EUROIMMUN ANTI BP 180-4X ELISA (IGG)

K083615 · Euroimmun Us, Inc. · OEG · Mar 16, 2009 · Immunology

Device Facts

• Record ID: K083615
• Device Name: EUROIMMUN ANTI BP 180-4X ELISA (IGG)
• Applicant: Euroimmun Us, Inc.
• Product Code: OEG · Immunology
• Decision Date: Mar 16, 2009
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5660
• Device Class: Class 2

Indications for Use

The EUROIMMUN Anti-BP180-4X ELISA (IgG) test kit is intended for the qualitative or semiquantitative determination of IgG class autoantibodies against BP180 in human serum and plasma. It is used as an aid in the diagnosis of bullous pemphigoid (PB), in conjunction with other laboratory and clinical findings.

Device Story

In vitro diagnostic ELISA test kit; detects IgG autoantibodies against BP180 antigen in human serum or plasma samples. Used by laboratory professionals to aid in clinical diagnosis of bullous pemphigoid. Operates via enzyme-linked immunosorbent assay principle; results interpreted by clinicians alongside other diagnostic findings to support patient management.

Clinical Evidence

No clinical data provided in the summary document; substantial equivalence based on analytical performance and comparison to predicate device.

Technological Characteristics

ELISA; recombinant tetramer protein of NC16A domain (E. coli); 48-well microtiter plate format; peroxidase-labelled rabbit anti-human IgG conjugate; TMB/H2O2 substrate; stop solution; manual/automated microtiter plate reader (450 nm/620-650 nm); qualitative/semi-quantitative output; 12-month shelf life at 2-8°C.

Indications for Use

Indicated for qualitative or semiquantitative detection of IgG autoantibodies against BP180 in human serum/plasma to aid in diagnosis of bullous pemphigoid (BP).

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

• MESACUP BP180 ELISA Kit (K071961)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k083615

B. Purpose for Submission:
New device

C. Measurand:
Anti-BP180 (IgG) autoantibodies

D. Type of Test:
Qualitative or Semi-Quantitative Enzyme immunoassay

E. Applicant:
EUROIMMUN US Inc.

F. Proprietary and Established Names:
Proprietary name - EUROIMMUN Anti-BP180-4X ELISA (IgG)

G. Regulatory Information:
1. Regulation
21 CFR 866.5660 Multiple autoantibodies immunological test system
2. Classification
Class II
3. Product Code
OEG Autoantibodies, skin (bullous pemphigoid 180 and bullous pemphigoid 230
4. Panel
Immunology 82

H. Intended Use:
1. Intended use(s):
The EUROIMMUN Anti-BP180-4X ELISA (IgG) test kit is intended for the qualitative or semi-quantitative determination of IgG class autoantibodies against BP180 in human serum and plasma. It is used as an aid in the diagnosis of bullous pemphigoid (PB), in conjunction with other laboratory and clinical findings.
2. Indication(s) for use:
Same as Intended Use
3. Special conditions for use statement(s):
Prescription use only
4. Special instrument requirements:
A microtiter plate reader capable of measuring optical densities at 450 nm and a reference wavelength of between 620 nm and 650 nm

I. Device Description:
The Anti-BP180-4X ELISA (IgG) test kit is composed of ready-to-use 6 microplate strips containing 8 individual break-off wells in a frame, three levels of calibrators (2 mL each, at 2, 20, and 200 RU/mL human IgG), positive and negative controls (2.0 mL each), 1×12 mL bottle peroxidase-labelled rabbit anti-human IgG, 1 × 100 mL bottle sample buffer, 1×12 mL bottle TMB/H₂O₂ chromogen/substrate, 1×12 mL bottle stop solution, and a 1×100 mL bottle of 10X concentrated wash solution.

J. Substantial Equivalence Information:
1. Predicate device name(s):

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MESACUP BP180 ELISA Kit manufactured by MBL International Corp.

