Axis-Shield Clinical Chemistry Anti-CCP Reagent Kit
Reag 1, 1 x 24.0 mL
FHCCP100
ENGLISH
FOR PROFESSIONAL USE ONLY
INTENDED USE
The Axis-Shield Anti-CCP test is a semi-quantitative assay for the detection of
autoantibodies specific to cyclic citrullinated peptide (CCP) in either human serum or
plasma specimens. Detection of anti-CCP antibodies is used as an aid in the diagnosis of
Rheumatoid Arthritis (RA), and should be used in conjunction with other clinical
information. Autoantibody levels represent one parameter in a multi-criterion diagnostic
process, encompassing both clinical and laboratory-based assessments.
SUMMARY
Rheumatoid Arthritis (RA) is a common, systemic autoimmune disease affecting 0.5-1.0%
of the adult population. RA is characterised by chronic inflammation of the synovium
which can lead to progressive joint destruction and in many cases lead to disability and
reduction of quality of life.1 It is generally accepted that early intervention is vital in
preventing irreversible joint damage and it is therefore important to diagnose RA as early
in the disease course as possible.2,3 The diagnosis of RA is primarily based on clinical,
radiological and immunological features. The most frequent serological test is the
measurement of rheumatoid factor (RF).4 Although the RF test has good sensitivity, it is
not specific for RA, as it is often present in healthy individuals and patients with other
rheumatic or inflammatory diseases, autoimmune diseases or chronic infections.5
For several years, it has been recognised that antibodies to anti-perinuclear factor (APF)
and keratin (AKA) are highly specific for RA. It was subsequently reported that both these
antibodies reacted with native filaggrin and now are referred to as anti-filaggrin
antibodies (AFA).6,7,8 Recent evidence has shown that all these antibodies are directed to
citrulline containing epitopes.9 Citrulline is a non-standard amino acid, as it is not
incorporated into proteins during protein synthesis. It can however be generated via post-
transitional modification of arginine residues by the enzyme peptidylarginine deiminase
(PAD).10 In 1998, Schellekens and colleagues reported that autoantibodies reactive with
linear synthetic peptides containing citrulline were highly specific for RA in an ELISA based
assay.11 Subsequent studies demonstrated that cyclic variants of these linear peptides,
termed cyclic citrullinated peptides (CCP) were as specific for RA but with a higher
sensitivity than the linear peptides.12 In an effort to further improve the sensitivity of the
CCP test, a dedicated library of citrulline-containing peptides was screened with RA sera
and a new set of peptides (CCP2) was discovered which gave superior performance
compared to the CCP1 test.13 Over the last few years many published reports have
confirmed the diagnostic performance of the CCP2 test.14 Anti-CCP antibodies, which are
also often termed as anti-citrullinated protein/peptide antibodies (ACPA’s), have been
found to be present very early in the disease, often with the absence of clinical symptoms
and many reports indicate that elevated levels of anti-CCP antibodies can predict the
development of erosive disease.15,16,17,18,19,20 These findings suggest an important role for
cyclic citrullinated peptides in the diagnosis of RA at an early stage of the disease course.
In 2010 the ACR / EULAR Rheumatoid Arthritis Classification Criteria were published and
replaced the "old" ACR criteria of 1987 which were widely considered not to be suitable
for the early diagnosis of RA. The revised classification criteria, jointly published by the
American College of Rheumatology (ACR) and the European League Against Rheumatism
(EULAR) recommend a point scoring system of between 0 and 10. The new classification
criteria are to be applied to every individual presenting with definitive synovitis
(undifferentiated inflammatory Arthritis). The four additional criteria were number of
joints involved, serologic abnormality, acute-phase response and duration of symptoms in
the involved joints. For the first time the serologic criteria included measurement of
ACPAs, such as anti-CCP, as well as some definition of a low positive and high positive
serology result.21
The Axis-Shield Anti-CCP assay is an immunoturbidimetric assay based on the detection of
autoantibodies in human serum or plasma towards a synthetic cyclic peptide containing
modified arginine residues (CCP2 peptides). The test provides an additional tool in the
diagnosis of patients with RA.
PRINCIPLE
The determination of anti-CCP antibodies is based on a specific turbidimetric reaction
which occurs between the antibodies present in the serum/plasma of patients affected by
Rheumatoid Arthritis (RA) and highly purified synthetic cyclic citrullinated peptide coated
on the surface of latex microparticles. Reaction occurs under optimal pH conditions and in
