Anti-CCP ELISA (IgG)

Test instruction

ORDER NO. ANTIBODIES AGAINST Ig CLASS SUBSTRATE FORMAT

cyclic citrullinated peptides Ag-coated
EA 1505-9601 G (CCP) IgG microplate wells 96 x 01 (96)

Indication: Rheumatoid arthritis

Principle of the test: The ELISA test kit provides a semi-quantitative or quantitative in vitro assay for
the determination of human autoantibodies of the IgG class against cyclic citrullinated peptides (CCP).
The test kit contains microtiter strips each with 8 individual break-off reagent wells coated with synthetic
cyclic citrullinated peptides. In the first reaction step, diluted patient samples (serum or EDTA, heparin or
citrate plasma) are incubated in the wells. In the case of positive samples, the specific IgG antibodies
(also IgA and IgM) will bind to the corresponding antigenic site. To detect the bound antibodies, a
second incubation is carried out using an enzyme-labelled anti-human IgG (enzyme conjugate), which is
capable of promoting a colour reaction.

Contents of the test kit:

Description Colour Format Symbol
1. Microplate wells coated with antigens
coated with antigens: 12 microplate strips each
--- 12 x 8 .STRIPS.
containing 8 individual break-off wells in a frame,
ready for use
2. Calibrators 1 to 5
red 5 x 2.0 ml .CAL 1-5.
1, 5, 20, 100, 200 RU/ml (IgG, human), ready for use
3. Positive control
blue 1 x 2.0 ml .POS CONTROL.
(IgG, human), ready for use
4. Negative control
green 1 x 2.0 ml .NEG CONTROL.
(IgG, human), ready for use
5. Enzyme conjugate
peroxidase-labelled anti-human IgG (rabbit), green 1 x 12 ml .CONJUGATE.
ready for use
6. Sample buffer
violet 1 x 100 ml .SAMPLEBUFFER.
ready for use
7. Wash buffer
colourless 1 x 100 ml .WASHBUFFER 10x.
10x concentrate
8. Chromogen/substrate solution
colourless 1 x 12 ml .SUBSTRATE.
TMB/H2O2, ready for use
9. Stop solution
colourless 1 x 12 ml .STOP SOLUTION.
0.5 M sulphuric acid, ready for use
10. Test instruction --- 1 booklet
11. Protocol with target values --- 1 protocol
.LOT. Lot description Storage temperature
.IVD. In vitro determination Unopened usable until

Storage and stability: The test kit has to be stored at a temperature between +2°C to +8°C, do not
freeze. Unopened, all test kit components are stable until the indicated expiry date.

Waste disposal: Undiluted patient samples and incubated microplate strips should be handled as
infectious waste. Other reagents do not need to be collected separately, unless stated otherwise in
official regulations.
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Preparation and stability of reagents

Note: All reagents must be brought to room temperature (+18°C to +25°C) around 30 minutes before
use. Unless stated otherwise, the reagents after initial opening are stable until the expiry date when
stored between +2°C and 8°C and protected from contamination.

- Coated wells: Ready for use. Tear open the resealable protective wrapping of the microplate at the
recesses above the grip seam. Do not open until the microplate has reached room temperature to
prevent the individual strips from moistening. Immediately replace the remaining wells of a partly used
microplate in the protective wrapping and tightly seal with the integrated grip seam (Do not remove
the desiccant bag).

- Calibrators and controls: Ready for use. The reagents must be mixed thoroughly before use.

- Enzyme conjugate: Ready for use. The enzyme conjugate must be mixed thoroughly before use.

- Sample buffer: Ready for use.

Note: The sample buffer provided in this test kit must only be used together with the Anti-CCP ELISA.

- Wash buffer: The wash buffer is a 10x concentrate. If crystallisation occurs in the concentrated
buffer, warm it to 37°C and mix well before diluting. The amount required should be removed from the
bottle using a clean pipette and diluted with deionised or distilled water (1 part reagent plus 9 parts
distilled water.
Example: For 1 microplate strip use 5 ml concentrate plus 45 ml water.
The ready-to-use diluted wash buffer is stable for 4 weeks when stored at +2°C to +8°C and handled
properly.

- Chromogen/substrate solution: Ready for use. Close bottle immediately after use, as the contents
are sensitive to light. The substrate solution must be clear on use. Do not use the solution if it is blue
coloured.

- Stop solution: Ready for use.

