | 81 | 81 | 100.0% | 95.5 - 100.0% |
| 4 | Sjögren's syndrome | 88 | 88 | 100.0% | 95.9 - 100.0% |
| 5 | Polymyositis/dermatomyositis | 151 | 151 | 100.0% | 97.6 - 100.0% |
| 6 | Mixed connective tissue diseases | 45 | 39 | 86.7% | 73.2 - 94.9% |
| 7 | Other autoimmune diseases* | 63 | 63 | 100.0% | 94.3 - 100.0% |
| 8 | Borreliosis | 30 | 30 | 100.0% | 88.4 - 100.0% |
| | Total | 622 | 616 | 99.0% | 97.9 - 99.6% |
#### b. Clinical specificity:
*from the following groups: AIH (n = 8), Grave's disease (n = 12), Hashimoto (n = 11), celiac disease (n = 11), Diabetes Type I (n = 12)
- Other clinical supportive data (when a. and b. are not applicable): C. Not applicable.
- 4. Clinical cut-off:
See Assay cut-off.
- ട. Expected values/Reference range:
The levels of anti-Sm antibodies (IgG) were analyzed in a panel of 200 samples from apparently healthy blood donors (120 men and 80 women with an average age of 40 y; age range: 19 - 68 y). The results are shown in the table below.
| n | 200 |
|---------------|-------|
| Positives | 0 |
| Negatives | 200 |
| Prevalence | 0.0% |
| | Ratio |
| Lowest value | 0.1 |
| Highest value | 0.3 |
| Mean value | 0.1 |
| Std deviation | 0.02 |
#### N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
#### 0. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence. decision.
Michael Locke
Signature
Dir. Regulatory Affairs
12 June 2013 Date
{10}------------------------------------------------
Image /page/10/Picture/1 description: The image shows a series of four dark, circular shapes positioned between two horizontal lines. The circular shapes are evenly spaced and appear to be touching both the top and bottom lines. The overall composition is simple and graphic, with a focus on the contrast between the dark shapes and the surrounding space.
### ATTACHMENT 1
### PREMARKET NOTIFICATION 510(K) SAFETY AND EFFECTIVENESS SUMMARY (as required by 21 CFR § 807.92)
- A. 510(k)Number: K123261
- Purpose for Submission: B. New device
- ். Measurand:
- Anti-SS-A autoantibodies ·
- D. Type of Test:
- Qualitative enzyme immunoassay ய் Applicant:
- EUROIMMUN US INC.
- Proprietary and Established Names: F. EUROIMMUN Anti-SS-A ELISA (IgG)
#### G. Regulatory Information:
- Regulation: 1.
- 21 CFR 866.5100 Antinuclear antibody immunological test system
- 2. Classification:
- Class II
- 3. Product code:
- LLL
- 4. Panel:
- Immunology H. Intended Use:
- Intended use(s): 1.
The EUROIMMUN Anti-SS-A ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against SS-A in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of Sjögren's syndrome and systemic lupus erythematosus, in conjunction with other laboratory and clinical findings.
- 2. Indication(s) for use: Same as intended use.
- 3. Special conditions for the use statement(s): For prescription use only.
- র্ব : Special instrument requirements:
Microwell plate reader capable of measuring OD at 450nm for dual wavelength readings. Device Description:
### l:
The EUROIMMUN Anti-SS-A ELISA (IgG) consists of a microwell ELISA plate coated with SS-A antigen, calibrator, positive and negative control, peroxidase-labelled anti-human IgG conjugate, sample buffer, wash buffer concentrate. TMB chromogen/substrate solution and stop solution.
#### Substantial Equivalence Information: J.
- Predicate device name (s): 1.
- Inova Quanta Lite SS-A ELISA
- 2. Predicate 510(k) number(s): K922830
{11}------------------------------------------------
Image /page/11/Picture/1 description: The image shows the number 3 followed by the text 'Comparison with predicate:'. The number 3 is on the left side of the image. The text is on the right side of the image. The text is underlined.