2. Predicate K number(s):
K071961

3. Comparison with predicate:

| | Similarities | |
| --- | --- | --- |
| Item | New Device | Predicate Device |
| Intended use | Detection of IgG antibodies to BP180 as an aid in diagnosis of bullous pemphigoid (PB). | Same |
| Technology | ELISA | Same |
| Assay platform | 48-well microtiter plates (6 strips x 8 wells) | Same |
| Calibration | Relative arbitrary units | Same |
| | Differences | |
| --- | --- | --- |
| Item | New Device | Predicate Device |
| Assay format | Qualitative or semi-quantitative (using either the 3 calibrators or 1 calibrator) | Semi-quantitative |
| Antigen | Recombinant tetramer protein of the NC16A domain (based on human cDNA), produced in E.coli. | Recombinant purified BP 180 NC16a. |
| Calibrators | 3 calibrators
2, 20, and 200 RU/mL | 2 calibrators
0 and 100 U/mL |
| Controls | 2 controls
1 positive, 1 negative | No controls (recommendation that user create own controls) |
| Samples | Serum or plasma
1:101 dilution | Serum
1:101 dilution |
| Reported units | RU/mL or Ratio | U/mL |
| Cut Off level | 20 RU/mL | 9 U/mL |

K. Standard/Guidance Document Referenced (if applicable):
None Listed

L. Test Principle:
The device utilizes direct binding enzyme immunoassay technology. Patient samples are diluted 1:101 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 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 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.

M. Performance Characteristics (where applicable):

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# 1. Analytical performance:

# a. Precision/Reproducibility:

The reproducibility of the test was investigated by determining the intra- and inter-assay coefficients of variation (CV) using 4 sera with values at different points on the calibration curve. The intra-assay CVs are based on 20 determinations and the inter-assay CVs on 4 determinations performed on 6 different days. Neither inter-assay variation nor intra-assay variation should result in a CV% of over $12\%$ . The following results were obtained:

Intra-assay reproducibility

| n = 20 | Sample 1 | Sample 2 | Sample 3 | Sample 4 |
| --- | --- | --- | --- | --- |
| | Concentration (RU/mL): | | | |
| Mean value (x): | 177 | 61 | 119 | 27 |
| Standard deviation (SD): | 3.6 | 0.7 | 1.9 | 0.8 |
| Coefficient of variation (CV, %): | 2.1 | 1.2 | 1.6 | 3.0 |
| Mean CV (%): | 2.0 | | | |

Inter-assay reproducibility

| n = 4 tests / 6 days = 24 | Sample 1 | Sample 2 | Sample 3 | Sample 4 |
| --- | --- | --- | --- | --- |
| | Concentration (RU/mL): | | | |
| Mean value (x): | 174 | 61 | 119 | 26 |
| Standard deviation (SD): | 4.6 | 1.9 | 5.0 | 1.3 |
| Coefficient of variation (CV, %): | 2.6 | 3.1 | 4.2 | 4.8 |
| Mean CV (%): | 3.7 | | | |

The Lot to Lot reproducibility is checked during the validation of the kit. 3 different lots are incubated with 3 different QC samples each. For each sample the coefficient of variation (CV) is calculated. Inter-lot variation should show results below $\mathrm{CV} = 12\%$ . The following results were obtained:

Lot to Lot reproducibility

| n = 3 lots x 2 runs = 6 | Sample 1 | Sample 2 | Sample 3 |
| --- | --- | --- | --- |
| | Concentration (RU/mL): | | |
| Mean value (x): | 145 | 48 | 99 |
| Standard deviation (SD): | 5.5 | 2.1 | 2.3 |
| Coefficient of variation (CV, %): | 3.8 | 4.4 | 2.4 |
| Mean CV (%): | 3.5 | | |

# b. Linearity/assay reportable range:

The linearity of the ELISA was determined by assaying two-fold serial dilutions of 8 serum samples (6 dilutions, covering the concentration range of 2 - 200 RU/mL). Four of the 8 samples were not linear in this range when evaluated by

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CLSI EP-6-A (Evaluation of linearity of quantitative measurement procedures). The deviation of 1 or more (up to 3 points in the supplied data) of the 6 points in the dilution series for the 4 samples was outside the 95% confidence interval for the linear line. A typical sample is shown as follows:


Since no reference standard material exists and units for the assay are arbitrary, semi-quantitative linearity may be of lesser importance than a qualitative result (positive or negative) during initial diagnosis.