Warning: Calibrators and controls used have tested negative for HBsAg, anti-HCV, anti-HIV-1 and anti-
HIV-2 using enzyme immunoassays and indirect immunofluorescence methods. Nonetheless, all
materials should be treated as being a potential infection hazard and should be handled with care. Some
of the reagents are contain the toxic agent sodium azide. Avoid contact with the skin.

Preparation and stability of the serum or plasma samples

Samples: Human serum or EDTA, heparin or citrate plasma. Do not use heat-inactivated samples, as
they can lead to false positive results.

Stability: Serum and plasma samples to be investigated can generally be stored at +2°C to +8°C for up
to 14 days. Provided that the cold storage is not interrupted, it is possible to store samples for up to 28
days. In these cases, the sample should be checked visually and should also be smelled (the
development of a smell indicates a bacterial contamination). Diluted samples must be incubated within
one working day.

Sample dilution: The serum or plasma samples to be investigated are diluted 1:101 with sample
buffer.
Example: Add 10 µl of serum to 1.0 ml sample buffer and mix well by vortexing (sample pipettes are not
suitable for mixing).

Note: Calibrators and controls are prediluted and ready for use, do not dilute them.

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Incubation
For qualitative/semiquantative analysis incubate calibrator 2 along with the positive and negative
controls and patient samples. For quantitative analysis incubate calibrators 1 to 5 along with the
positive and negative controls and patient samples.

Sample incubation: Transfer 100 µl calibrators, positive and negative controls or diluted patient
(1. step) samples into the individual microplate wells according to the pipetting
protocol. The pipetting should not take longer than 15 minutes. Incubate for
60 minutes at room temperature (+18°C to 25°C).

Wash: Manual: Empty the wells and subsequently wash 3 times using 300 µl of
working strength wash buffer for each wash.
Automatic: Wash reagent wells 3 times with 450 µl working strength wash
buffer (programme setting: e.g. TECAN Columbus Washer “Overflow Mode”).

Leave the wash buffer in each well for 30 to 60 seconds per washing cycle,
then empty the wells. After washing (manual and automated tests),
thoroughly dispose of all liquid from the microplate by tapping it on absorbent
paper with the openings facing downwards to remove all residual wash buffer.

Attention: Residual liquid (> 10 µl) remaining in the reagent wells after
washing can interfere with the substrate and lead to falsely low extinction
values.
Insufficient washing (e.g. less than 3 wash cycles, too small wash buffer
volumes, or too short reaction times) can lead to falsely high extinction
values.

Conjugate incubation Pipette 100 µl of enzyme conjugate (peroxidase-labelled anti-human IgG) into
(2. step) each of the microplate wells. Incubate for 30 minutes at room temperature
(+18°C to 25°C).

Wash: Empty the wells. Wash as described above.

Substrate incubation: Pipette 100 µl of chromogen/substrate solution into each of the microplate
(3. step) wells. Incubate for 30 minutes at room temperature (+18°C to 25°C), protect
from direct sunlight.

Stop: Pipette 100 µl of stop solution into each of the microplate wells in the same
order and at the same speed as the chromogen/substrate solution was intro-
duced.

Measurement: Photometric measurement of the colour intensity should be made at a

wavelength of 450 nm and a reference wavelength of between 620 nm and
650 nm within 30 minutes of adding the stop solution. Prior to measuring,
carefully shake the microplate to ensure a homogeneous distribution of the
solution.

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Pipetting protocol
1 2 3 4 5 6 7 8 9 10 11 12
A C2 P 6 P 14 P 22 C1 P 2 P 10 P 18

B pos. P 7 P 15 P 23 C2 P 3 P 11 P 19

C neg. P 8 P 16 P 24 C3 P 4 P 12 P 20

D P 1 P 9 P 17 P 25 C4 P 5 P 13 P 21

E P 2 P 10 P 18 C5 P 6 P 14 P 22

F P 3 P 11 P 19 pos. P 7 P 15 P 23

G P 4 P 12 P 20 neg. P 8 P 16 P 24

H P 5 P 13 P 21 P 1 P 9 P 17 P 25

The pipetting protocol for microtiter strips 1-4 is an example for the qualitative/semiquantitative
analysis of 25 patient sera (P 1 to P 25).
The pipetting protocol for microtiter strips 7-10 is an example for the quantitative analysis of 25 patient
sera (P 1 to P 25).
The calibrators (C 1 to C 5), the positive (pos.) and negative (neg.) controls, and the patient samples
have each been incubated in one well. The reliability of the ELISA test can be further improved by
duplicate determinations for each sample. Both positive and negative controls serve as internal controls
for the reliability of the test procedure. They should be assayed with each test run.