the presence of an accelerator. The turbidity of the immune-complex is proportional to
the concentration of the analyte in the examined sample.
KIT COMPONENTS
1 x 24.0 mL MES buffer, accelerator, Bovine
Albumin 0.2%, sodium azide
< 0.1%, detergents and
stabilizers. READY TO USE
1 x 7.6 mL Phosphate buffer, Bovine
Albumin 2%, Synthetic
Citrullinated peptide coated on
microparticles surface, sodium
azide < 0.1%, detergents and
stabilizers. READY TO USE
Reag 2, 1 x 7.6 mL
100 Tests
EQUIPMENT AND MATERIALS REQUIRED BUT NOT PROVIDED
• Clinical Chemistry system capable of measuring up to 800 nm with 37°C incubation.
Refer to specific Axis-Shield Clinical Chemistry Anti-CCP Application Guide.
• Some instruments may require appropriate reagent holders.
• Axis-Shield Clinical Chemistry Anti-CCP Control Kit, Product Code FHCCP200
• Axis-Shield Clinical Chemistry Anti-CCP Calibrator Kit, Product Code FHCCP300
WARNINGS AND PRECAUTIONS
1. For in vitro diagnostic use.
2. Disposal of all waste material must be in accordance with local guidelines.
3. Reagents contain sodium azide which can react with lead and copper plumbing to
form highly explosive metal azides. On disposal, drain with large quantities of
water to prevent azide build-up.
4. Material safety data sheets are available upon request from Axis-Shield.
5. Caution: Federal law restricts this device to sale by or on the order of a physician.
REAGENT STORAGE AND STABILITY
Opened (in-Use) Kit Stability
• Reag 1 and Reag 2:
• For on-board storage capabilities and any limitations refer to specific Axis-Shield
Clinical Chemistry Anti-CCP Application Guide.
• If reagents are removed from the analyser, return to storage at 2-8oC in the
original packaging.
Unopened kit stability
All components are stable until expiration as directed on the label at 2-8oC.
Reagents must not be frozen.
Handling and Procedural Notes
• Store kit components at 2-8°C.
• Reagents are supplied ready for use, but for some instruments, these may require
transfer to appropriate reagent holders. Refer to specific Axis-Shield Clinical
Chemistry Anti-CCP Application Guide.
• ENSURE HOMOGENEITY OF REAGENT 2 BEFORE USE BY GENTLE INVERSION OF
THE VIAL.
• Do not use beyond the expiration date.
• DO NOT FREEZE REAGENTS.
• Reagents must be returned to 2-8°C storage after use if they cannot be stored
on-board under refrigerated conditions in the original packaging.
• Do not mix reagents between different lot numbers.
• Use a new disposable pipette tip for each reagent or sample manipulation to
avoid contamination.
• Un-mixed reagents should be clear of particulate material and should be
discarded if they become turbid.
Indications of Deterioration
Values outside the recommended acceptable range for the Axis-Shield Clinical
Chemistry Anti-CCP controls may be an indication of reagent instability and associated
results are invalid. The kit should be discarded and the samples retested.
SAMPLE STORAGE, COLLECTION AND HANDLING
Collection and Handling
• For sample collection and preparation, only use suitable tubes or collection
containers.
• Only the sample matrices listed are suitable for use:
- Human serum (including serum separator tubes (SST)).
- Plasma (K2 EDTA or Lithium Heparin).
• Other sample collection tubes have not been verified for use.
• Do not use grossly haemolysed or turbid samples.
• Thoroughly mix thawed samples before assay and avoid repeated freeze/thawing.
• Do not use heat-inactivated samples, this may yield incorrect results.
• Use caution when handling patient samples in order to prevent cross
contamination. Use of disposable pipettes or pipette tips is recommended.
Remove bubbles with an applicator stick before analysis. Use a new applicator
stick for each sample to prevent cross contamination.22
Preparation for analysis
• When processing samples follow the instructions provided by the collection tube
manufacturer.
• All human samples should be considered potentially infectious. It is
recommended that these materials be handled in accordance with local/national
guidelines on laboratory safety procedures.
• Fresh/Non-Frozen Samples:
- Samples must be mixed thoroughly prior to use.
• Frozen Samples:
- Thaw samples thoroughly for a minimum of 30 minutes.
- Mix thawed samples thoroughly by inverting a minimum of 5 times.
Sample Storage and Stability
This procedure can be performed with human serum or plasma specimens. Grossly
lipaemic, haemolysed or microbial contaminated samples may give poor results and
should not be used.
Sample stability can be supported at the following storage conditions:
PROZONE
Prozone effect is whereby specimens with high levels of analyte may assay within the
measuring interval of the assay. Anti-CCP levels up to 200 U/mL did not display any
prozone effect. Concentrations above this level did display a prozone effect however
did not affect the interpretation relative to the cut-off.

