IMMUNOSCAN RA ANTI-CCP TEST SYSTEM

K052133 · Eurodiagnostica · NHX · Dec 12, 2005 · Immunology

Device Facts

• Record ID: K052133
• Device Name: IMMUNOSCAN RA ANTI-CCP TEST SYSTEM
• Applicant: Eurodiagnostica
• Product Code: NHX · Immunology
• Decision Date: Dec 12, 2005
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5775
• Device Class: Class 2

Indications for Use

The Immunoscan RA anti-CCP test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and semi-quantitation of IgG antibodies to Cyclic Citrullinated Peptides (CCP) in human sera. The assay is 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 Rheumatoid Arthritis (RA), in conjunction with other laboratory and clinical findings. The analysis should be performed by trained laboratory professionals.

Device Story

Immunoscan RA anti-CCP test kit is an ELISA-based in vitro diagnostic assay; detects IgG antibodies to Cyclic Citrullinated Peptides (CCP) in human serum samples. Performed by trained laboratory professionals in clinical laboratory settings. Input: single serum specimen. Process: enzyme-linked immunosorbent assay; semi-quantitation of antibody levels. Output: semi-quantitative result used by clinicians as an aid in diagnosing Rheumatoid Arthritis (RA) alongside other clinical and laboratory findings. Benefits: assists in early or differential diagnosis of RA.

Clinical Evidence

No clinical data provided in the summary document; relies on bench testing and performance validation of the ELISA assay.

Technological Characteristics

ELISA-based immunoassay; 96-well microtiter plate format. Antigen: purified citrullinated synthetic peptides. Detection: HRP-labeled anti-human IgG conjugate with TMB substrate. Energy source: photometric measurement at 450 nm. Requires microplate reader and washer. Qualitative and semi-quantitative output.

Indications for Use

Indicated for the detection and semi-quantitation of IgG antibodies to Cyclic Citrullinated Peptides (CCP) in human serum as an aid in the diagnosis of Rheumatoid Arthritis (RA). For use by trained laboratory professionals.

Regulatory Classification

Identification

A rheumatoid factor immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the rheumatoid factor (antibodies to immunoglobulins) in serum, other body fluids, and tissues. Measurement of rheumatoid factor may aid in the diagnosis of rheumatoid arthritis.

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k052133

B. Purpose for Submission:
New Device

C. Measurand:
Anti-CCP antibodies

D. Type of Test:
Qualitative and Semi-quantitative ELISA

E. Applicant:
Eurodiagnostica

F. Proprietary and Established Names:
Immunoscan RA anti-CCP Test Kit

G. Regulatory Information:
1. Regulation section:
21 CFR 866.5775, Rheumatoid factor immunological test system
2. Classification:
II
3. Product codes:
NHX, Antibodies, Anti-Cyclic Citrullinated Peptide (CCP)
4. Panel:
Immunology 82

H. Intended Use:
1. Intended use(s):
The Immunoscan RA anti-CCP test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and semi-quantitation of IgG antibodies to Cyclic Citrullinated (CCP) in human sera. The assay is 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 Rheumatoid Arthritis (RA), in conjunction with other laboratory and clinical findings. The analysis should be performed by trained laboratory professionals.
For In Vitro Diagnostic Use.
2. Indication(s) for use:
Same as intended use.
3. Special conditions for use statement(s):
The device is for prescription use only.
4. Special instrument requirements:
Microplate reader capable of measuring OD at 450 nm.
Microplate washer (300μL volume).

I. Device Description:
Each device contains the following: microplate wells coated with citrullinated synthetic peptides; five levels calibrators (25, 50, 200, 800, 1600 units/mL); reference, positive and negative controls (human serum in diluent); wash buffer 20X concentrate; diluent buffer; rabbit anti-human IgG horseradish peroxidase conjugate; TMB (3,3', 5'-tetramethylbenzidin) substrate.