| Similarities | | |
|---------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|
| Item | New Device | Predicate Device |
| Intended use | Detection of IgG antibodies to SS-A | Same |
| Technology | ELISA | Same |
| Assay platform | 96-well microtiter plates | Same |
| Calibration | Relative units | Same |
| Antigen | Purified SS-A antigen | Same |
| Conjugate | Anti-human IgG labeled with horseradish peroxidase | Same |
| Substrate | TMB | Same |
| Reagent preparation | All reagents, calibrator and controls are ready to use,<br>except for the wash buffer. | Same |
| Procedure | Sample incubation with micro-well antigen coated plate,<br>followed by a wash step, incubation with an anti-human<br>IgG enzyme conjugate; wash step, incubation with<br>substrate; then the addition of a stop solution and<br>reading at 450nm. | Same |
| Differences | | |
| Item | New Device | Predicate Device |
| Assay format | Qualitative | "Semi-quantitative" (using low positive control) |
| Sample dilution | 1:201 | 1:101 |
| Calibrators and | 1 calibrator | 3 controls: 1 high positive, 1 low positive (used for |
| controls | 2 controls: 1 positive, 1 negative | calculation of results), 1 negative |
| Wash buffer | 10x concentrate | 40x concentrate |
| Stop solution | 0.5 M sulphuric acid | 0.344 M sulphuric acid |
| Sample types | Serum or plasma (EDTA, Li-heparin, Citrate) | Serum |
| Reported results | Ratio | Units |
| Cut off level | Ratio 1.0 | 20 Units |
#### Standard/Guidance Document Referenced (if applicable): K.
Guidance for Industry and FDA Staff: Recommendations for Antibody (ANA) Test System Premarket (510(k)) Submissions (January 22, 2009)
#### ட. Test Principle:
1.
Patient samples are diluted 1:201 in sample buffer, 100 µl of each diluted patient sample and pre-diluted controls and calibrators are added to the antigen coated microtiter wells and incubated for 30 minutes at room temperature. After incubation the microtiter well strips are washed with wash buffer to remove unbound antibodies and 100 µl of the anti-human IgG enzyme conjugate reagent is added to each microtiter well. After an additional 30-minutes incubation at room temperature, the microtiter wells are again washed 3 times with 300 ul of wash buffer to remove any unbound enzyme conjugate and 100 µl of the chromogen substrate is added. The strips are incubated for 15 minutes at room temperature and 100 µl stop solution is added. The microtiter plates are placed in an ELISA reader and read at a wavelength of 450 nm and a reference wavelength of between 620 nm and 650 nm within 30 minutes.
#### Performance Characteristics (where applicable): M.
- Analytical performance:
- Precision/Reproducibility: ·
- The reproducibility of the test was investigated using sera with different concentrations. Intra-assay reproducibility is based on 20 determinations and inter-assay reproducibility on 40 determinations performed in 10 different runs on 5 days with 2 runs per day, each run performed according to the package insert. The following results were obtained:
Image /page/11/Picture/13 description: The image shows a pattern of alternating black and white shapes. The black shapes are rectangular bars, and the white shapes are oval-like shapes. The black bars are arranged horizontally, and the white shapes are arranged vertically between the bars. There are four white shapes in the image.
{12}------------------------------------------------
Image /page/12/Picture/0 description: The image shows a pattern of black shapes against a white background. The shapes are arranged in a row, with each shape consisting of two horizontal lines connected by a rounded, oval-like form in the middle. The pattern is symmetrical and evenly spaced, creating a repeating design.
### Intra-assay reproducibility
| n = 20 | Anti-SS-A ELISA (IgG)<br>Ratio | | | | | |
|------------------|--------------------------------|-----------|-----------|-----------|-----------|-----------|
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| Mean value (x): | 6.1 | 4.2 | 2.0 | 1.3 | 0.8 | 0.3 |
| Range of values: | 5.9 - 6.4 | 4.0 - 4.5 | 1.9 - 2.1 | 1.2 - 1.4 | 0.7 - 0.8 | 0.3 - 0.3 |
| Expected result: | positive | positive | positive | positive | negative | negative |
| % positive: | 100% | 100% | 100% | 100% | 0% | 0% |
| % negative: | 0% | 0% | 0% | 0% | 100% | 100% |
Inter-assay reproducibility
| n = 10 x 4 | Anti-SS-A ELISA (IgG)<br>Ratio | | | | | |
|------------------|--------------------------------|-----------|-----------|-----------|-----------|-----------|