Storage Claim LIMIT OF DETECTION

Room Temperature (20°C) Up to 4 hours The limit of detection (LOD) of the Axis-Shield Clinical Chemistry anti-CCP assay was
2-8°C Up to 4 hours determined to be 10.5 U/mL.
On-board Up to 4 hours The LOD is defined as the lowest concentration of analyte that can be detected with
Freeze/thaw cycles Up to 3 cycles greater than or equal to 95% probability as described in CLSI Document EP17-A2.24

For longer term storage, store frozen at -20°C or lower. CLINICAL SENSITIVITY AND SPECIFICITY
Dispose of any used materials in accordance with local waste management regulations. Clinical sensitivity and specificity of the Axis-Shield Clinical Chemistry Anti-CCP assay was
determined using a total of 547 patient samples (196 RA, 156 other disease states and
ASSAY PROCEDURE 195 healthy asymptomatic). Applying a cut-off of 20 U/mL, the following results were
• Program the instrument using the appropriate instrument-specific protocol. Refer obtained across 3 lots tested:
to specific Axis-Shield Clinical Chemistry Anti-CCP Application Guide.
Specimen Category Total Positive Negative % Sensitivity
• Invert Reagents to facilitate mixing and then load reagents and samples as per
N N N
specific Axis-Shield Clinical Chemistry Anti-CCP Application Guide.
RA 196 134 62 68.4
STANDARDIZATION Specimen Category Total Positive Negative % Specificity
Currently there is no International reference standard for this assay. The Axis-Shield N N N
Clinical Chemistry Anti-CCP assay is traceable to an Axis-Shield Reference Standard.
Non-RA Specimens 351 14 337 96
Assigned values for calibrators are traceable to this standard.
CLINICAL SPECIFICITY
CALIBRATION
Clinical specificity was assessed using 156 specimens from potentially cross-reacting
For assay calibration use the Axis-Shield Clinical Chemistry Calibration materials as listed
conditions. The following conditions and the groups where more than 1 specimen was
in the “Equipment and Materials required but not Provided” section.
tested are tabulated below:
The Calibrator values are lot specific as directed on the labels.
Number tested
Calibration Frequency:
Number positive in Axis-
Calibration curve stability is instrument specific. Refer to specific Axis-Shield Clinical
Non-RA Disease Specimens Tested Shield Clinical
Chemistry Anti-CCP Application Guide.
(N) Chemistry
Recalibration is also recommended after a change in reagent lot, if a control reads out-of-
Anti-CCP assay
range or as required following quality control procedures.
Ankylosing Spondilitis 5 0
QUALITY CONTROL Antiphospholipid Syndrome 4 0
For quality control, use Axis-Shield Clinical Chemistry Control materials as listed in the Centrifugal Annular Erythema 2 0
“Equipment and Materials required but not provided” section. Maintenance and CREST syndrome 4 2
calibration of the instrument must be performed according to the operator’s instruction
Fibromyalgia 7 1
manual for the specific analyser.
Users should ensure they understand the instructions of this assay, particularly the Non-inflammatory gut problems 2 0
Warnings and Precautions, Sample Handling and Procedure Limitations sections. It is Multiple Sclerosis 2 0
recommended that Axis-Shield Clinical Chemistry Anti-CCP Controls and Calibrators are Myositis 4 0
run in duplicate each day of use. 1