The reportable range of the assay is 2 RU/mL (the lowest calibrator) to 200 RU/mL (the highest calibrator).

c. Traceability, Stability, Expected values (controls, calibrators, or methods): No reference standard material exists for antibody to NC16A domain of BP180. Units for the assay are arbitrary and linked to value assignment using a recombinant protein produced in bacteria (E. coli). Value assignment of calibrators is made for a new lot against a current lot using a panel of reference sera. Target values are adjusted by dilution or spiking. The single value positive and single value negative controls used in the assay are derived from human blood donor material. The positive control is assigned an acceptable range using the same criteria (± a stated percentage) regardless of actual concentration.

Stability studies are conducted in accordance the international standard DIN EN 13640:2002: Stability testing of in vitro diagnostics reagents. Three production lots of all kit reagents are tested. For sealed original reagents, real-time stability testing currently supports 12 months duration at 2-8°C. Once opened, real-time stability currently supports 12 months duration at 2-8°C, however upon reconstitution, stability of the wash buffer is currently only 28 days when stored at 2-8°C.

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d. Detection limit:

The limit of blank is the lowest arbitrary concentration from a replicate series of samples without analyte. It is defined for this assay as a value of three times the standard deviation of the sample buffer. The detection limit of the Anti-BP180-4X ELISA (IgG) is 0.6 RU/mL determined from 20 replicate determinations of sample buffer only. Due to the high dilution factor of the samples, no influence of the sample material (serum or plasma) is expected. The lowest quantified concentration is the lowest calibrator used in the assay (2 RU/mL).

e. Analytical specificity:

Cross reactivity with serum from related autoimmune diseases was investigated using a panel of 27 sera from different sources positive for Pemphigus vulgaris (n = 8), Pemphigus folicaeus (n = 2) and Goodpasture syndrome (n = 17). All 27 sera were negative in the Anti-BP180-4X ELISA (IgG).

To investigate interference from hemoglobin, triglycerides, and bilirubin, 4 different specimens at 4 different anti-BP180 concentrations (29, 73, 123, and 186 RU/mL) were spiked with potential interfering substances and were incubated with the test system. The recovery in relation to the unspiked sample without interferent was calculated. The individual value of the recovery was within the acceptable range of 75-125%. No interference was observed for concentrations of up to 1000 mg/dl for hemoglobin, 2000 mg/dl for triglyceride and 40 mg/dl for bilirubin.

f. Assay cut-off:

Receiver operator characteristics curve analysis was utilized to determine a cutoff. Disease subject samples were obtained from 118 patients with Bullous pemphigoid or 20 patients with Pemphigoid gestationis from 2 sites not in the United States. Non-diseased subject samples were obtained from the following patients groups from various sites not in the United States:

| Panel | N
(men, women) | Mean age
(age range) |
| --- | --- | --- |
| Rheumatoid arthritis | 107
(21, 50)
(36 unknown) | 56 y
(30 – 76)
(36 unknown) |
| Progressive systemic sclerosis | 50 | 55 y
(22 – 82)
(1 unknown) |
| Systemic lupus erythematosus | 72
(1, 29)
(42 unknown) | 54 y
(26 – 87 y)
(40 unknown) |
| Asymptomatic blood donors | 494
(309, 185) | 39 y
(18 – 67 y) |
| Total | 723 | |

At a cutoff of 20 RU/mL, 98% of non-disease subjects were negative in the assay. Qualitative results are evaluated by calculating a ratio of the Optical Density value of the control or patient sample over the Optical Density value of the 20 RU/mL calibrator. A ratio greater than or equal to 1.0 is considered positive.

2. Comparison studies:

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# a. Method comparison with predicate device:

An external study was performed in collaboration with several hospitals outside the United States on 347 samples tested with the proposed assay and with the predicate device, using the cut-offs recommended by the respective test instructions. The panel consisted of 61 men and 158 women (128 unknown gender). Age ranged from 22 to 99 years with an average age of 65 years (with 80 unknown). The results were categorized as positive or negative and are shown in the table below.

| Predicate | | | |
| --- | --- | --- | --- |
| EUROIMMUN | >9 U/mL | <9 U/mL | |
| >20 RU/mL | 107 | 5 | 112 |
| <20 RU/mL | 9 | 226 | 235 |
| | 116 | 231 | 347 |
| | estimate | standard error | lower 95% CI | upper 95% CI |
| --- | --- | --- | --- | --- |
| positive agreement | 92.2% (107/116) | ± 2.5% | 85.8% | 96.4% |
| negative agreement | 97.8% (226/231) | ± 1.0% | 95.0% | 99.3% |
| total agreement | 96.0% (333/347) | ± 2.5% | 93.3% | 97.8% |