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| Mean value (x): | 6.0 | 4.0 | 1.8 | 1.2 | 0.8 | 0.3 |
| Range of values: | 5.0 - 6.4 | 3.7 - 4.5 | 1.6 - 1.9 | 1.0 - 1.4 | 0.7 - 0.9 | 0.3 - 0.4 |
| Expected result: | positive | positive | positive | positive | negative | negative |
| % positive: | 100% | 100% | 100% | 100% | 0% | 0% |
| % negative: | 0% | 0% | 0% | 0% | 100% | 100% |
The lot to lot reproducibility was investigated during the validaton and quality control of the kit using different lots with QC samples distributed over the measurement range. The following results were obtained:
Lot to lot reproducibility
| | Anti-SS-A ELISA (IgG)<br>Ratio | | | | | |
|------------------|--------------------------------|-----------|-----------|-----------|-----------|-----------|
| | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| n | 6* | 6* | 6* | 6* | 6* | 6* |
| Mean value (x): | 4.6 | 5.6 | 5.6 | 0.3 | 0.9 | 1.1 |
| Range of values: | 4.4 - 4.8 | 5.1 - 6.2 | 5.1 - 6.0 | 0.3 - 0.4 | 0.9 - 0.9 | 1.1 - 1.2 |
| Expected result: | positive | positive | positive | negative | negative | positive |
| % positive: | 100% | 100% | 100% | 0% | 0% | 100% |
| % negative: | 0% | 0% | 0% | 100% | 100% | 0% |
| | Anti-SS-A ELISA (IgG)<br>Ratio | | | | |
|------------------|--------------------------------|-----------|-----------|-----------|-----------|
| | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 |
| n | 10** | 11** | 10** | 9** | 11** |
| Mean value (x): | 0.1 | 1.6 | 2.7 | 4.6 | 7.8 |
| Range of values: | 0.1 - 0.2 | 1.3 - 1.8 | 2.3 - 3.1 | 4.3 - 5.2 | 7.4 - 8.3 |
| Expected result: | negative | positive | positive | positive | positive |
| % positive: | 0% | 100% | 100% | 100% | 100% |
| % negative: | 100% | 0% | 0% | 0% | 0% |
*3 lots x 2 runs ** n lots x 1 run
> b. Linearity/assay reportable range: Not applicable.
Hiah dose Hook effect C.
Potential for a high dose Hook effect is a phenomenon that is inherent with one step "sandwich" assay designs: Very high concentrations of antigen in the patient sample bind to all available sites saturating them - on both the antibody-solid phase and the antibody-labeled conjugate and thereby prevent the "sandwich" formation. Under these conditions, the measured level of analyte may be significantly lower than the actual level present in the sample. The two-step immunoassay design of the Anti-SS-A ELISA (IgG) eliminates the adverse contribution of binding proteins, endogenous interfering substances and general matrix effects due to the extra wash step.
{13}------------------------------------------------
Image /page/13/Picture/1 description: The image shows a pattern of alternating black and white shapes. There are four black, oval-like shapes evenly spaced between two horizontal black lines. The white space between the black shapes and lines creates a contrasting pattern.
Traceability, Stability, Expected values (controls, calibrators or methods): d.
A recognized standard or reference material for anti-nuclear antibodies is not available. Results of this assay are given in ratios. The reactivity of the Anti-SS-A ELISA was verified using the ANA reference panel of the CDC Centers for Disease Control and Prevention, Atlanta, USA.
- e. Limit of detection:
- Not applicable.
- f. Analytical specificity:
Cross-reactivity: The quality of the antigen coated on the plates ensures a high specificity of the ELISA. Cross reactivity was investigated using a panel of 30 sera serologically positive for antibodies against rib.P-P, nRNP/Sm, Sm, Scl-70, Jo-1, centromeres, Rubella virus, Measles virus, Herpes simplex virus type 1 and Borrelia burgdorferi. All 30 sera were negative in the Anti-SS-A ELISA (IgG), so no cross reactivity is expected.
Interference: To investigate the influence from hemoglobin, triglycerides and bilirubin, 5 different sera at different anti-SS-A concentrations (15 - 124 RU/ml) were spiked with potential interfering substances and were incubated with the test system according to the package insert. Interferences from rheumatoid factor were investigated similarly by spiking with a RF positive serum. The recovery in relation to the unspiked sample without interferent was calculated. The individual recovery was within the range of 93 - 107 %. No significant interference was observed for concentrations of up to 1000 mg/dl for hemoglobin, 2000 mg/dl for triglyceride, 40 mg/dl for billirubin and 500 IU/ml for rheumatoid factor.
- ದ್ರ. Assay cut-off:
. .
Ratio 1.0
#### 2. Comparison studies:
- Method comparison with predicate device: a.