Osteoarthritis 16
The control limits should be established by individual laboratories and in accordance with
Pemphigus 3 0
laboratories quality control procedures and/or any local or government guidelines.
Primary Vasculitis 4 0
RESULTS Scleroderma 3 0
Unit of Measure Sjogren’s Syndrome 13 3
The default result unit for the assay is U/mL. Systemic lupus erythematosus 50 2
Solar Dermatitis 2 0
Interpretation of Results
The proposed clinical cut-off value was determined by a Receiver Operating Characteristic Thyroiditis 7 0
(ROC) analysis with a balanced consideration of sensitivity and specificity according to the Undifferentiated connective tissue disease 4 0
CLSI document EP24-A2.23 The internal study analysed 354 patient samples (100 Urticaria 3 0
confirmed positive for Rheumatoid arthritis (RA) and 254 asymptomatic) using the
Venous Thrombosis 2 0
Axis-Shield Clinical Chemistry Anti-CCP assay. The area under the ROC Curve (AUC) for the
Axis-Shield Clinical Chemistry Anti-CCP assay was 0.81 with a 95% confidence interval of
0.76-0.87. ASSAY PRECISION
A study was performed according to CLSI Document EP9-A3.25 Six patient samples were
The proposed clinical cut-off value is 20 U/mL.
analysed using 3 lots of reagents. Each sample was tested in replicates of 2 at two
This cut-off is suggested as a guideline only and each laboratory should establish a cut-off, separate times per day for 20 days (n=80). Data from one lot tested is summarised
which may be unique to the population it serves depending upon geographical, patient, below as representative data.
dietary, environmental factors or clinical practice. Please note that RA is approximately Within Run Total
twice as prevalent in females as in males. Mean CCP
(Repeatability) (Within Lab)
Sample Conc N
SD CV SD CV
EXPECTED VALUES (U/mL)
(U/mL) (%) (U/mL) (%)
In a representative study, 120 serum samples from an asymptomatic apparently healthy Sample 1 0.85 80 0.72 N/A 1.31 N/A
population were tested. All samples were reported as anti-CCP negative and the upper Sample 2 34.11 80 1.70 5.0 3.77 11.0
limit (based on the 97.5th percentile of the data) reported (7.7 U/mL) less than the limit of Sample 3 48.08 80 2.18 4.5 4.90 10.2
detection (10.5 U/mL). As with all in vitro diagnostic assays, each laboratory should
Sample 4 71.76 80 2.08 2.9 5.13 7.1
determine its own reference range (s). Consider these values as guidelines only.
Sample 5 83.19 80 2.53 3.0 7.00 8.4
Sample 6 10.04 80 1.11 N/A 2.64 N/A
PERFORMANCE CHARACTERISTICS
Representative data from testing on the Horiba ABX Pentra 400 analyser is presented;
results from individual laboratories may vary. Other systems are not supported unless an
SAMPLE CARRYOVER
instrument guide is provided. Within assay sample carryover was assessed using a high anti-CCP positive sample
greater than 1000 U/mL. A bias (%) of less than 10% on the value obtained for a
MEASURING INTERVAL negative anti-CCP sample was observed.
The Axis-Shield Clinical Chemistry Anti-CCP assay measures anti-CCP concentrations from
the limit of detection up to the concentration of the highest calibrator (U/mL).
Specimens with anti-CCP concentrations above the highest calibrator must be reported as
> 200 U/mL. Sample dilutions have not been verified.
The Calibrator values are lot specific as directed on the labels.