Calculation of results

Qualitative/semiquantitative: Results can be evaluated semiquantitatively by calculating a ratio of the

extinction value of the control or patient sample over the extinction value of calibrator 2. Calculate the
ratio according to the following formula:

Extinction of the control or patient sample

= Ratio
Extinction of calibrator 2

EUROIMMUN recommends interpreting results as follows:

Ratio 1.0: negative

Ratio >1.0: positive

Quantitative: The standard curve from which the concentration of anti-CCP antibodies in the serum
samples can be taken is obtained by plotting the extinction values measured for the 5 calibration sera
(linear, y-axis) against the corresponding concentrations (logarithmic, x-axis). The standard curve can be
calculated by computer using one of the following curve-fitting techniques: 4-parameter logistic, 5-
parameter logistic, spline fits, log/logit curve and lim/limit curve. The following plot is an example of a
typical calibration curve. Please do not use this curve for the determination of antibody concentrations in
patient samples.

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If the extinction for a patient sample lies above that of calibrator 5 (200 RU/ml), the result should be
reported as “>200 RU/ml”. It is recommended that the sample be remeasured in a new test run at a
dilution of e.g. 1:400. The result in RU/ml read from the calibration curve for this sample must then be
multiplied by a factor of 4. The upper limit of the normal range (cut-off) recommended by EUROIMMUN
is 5 relative units (RU) /ml. EUROIMMUN recommends interpreting results as follows:

5 RU/ml: negative
>5 RU/ml: positive

The recommendation is based on data yielded in a ROC analysis using the results of 419 samples of
patients with rheumatoid arthritis and 1144 control samples. According to the analysis, the specificity
was 98% at a cut-off of 4.2 RU/ml. The 99th percentile based on 400 healthy blood donors was also 4.2
RU/ml (q.v. respective paragraphs under “Test characteristics”).

Please note that 11 of 21 (52%) positive results from the control panel samples (n = 1144) but only 16 of
329 (5%) positive results in patients with rheumatoid arthritis ranged between 5 RU/ml and 10 RU/ml.
Results from a weak positive range of 5 RU/ml to 10 RU/ml should be interpreted with prudence and
possibly verified with a new patient sample taken several weeks later.

For duplicate determinations the mean of the two values should be taken. If the two values deviate
substantially from one another the sample should be retested.

For diagnosis, the clinical symptoms of the patient should always be taken into account alongside the
serological results.

Test characteristics
Calibration: As no international reference serum exists for antibodies against CCP, the calibration is
performed in relative units (RU/ml).

For every group of tests performed, the relative units or ratio values determined for the positive and
negative controls must lie within the limits stated for the relevant test kit lot. A protocol containing these
target values is included. If the values specified for the controls are not achieved, the test results may be
inaccurate and the test should be repeated.

The binding activity of the antibodies and the activity of the enzyme used are temperature-dependent. It
is therefore recommended using a thermostat in all three incubation steps. The higher the room
temperature during substrate incubation, the greater will be the extinction values. Corresponding
variations apply also to the incubation times. However, the calibrators are subject to the same
influences, with the result that such variations will be largely compensated in the calculation of the result.

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Antigen: The reagent wells are coated with synthetic cyclic citrullinated peptides (CCP) which contains
modified arginine residues.

Linearity: The linearity of the ELISA was determined by assaying serial dilutions of 6 serum samples.
The average concordance of dilution-factor-corrected results for the serum samples amounted to 103%
(86% – 125%). The Anti-CCP ELISA is linear in the tested concentration range (3 RU/ml to 196 RU/ml).

Detection limit: The detection limit is defined as a value of three times the standard deviation of an
analyte-free sample and is the smallest detectable antibody titer. The lower detection limit of the Anti-
CCP ELISA is 0.3 RU/ml.

Cross reactivity: The ELISA presented here specifically detects IgG class antibodies directed against
CCP. There were no cross reactions with other autoantibodies in samples of patients with the following
diseases: SLE (n = 6), Scleroderma (n = 5), Sjögren’s syndrome (n = 5) and IgM rheumatoid factor-
positive rheumatoid arthritis (n = 10).