A comparison study was performed using 305 clinically characterized samples (63 SLE, 77 Sjögren's syndrome, 23 systemic sclerosis, 1 SSc/SS, 15 fibromyalgia, 26 myositis, 35 RA, 30 borreliosis, 20 healthy), obtained from different sources from Europe and North America. The panel consisted of 54 men and 251 women. Age ranged from 14 to 85 years with an average age of 48 years. Anti-SS-A antibodies are expected in either Siögren's syndrome or SLE. The other disease groups serve as control cohorts. The samples were tested with the EUROIMMUN Anti-SS-A ELISA (IgG) and with the Inova Quanta Lite SS-A ELISA as the predicate device. Of the 9 discrepant samples negative in the EUROIMMUN test. 2 were from patients with Siögren's syndrome and one from a SLE patient, the other 6 were from controls. All 3 discrepant samples positive in the EUROIMMUN test were from SLE patients.
| All samples<br>n = 305 | | Predicate ELISA | |
|-----------------------------------------------------------------------|----------|-----------------|----------|
| | | positive | negative |
| EUROIMMUN<br>Anti-SS-A ELISA (IgG) | positive | 116 | 3 |
| | negative | 9 | 177 |
| Negative agreement 177 / 180 = 98.3% 95% C.I.: 95.2% - 99.7% | | | |
| Positive agreement 116 / 125 = 92.8% 95% C.I.: 86.8% - 96.7% | | | |
| Overall agreement 293 / 305 = 96.1% 95% C.I.: 93.2% - 98.0% | | | |
- b. Matrix comparison:
The usability of plasma was investigated using sample pairs each of serum and corresponding plasma (EDTA, Li-heparin, Citrate). Passing-Bablok regression was calculated for the comparison of serum to plasma. The regression equation is near the ideal correlation (intercept 0; slope 1.0) indicating equivalence of concentrations between serum and the corresponding plasma matrices. Coefficients of determination were found to be above 0.99 and %recovery compared to serum was in the range of 85 to 104 % (serum = 100 %).
| | EDTA plasma | Li-heparin plasma | Citrate plasma |
|------------------------------------------------|----------------------|----------------------|--------------------|
| n | 16 | 16 | 16 |
| Regression equation<br>(y = plasma, x = serum) | $y = -1.59 + 0.99 x$ | $y = -1.47 + 0.99 x$ | $y = -0.54 + 0.98$ |
| 95% C.I. of intercept | -4.49 - 3.01 | -4.62 - 1.36 | -4.65 - 3.32 |
| 95% C.I. of slope | 0.95 - 1.02 | 0.95 - 1.02 | 0.94 - 1.04 |
| Coefficient of determination R² | 0.9972 | 0.9929 | 0.9956 |
| Mean %recovery | 97 % | 95 % | 98 % |
| Range of %recovery | 90 - 102 % | 85 - 102 % | 93 - 104 % |
{14}------------------------------------------------
### Clinical studies:
Clinical studies were performed in cooperation with different sites. In total 1026 clinically characterized samples (88 from SS patients, 404 from SLE patients and 534 from control groups) were investigated for anti-SS-A antibodies (IgG). With the EUROIMMUN Anti-SS-A ELISA (IgG) a prevalence of 73.9% (95% C.I.: 63.4 – 82.7%) was found in Sjögren's syndrome (clinical sensitivity) and a prevalence of 40.6% (95% C.I.: 35.8 - 45.6%) was found in SLE with a specificity of 94.8% (95% C.I.: 92.5 - 96.5%). Systemic sclerosis and myositis panels were considered for calculation of specificity, however, it has been reported that anti-SS-A antibodies may occur in these diseases [Antonioli 2002, Ghirardello 2005]. The results are shown in the table below. 95% C.I. are calculated by the exact method.
- Clinical sensitivity: a.