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INTERFERENCES BIBLIOGRAPHY
Interfering substances were tested as per CLSI Document EP7-A226 in the Axis-Shield 1. Feldmann M, Brennan FM, Maini RN. Rheumatoid Arthritis. Cell 1996;85:307-10.
Clinical Chemistry Anti-CCP assay. The levels of substances tested as indicated below did 2. Landewé RB. The benefits of early treatment in rheumatoid arthritis: confounding
not interfere in the assay as defined by a bias (%) of less than 10% observed. by indication, and the issue of timing. Arthritis Rheum 2003;48(1):1-5
3. Lard LR, Visser H, Speyer I, et al. Early versus delayed treatment in patients with
Interfering Substances Test Concentration recent-onset rheumatoid arthritis: comparison of two cohorts who received
Bilirubin (Conjugated) up to 56 mg/dL different treatment strategies. Am J Med 2001;111:446-51.
Bilirubin (Unconjugated) up to 60 mg/dL 4. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association
Haemoglobin up to 20 g/L 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum
Human IgG up to 54 g/L 1988;31(3):315-24.
Triglycerides (Native) up to 1437 mg/dL 5. van Venrooij WJ, Hazes JM, Visser H. Anticitrullinated protein/peptide antibody
Triglycerides (Intralipid) up to 564 mg/dL and its role in the diagnosis and prognosis of early rheumatoid arthritis. Neth J Med
Rheumatoid Factors up to 702 IU/mL 2002;60(10):383-8.
Total Proteins up to 132 g/L 6. Nienhuis RL, Mandema E, Smids C. A new serum factor in patients with rheumatoid
arthritis. The antiperinuclear factor. Ann Rheum Dis 1964;23:302-05.
LIMITATIONS OF USE 7. Young BJ, Mallya RK, Leslie RD, et al. Anti-keratin antibodies in rheumatoid
1. Although the presence of antibodies to CCP is associated with Rheumatoid Arthritis, arthritis. Br Med J 1979;2:97-9.
a positive result is not in itself diagnostic, the data must be considered in light of 8. Hoet RM, Boerbooms AM, Arends M, et al. Antiperinuclear factor, a marker
other clinical and laboratory findings. autoantibody for rheumatoid arthritis: colocalisation of the perinuclear factor and
2. Some individuals may have high levels of anti-CCP antibodies with little or no profilaggrin. Ann Rheum Dis 1991;50:611-8.
evidence of clinical disease. By contrast, some patients with active disease may have 9. Sebbag M, Simon M, Vincent C, et al. The antiperinuclear factor and the so-called
undetectable levels of these antibodies. The clinical significance of this information antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies. J
is currently unclear. Clin Invest 1995;95:2672-9.
3. As the result of an anti-CCP assay is not diagnostic proof of the presence or absence 10. Vossenaar ER, Zendman AJ, van Venrooij WJ, et al. PAD, a growing family of
of clinical disease, therapy should not be started on the basis of an anti-CCP positive citrullinating enzymes: genes, features and involvement in disease. BioEssays
result alone.
2003;25:1106-18.
4. Initiation or changes in treatment should not be based on changes in anti-CCP
11. Schellekens GA, de Jong BA, van den Hoogen FH, et al. Citrulline is an essential
autoantibody concentration but rather on clinical observation(s).
constituent of antigenic determinants recognized by rheumatoid arthritis-specific
5. The clinical effectiveness of monitoring CCP autoantibody levels as an indication of
progression/remission of Rheumatoid Arthritis has not been defined. autoantibodies. J Clin Invest 1998;101(1):273-81.
6. The value of anti-CCP in juvenile Arthritis has not been determined. 12. Schellekens GA, Visser H, de Jong BA, et al. The diagnostic properties of
7. Since patient sera contain heterogeneous antibody populations, some samples may rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis
exhibit non-linearity, especially at very high sample dilutions. Linearity of this Rheum 2000;43(1):155-63.