Interference: Haemolytic, lipaemic and icteric samples showed no influence on the result up to a
concentration of 10 mg/ml for haemoglobin, 20 mg/ml for triglycerides and 0.4 mg/ml for bilirubin in this
ELISA.

Reproducibility: The reproducibility of the test was investigated by determining the intra- and inter-
assay coefficients of variation using 4 sera. The intra-assay CVs are based on 20 determinations and the
inter-assay CVs on 4 determinations performed in 6 different test runs.

Intra-assay variation, n = 20 Inter-assay variation, n = 4 x 6

Serum Mean value CV Serum Mean value CV
(RU/ml) (%) (RU/ml) (%)
1 18 5.9 1 19 6.3
2 20 4.0 2 23 6.5
3 26 3.6 3 35 7.2
4 52 3.4 4 63 6.8

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Clinical sensitivity and specificity: Sera from 419 patients with rheumatoid arthritis, a control panel of
744 patients with other diseases and 400 healthy blood donors were analysed using the EUROIMMUN
Anti-CCP ELISA. The sensitivity of the ELISA for rheumatoid arthritis was 78.5% with a specificity of
98.2%.

Anti-CCP ELISA
Panel
n positive
Sensitivity for rheumatoid arthritis 419 329 (78.5%)

Asymptomatic blood donors 400 2 (0.5%)

Psoriatic arthritis 28 0
Other arthritides 35 3 (8.6%)
SLE 108 3 (2.8%)
Sjögren’s syndrome 106 2 (1.9%)
Scleroderma 98 3 (3.1%)
Autoimmune thyroiditis 159 4 (2.5%)
Wegener’s granulomatosis 25 1 (4.0%)
Anti-parvovirus B19-positive 126 3 (2.4%)
Viral hepatitides 54 0
Anti-HIV-positive 5 0
Specificity for rheumatoid arthritis 1144 21 (98.2%)

In the case of anti-CCP positive results in the control panel it can neither be excluded that the patients
have rheumatoid arthritis in addition to the underlying illness nor that they are at an early, symptom-free
stage of RA. Various studies have shown that a majority of anti-CCP positive test persons without
characteristic RA symptoms developed RA within a few years after the serological analysis.

In a ROC analysis of the results of 419 RA patients samples and 1144 control samples listed in the
above table the following characteristics were determined:

cut-off specificity sensitivity

2.6 RU/ml 95.0% 81.4%
4.2 RU/ml 98.0% 79.0%
8.0 RU/ml 99.0% 75.4%

Reference range: Levels of anti-CCP antibodies were analysed in 400 sera from healthy blood donors
of between 18 and 68 years of age (149 women, 251 men) using the EUROIMMUN ELISA. No
differences with respect to age or gender were observed. The mean concentration of antibodies against
CCP was 1.2 RU/ml (± 0.8 RU/ml of standard deviation) and the values ranged from 0.2 to 8.0 RU/ml.
With a cut-off of 5 RU/ml, 0.5% of the blood donors were anti-CCP positive.