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J. Substantial Equivalence Information:

1. Predicate device name(s):
Axis Shield Diastat™ Anti-CCP test Kit

2. Predicate 510(k) number(s):
k021516

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | New Device | Predicate Device |
| Intended use | To aid in the diagnosis of Rheumatoid Arthritis (RA) | Same |
| Technology | ELISA | Same |
| Assay Format | Qualitative and semi-quantitative | Same |
| Reference control | Ready to use | Same |
| Sample volume required | 10 μL | Same |
| Incubation times | 30-30-60 minutes | Same |
| Platform | 96 well microtiter plates | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Antigen | Purified citrullinated synthetic peptides | Purified synthetic cyclic peptide containing modified arginine residues |
| Sample type and dilution | Serum at 1:50 | Serum at 1:100 |
| Positive and Negative controls | Ready to use | Dilute 1:100 with diluted sample diluent |
| Calibrator | Five Levels (25, 50, 200, 800, 1600 units/mL); ready to use | Five levels (0, 2, 8, 30, 100 U/mL); ready to use |
| Enzyme-Conjugate | Horseradish peroxidase | Alkaline Phosphatase |
| Substrate | TMB | PMP (Phenolphthalein monophosphate, Mg²⁺) |
| Wash buffer | 20x Concentrate | 16x Concentrate |
| Stop solution | 0.5M sulphuric acid | Sodium hydroxide |
| OD reading | 450 nm | 550 nm (540-565nm is acceptable) |
| Anti-CCP Antibody Results Interpretation | Negative: < 25 units/mL
Positive: ≥ 25 units/mL | Negative: ≤ 5U/mL
Positive: > 5U/mL |

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

L. Test Principle:
The Immunoscan RA anti-CCP test kit is an enzyme-linked immunosorbent assay (ELISA) for detection and semi-quantitation of IgG antibodies to Rheumatoid Arthritis in human serum. The wells of the microtiter plate wells are coated with purified citrullinated synthetic peptides antigen. Diluted serum is applied to the wells and incubated. If specific antibodies are present, they will bind to the antigen in the wells. Unbound material is washed away and any bound antibody is detected

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by adding horse radish peroxidase (HRP) labeled anti-human IgG, followed by a second washing step, and an incubation with substrate. The presence of reacting antibodies will result in the development of colour which is proportional to the quantity of bound antibody, and this is determined photometrically.

## M. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

a. **Precision/Reproducibility:**

The intra-assay reproducibility was determined by testing six samples eight times. Four samples with high anti-CCP concentrations (88.1-1007.4 U/mL) had a CV of 4.3-12.8% and two samples close to the assay cut-off (33.6-51.7 U/mL) had a CV of 8.1-8.4%.

The inter-assay reproducibility was determined by testing six samples eight times. Four samples with high anti-CCP concentrations (93.1-1105.9 U/mL) had a CV of 6.0-17.7%. Two samples close to the assay cut-off (33.3-52.5 U/mL) had a CV of 7.8-14.5%.

b. **Linearity/assay reportable range:**

Three positive sera were diluted serially from neat, 1:2, 1:4, 1:8, and 1:16 dilutions. The values were compared to log 2 of dilution by standard regression. The values indicate that the assay has a linear relationship with serum dilutions. The assay reportable range is from 25 units/mL to 1600 units/mL.

c. **Traceability, Stability, Expected values (controls, calibrators, or methods):**

There is no reference standard for anti-CCP. The calibrators and controls (positive and negative) are prepared in-house and arbitrary units are assigned during the development process.

d. **Detection limit:**

The detection limit was determined by running the zero standard fourteen times on three different lots. The detection limit of 1.6 Units/mL was calculated by finding the mean plus two standard deviations.

e. **Analytical specificity:**

**Interference study:** Three low positive samples were spiked with bilirubin at 0.2 mg/mL, haemoglobin at 400 mg/dL, lipid at 15 mg/mL and rheumatoid factor at 200 IU/mL. The data indicates that these interferences at the assayed concentrations do not interfere with the anti-CCP results.

f. **Assay cut-off:**

The cut-off value of &gt;25 U/mL was established based on testing of 63 normal blood donor sera, 167 non-RA sera and 219 RA sera. With this cut-off value, 62 normal donor sera (98.4%), 165 non-RA sera (98.8%) and 17 RA sera (7.8%) were negative.

### 2. Comparison studies:

a. **Method comparison with predicate device:**

Testing was performed on 320 samples which included 175 samples from RA patients and 145 samples from normal blood donors. The positive percent agreement was 99.3% (135/136); the negative percent agreement was 96.7% (178/184) and the Overall Agreement was 97.8% (313/320).

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| | Axis Shield Diastat™ Anti-CCP | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| Immunoscan RA CCP Kit | Positive | 135 | 6 | 141 |
| | Negative | 1 | 178 | 179 |
| | Total | 136 | 184 | 320 |

b. Matrix comparison:
Not applicable.