procedure has not been verified. 13. Vossenaar ER, van Venrooij WJ. Anti-CCP antibodies, a highly specific marker for
8. Return any unused reagents stored in original packaging to 2-8oC storage after use. (early) rheumatoid arthritis. Clin Applied Immunol Rev 2004;4:239-62.
9. No reagent carryover studies have been performed for this assay. 14. Pruijn GJ, Vossenaar ER, Drijfhout JW, et al. Anti-CCP antibody detection facilitates
early diagnosis and prognosis of rheumatoid arthritis. Current Rheumatology
TECHNICAL ASSISSTANCE Reviews 2005;1(1):1-7.
For any technical assistance contact Axis-Shield Diagnostics Limited. 15. Rantapaa-Dahlqvist S, de Jong BA, Berglin E, et al. Antibodies against cyclic
e-mail: AXD.sales@alere.com citullinated peptide and IgA rheumatoid factor predict the development of
rheumatoid arthritis. Arthirtis Rheum 2003: 48(10):2741-49.
ANALYTICAL PROCEDURE 16. Nielen MM, van Schaardenbur D, Reesink HW, et al. Specific autoantibodies
Refer to specific Axis-Shield Clinical Chemistry Anti-CCP Application Guide. precede the symptoms of rheumatoid arthritis. Arthritis Rheum 2004: 50 (2):380-
386.
17. van Gaalen, Linn-Rasker SP, van Venrooij WJ, et al. Autoantibodies to cyclic
citrullinated peptides predict progression to rheumatoid arthritis in patients with
undifferentiated arthritis. Arthritis Rheum 2004: 50(3):709-15.
18. Meyer O, Labarre C, Dougados M, et al. Anticitrullinated protein / peptide antibody
assays in early Rheumatoid Arthritis for predicting five year radiographic damage.
Ann Rheum Dis 2003: 62:120-26.
19. Forslind K, Ahlmén M, Eberhardt K, et al. Prediction of radiological outcome in
early rheumatoid arthritis in clinical practice: role of antibodies to citrullinated
peptides (anti-CCP). Ann Rheum Dis 2004: 63:1090-95.
20. Kastbom A, Strandberg G, Lindroos A, et al. Anti-CCP antibody test predicts the
disease course during 3 years in early rheumatoid arthritis (the Swedish TIRA
project). Ann Rheum Dis 2004: 63:1085-89.
21. Aletaha D, Neogi T, Silman AJ, et al. 2010 Rheumatoid Arthritis Classification
Ciriteria. An American College of Rheumatology/European League against
Rheumatism Collaborative Initiative. Arthritis Rheum 2010: 62 (9):2569-81.
22. Clinical and Laboratory Standards Institute. Protection of Laboratory Workers from
Occupationally Acquired Infections; Approved Guideline – Fourth Edition. CLSI
document M29-A4. Wayne, PA: Clinical and Laboratory Standards Institute, 2014.
23. Clinical and Laboratory Standards Institute. Assessment of the Diagnostic Accuracy
of Laboratory tests Using Receiver Operating Characteristic Curves; Approved
Guideline. CLSI document EP24-A2. Wayne, PA: Clinical and Laboratory Standards
Institute, 2011.
24. Clinical and Laboratory Standards Institute. Evaluation of Detection Capability for
Clinical Laboratory Measurement Procedures; Approved Guideline—Second Edition.
CLSI document EP17-A2 Wayne, PA: Clinical and Laboratory Standards Institute,
2012.
25. Clinical and Laboratory Standards Institute. Measurement Procedure Comparison
and Bias Estimation Using Patient; Approved Guideline—Third Edition. CLSI
document EP5-A3. Wayne, PA: Clinical and Laboratory Standards Institute, 2013.
26. Clinical and Laboratory Standards Institute. Interference Testing in Clinical
Chemistry; Approved Guideline—Second Edition. CLSI document EP7-A2. Wayne,
PA: Clinical and Laboratory Standards Institute, 2005.

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 In vitro diagnostic medical device

Product Code
Lot Number

Number of tests

Consult Instructions For Use

Manufacturer

Use by

Store at 2-8 °C

Reagent 1

Reagent 2
For Prescription Use Only
Rx Only
Axis-Shield Diagnostics Ltd.,
Luna Place, The Technology Park,
Dundee, DD2 1XA
United Kingdom
Tel: +44 (0) 1382 422000
Fax: +44 (0) 1382 422088
Web:www.axis-shield.com
E-mail: AXD.sales@alere.com

RPBL1203/R1
Ver: 2016/09

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