# b. Matrix comparison:

Sixteen samples of serum and the corresponding EDTA, heparin and citrate plasma were tested in the Anti-BP180-4X ELISA. The samples cover concentrations in the diagnostically important range. Passing-Bablok regression was calculated for the comparison of serum to each type of plasma. The slopes and $95\%$ confidence intervals for each comparison are as follows:

| Parameter | Serum vs. EDTA-Plasma | |
| --- | --- | --- |
| Intercept | 1.00 | 95% C.I.: -2.18 to 3.54 |
| Slope | 1.00 | 95% C.I.: 0.96 to 1.04 |
| | Serum vs. Heparin-Plasma | |
| Intercept | -3.24 | 95% C.I.: -5.71 to 0.32 |
| Slope | 1.02 | 95% C.I.: 0.98 to 1.05 |
| | Serum vs. Citrate-Plasma | |
| Intercept | 2.03 | 95% C.I.: -2.94 to 5.84 |
| Slope | 0.98 | 95% C.I.: 0.91 to 1.06 |

A comparison in which the $95\%$ confidence interval of the slope contains 1.0 and the $95\%$ confidence interval of the intercept contains 0 indicates equivalence of concentration between serum and the corresponding plasma matrices. The comparisons above satisfy this condition.

# 3. Clinical studies:

In a clinical study performed in cooperation with several hospitals outside the United States, a total of 841 clinically characterized samples were investigated for anti-

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BP180 antibodies using the proposed assay. The EUROIMMUN Anti-BP180-4X ELISA (IgG) showed a sensitivity of 89.8% for Bullous pemphigoid and a specificity of 97.9%. The results are shown in the tables below. 95% C.I. are calculated by the exact method.

a. Clinical Sensitivity:

| Panel | n (men, women) | Mean age (age range) | Anti-BP180-4X ELISA (IgG) | | |
| --- | --- | --- | --- | --- | --- |
| | | | positive | % | 95% C.I. |
| Bullous pemphigoid | 118 (39, 79) | 76 y (25 – 99 y) (1 unknown) | 106 | 89.8% | 82.9 – 94.6% |
| Pemphigoid gestationis | 20 (0, 18) (2 unknown) | 32 y (19 – 40) (9 unknown) | 20 | 100.0% | 83.2 – 100.0% |

b. Clinical specificity:

| Panel | n (men, women) | Mean age (age range) | Anti-BP180-4X ELISA (IgG) | | |
| --- | --- | --- | --- | --- | --- |
| | | | negative | % | 95% C.I. |
| Rheumatoid arthritis | 107 (21, 50) (36 unknown) | 56 y (30 – 76) (36 unknown) | 105 | 98.1% | 93.4 – 99.8% |
| Progressive systemic sclerosis | 50 | 55 y (22 – 82) (1 unknown) | 48 | 96.0% | 86.3 – 99.5% |
| Systemic lupus erythematosus | 72 (1, 29) (42 unknown) | 54 y (26 – 87 y) (40 unknown) | 71 | 98.6% | 92.5 – 100.0% |
| Asymptomatic blood donors | 494 (309, 185) | 39 y (18 – 67 y) | 484 | 98.0% | 96.3 – 99.0% |
| Total | 723 | | 708 | 97.9% | 96.6 – 98.8% |

c. Other clinical supportive data (when a. and b. are not applicable):

4. Clinical cut-off:
No cutoff has been established.

5. Expected values/Reference range:
The levels of anti-BP180 antibodies were analyzed with the EUROIMMUN Anti-BP180-4X ELISA (IgG) in a panel of 494 apparently healthy blood donors. The subjects consisting of 309 men and 185 women with an age range of 18-67 years (average age: 39 years). The 98th percentile of the healthy blood donors was 27.4 RU/mL.

| Positives | 10 |
| --- | --- |
| Negatives | 484 |
| Lowest value | 0.01 RU/mL |
| Highest value | 232.16 RU/mL |
| Mean value | 4.73 RU/mL |
| Std dev. (SD) | 15.87 RU/mL |

N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

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O. Conclusion:

1. The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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