3. Clinical studies:

a. Clinical sensitivity and specificity:

The clinical sensitivity and specificity study were evaluated on 1052 frozen retrospective sera with clinically characterized sera from patients with the following diagnosis: 338 RA, 20 WG; 20 microscopic polyangiitis (MP); 70 SLE; 17 Sjogren's; 100 IBD; 21 Osteoarthritis; 20 Thyroiditis; 5 EBV; 5 Parvovirus; 9 Mycoplasma; 5 Tuberculosis; 8 Yersinia; 3 Salmonella; 5 Chlamydia; 3 Malaria; 9 Borrelia; 5 Syphilis; 3 Infectious endocarditis; 4 Legionella; 3 AST; Schistomiasis; 5 Rubella; 3 Chaga's syndrome; 17 Scleroderma; 20 Multiple sclerosis; 20 IDDM; 20 PM/DM; 20 MCTD; and routine samples. Sensitivity for RA was 75.1%. The overall specificity of the new device for healthy blood donors and disease controls was 98.7%.

| N= 1052 | | Anti-CCP results | |
| --- | --- | --- | --- |
| Patient Group | n= | negative | positive |
| RA | 338 | 84 | 254 |
| WG | 20 | 20 | 0 |
| MP | 20 | 20 | 0 |
| Healthy controls | 184 | 182 | 2 |
| Disease controls | | | |
| SLE | 70 | 68 | 2 |
| Sjogren's | 17 | 16 | 1 |
| IBD | 100 | 97 | 3 |
| Osteoarthritis | 21 | 21 | 0 |
| Thyroiditis | 20 | 20 | 0 |
| EBV | 5 | 5 | 0 |
| Parvovirus | 5 | 5 | 0 |
| Mycoplasma | 9 | 9 | 0 |
| Toxoplasma | 5 | 5 | 0 |
| Tuberculosis | 5 | 5 | 0 |
| Yersinia | 8 | 8 | 0 |
| Salmonella | 3 | 3 | 0 |
| Chlamydia | 5 | 4 | 0 |
| Malaria | 4 | 4 | 0 |
| Borrelia | 9 | 9 | 0 |
| Syphilis | 5 | 5 | 0 |
| Infectious endocarditis | 3 | 3 | 0 |

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| Legionella | 4 | 4 | 0 |
| --- | --- | --- | --- |
| AST | 3 | 3 | 0 |
| Schistomiasis | 4 | 4 | 0 |
| Rubella | 5 | 5 | 0 |
| Chaga’s syndrome | 3 | 3 | 0 |
| Scleroderma | 17 | 16 | 1 |
| Multiple sclerosis | 21 | 21 | 0 |
| IDDM | 20 | 20 | 0 |
| PM/DM | 20 | 20 | 0 |
| MCTD | 20 | 19 | 1 |
| Routine samples | 80 | 78 | 2 |

Sensitivity:

RA: 75.1% (254/338) 95% CI: 70.5% to 79.8%

Specificity:

Healthy controls: 98.9% (182/184) 95% CI: 96.1% to 99.9%

WG: 100% (20/20) 95% CI: 83.2% to 100%

MP: 100% (20/20) 95% CI: 83.2% to 100%

SLE: 97.1% (68/70) 95% CI: 90.1% to 99.7%

Sjogren’s: 94.1% (16/17) 95% CI: 71.3% to 99.8%)

IBD: 97.0% (97/100) 95% CI: 91.5% to 99.4%)

Osteoarthritis: 100% (21/21) 95% CI: 83.9% to 100%

Thyroiditis: 100% (20/20) 95% CI: 83.2% to 100%

Infectious Disease: 98.8% (84/85) 95% CI: 93.6% to 100%

Scleroderma: 94.1% (16/17) 95% CI: 71.3% to 99.8%

Multiple sclerosis: 100% (20/20) 95% CI: 83.2% to 100%

IDDM: 100% (20/20) 95% CI: 83.2% to 100%

PM/DM: 100% (20/20) 95% CI: 83.2% to 100%

MCTD: 95.0% (19/20) 95% CI: 75.1% to 99.9%

Routine samples: 97.5% (78/80) 95% CI: 91.3% to 99.7%

b. Other clinical supportive data (when a. is not applicable):

Not Applicable.

4. Clinical cut-off:

Same as assay cut-off.

5. Expected values/Reference range:

Expected values in the normal population should be negative.

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.