| No. | Panel | n | positive | % | 95% C.I. |
|-----|------------------------------|-----|----------|-------|--------------|
| 1 | Sjögren's syndrome | 88 | 65 | 73.9% | 63.4 - 82.7% |
| 2 | Systemic lupus erythematosus | 404 | 164 | 40.6% | 35.8 - 45.6% |
#### Clinical specificity: b.
| No. | Panel | n | negative | % | 95% C.I. |
|-----|----------------------------------|-----|----------|--------|---------------|
| 3 | Systemic sclerosis | 81 | 75 | 92.6% | 84.6 - 97.2% |
| 4 | Polymyositis/dermatomyositis | 151 | 138 | 91.4% | 85.7 - 95.3% |
| 5 | Rheumatoid arthritis | 164 | 159 | 97.0% | 93.0 - 99.0% |
| 6 | Mixed connective tissue diseases | 45 | 41 | 91.1% | 78.8 - 97.5% |
| 7 | Other autoimmune diseases* | 63 | 63 | 100.0% | 94.3 - 100.0% |
| 8 | Borreliosis | 30 | 30 | 100.0% | 88.4 - 100.0% |
| | Total | 534 | 506 | 94.8% | 92.5 - 96.5% |
ffrom the following groups: AIH (n = 8), Grave's disease (n = 12), Hashimoto (n = 11), celiac disease (n = 11), Diabetes Type I (n = 12)
- Other clinical supportive data (when a. and b. are not applicable). C. Not applicable.
- 4. Clinical cut-off:
See Assay cut-off.
- 5. Expected values/Reference range:
The levels of anti-SS-A antibodies (IgG) were analyzed in a panel of 200 samples from apparently healthy blood donors (120 men and 80 women with an average age of 40 y; age range: 19 - 68 y). The results are shown in the table below.
| n | 200 |
|---------------|-------|
| Positives | 2 |
| Negatives | 198 |
| Prevalence | 1.0% |
| | Ratio |
| Lowest value | 0.0 |
| Highest value | 4.4 |
| Mean value | 0.1 |
| Std deviation | 0.33 |
#### 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.
*Michael Locke*
*Signature*
Dir. Regulatory Affairs Title
12 June 2013 Date
{15}------------------------------------------------
Image /page/15/Picture/1 description: The image shows a pattern of alternating black and white shapes. The black shapes are arranged in a row and are separated by white spaces. The black shapes are rounded and bulbous, while the white spaces are rectangular. The pattern is symmetrical and repeats several times across the image.
### ATTACHMENT 1
### PREMARKET NOTIFICATION 210(K) SAFETY AND EFFECTIVENESS SUMMARY (as required by 21 CFR § 807.92)
- A. 510(k)Number: K123261
- Purpose for Submission: в. New device
- C. Measurand:
- Anti-SS-B autoantibodies
- D. Type of Test:
- Qualitative enzyme immunoassay
- E. Applicant:
- EUROIMMUN US INC.
- F. Proprietary and Established Names: EUROIMMUN Anti-SS-B ELISA (IqG)
#### Regulatory Information: G.
- 1. Regulation:
- 21 CFR 866.5110 Antinuclear antibody immunological test system
- 2. Classification: Class II
- 3. Product code: LLL
- র্ব Panel:
- Immunology
- H. Intended Use:
- Intended use(s): 1.
The EUROIMMUN Anti-SS-B ELISA (IgG) test kit is intended for the qualitative determination of IgG class autoantibodies against SS-B in human serum and plasma (EDTA, Li-heparin, Citrate). It is used as an aid in the diagnosis of Sjögren's syndrome and systemic lupus erythematosus, in conjunction with other laboratory and clinical findings.
- 2. Indication(s) for use: Same as intended use.
- ന Special conditions for the use statement(s): For prescription use only .
Special instrument requirements: 4.
Microwell plate reader capable of measuring OD at 450nm for dual wavelength readings. Device Description:
## l.
The EUROIMMUN Anti-SS-B ELISA (IgG) consists of a microwell ELISA plate coated with SS-B antigen, calibrator, positive and negative control, peroxidase-labelled anti-human IgG conjugate, sample buffer, wash buffer concentrate, TMB chromogen/substrate solution and stop solution.
#### Substantial Equivalence Information: J.
- Predicate device name (s): 1.
Inova Quanta Lite SS-B ELISA
- 2. Predicate 510(k) number(s): K922832
{16}------------------------------------------------
#### റ്റ് Comparison with predicate:
| Similarities | | |
|---------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|
| Item | New Device | Predicate Device |
| Intended use | Detection of IgG antibodies to SS-B | Same |
| Technology | ELISA | Same |
| Assay platform | 96-well microtiter plates | Same |
| Calibration | Relative units | Same |
| Antigen | Purified SS-B antigen | Same |
| Conjugate | Anti-human IgG labeled with horseradish peroxidase | Same |
| Substrate | TMB | Same |
| Reagent preparation | All reagents, calibrator and controls are ready to use,<br>except for the wash buffer. | Same |
| Procedure | Sample incubation with micro-well antigen coated plate,<br>followed by a wash step, incubation with an anti-human<br>IgG enzyme conjugate; wash step, incubation with<br>su…