cut-off percentile
2.6 RU/ml 95.0%
3.3 RU/ml 98.0%
4.2 RU/ml 99.0%

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Clinical significance
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and also the most frequent
chronic inflammatory arthropathy. The disease affects around 1% of the world population, 75% of which
are female. It is characterised by inflammation of the synovial membrane, which spreads symmetrically
from the small to large joints leading to the destruction of the joints in the late phase accompanied by a
systemic involvement of the soft tissue. Initial symptoms include painful swelling of the
metacarpophalangeal joints with morning stiffness in the joints [1]. Reliable and earliest possible
diagnosis is indispensable to keep the disease under control with suitable therapy and to avoid
irreversible joint damage [2, 3, 4, 5].
The most commonly performed serological test in suspected RA cases was until now the determination
of rheumatoid factors (RF) in addition to general inflammatory parameters. RF are antibodies
(predominantly of class IgM) which react with gamma globulins and occur in 60-80% of RA patients. RF
are a sensitive, but not very specific marker for RA since they also occur in healthy individuals, e.g. in
various infections or other autoimmune diseases such as systemic lupus erythematosus, Sjögren’s
syndrome and scleroderma [6, 7, 8, 9].
40-60% of RA patients also exhibit autoantibodies against epidermal filaggrin (RA keratin, anti-
perinuclear factor) in their serum. Filaggrin is a protein of the epidermis, which links keratin filaments to
one another. Autoantibodies against filaggrin are detected by indirect immunofluorescence: the antigen
substrate rat oesophagus shows staining of the stratum corneum (RA keratin) on the luminal side; anti-
perinuclear factors (APF) are apparent in the cytoplasmic inclusion bodies of human epithelial cells of
the oral mucosa [2, 10, 11, 12].
In recent years it has been shown that the rare amino acid citrulline, which is present in filaggrin, is a
substantial component of the antigenic epitope. A direct comparison study demonstrated that the
sensitivity can be increased from 49% to 68% by using cyclic citrullinated peptides instead of linear
citrullinated peptides as an ELISA substrate [13]. Autoantibodies against cyclic citrullinated peptides
(CCP) are therefore a new, highly specific marker for rheumatoid arthritis [2, 13, 14, 15, 16].
Antibodies against CCP occur independently of rheumatoid factors. The term “seronegative RA” for RF-
negative cases is outdated and should not be used anymore. It could be demonstrated in many studies
that 20% to 57% of all RF-negative RA patients have antibodies against CCP. Thus, the parallel
determination of both antibodies increases the serological hit rate in RA patients [8, 13, 17, 18, 19, 20].
The CCP titer generally correlates with the activity of the disease [2, 14, 15, 16, 21, 22].
Antibodies against CCP are predominantly of class IgG and have a specificity of 95% for RA [9, 13].
They are a predictive marker since they can be found in the serum and the synovial liquid of 70%-80% of
patients very early during the development of the disease, often even many years before the onset of the
first symptoms [8, 18, 22, 23, 24, 25, 26, 27, 28]. The earlier the diagnosis is established, the earlier a
suitable therapy can be started. With respect to disease prognosis, radiological examinations show that
severe joint damage is found significantly more often in patients with anti-CCP antibodies than in anti-
CCP negative patients [13, 18, 19, 20, 22, 27, 29, 30, 31, 32, 33]. This fact emphasises the importance
of CCP antibodies as prognostic marker for the development and progression of the disease.
The great importance of the Anti-CCP antibody determination in the diagnosis of rheumatoid arthritis is
limited in suspected cases of juvenile idiopathic arthritis (JIA) and in the monitoring of RA therapy: The
prevalence of antibodies against CCP in patients with JIA is as low as 2% to 12%. Therefore, the
determination of antibodies against CCP in suspected JIA cases plays only a minor role [34, 35, 36, 37].
Due to partially discrepant research results, the determination of antibodies against CCP can only be of
limited use in the monitoring of therapy measures [38, 39, 40, 41].
The importance of antibodies against CCP as a serological marker becomes apparent in comparison
with rheumatoid factors (RF) which have a significantly lower specificity (anti-CCP: 96%-100%, RF: 63%)
at almost the same sensitivity (anti-CCP: 80%, RF: 79%) [13, 42]. Antibodies against CCP can also be
used as a marker in differential diagnostics, e.g. in the differentiation of hepatitis-associated
arthropathies from rheumatoid arthritis (e.g. anti-CCP negative and RF positive in HCV infections) [43,
44].

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Literature references

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disease-modifying therapy on radiographic outcome in inflammatory polyarthritis at five
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Frankfort C, de Jong BA, van Venrooij WJ, Bijlsma JW. The prognostic value of the
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rheumatoid arthritis. Clin Exp Rheumatol 17 (1999) 689-697.
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early: a prediction model for persistent (erosive) arthritis. Arthritis Rheum 46 (2002) 357-365.
31. Forslind K, Ahlmen M, Eberhardt K, Hafstrom I, Svensson B; BARFOT Study Group. Prediction of
radiological outcome in early rheumatoid arthritis in clinical practice: role of antibodies to
citrullinated peptides (anti-CCP). Ann Rheum Dis 63 (2004) 1090-1095.
32. Lindqvist E, Eberhardt K, Bendtzen K, Heinegard D, Saxne T. Prognostic laboratory markers of
joint damage in rheumatoid arthritis. Ann Rheum Dis 64 (2005) 196-201.
33. Lodder MC, de Jong Z, Kostense PJ, Molenaar ET, Staal K, Voskuyl AE, Hazes JM, Dijkmans BA,
Lems WF. Bone mineral density in patients with rheumatoid arthritis: relation between disease
severity and low bone mineral density. Ann Rheum Dis 63 (2004) 1576-1580.
34. Avcin T, Cimaz R, Falcini F, Zulian F, Martini G, Simonini G, Porenta-Besic V, Borghi MO, Meroni
PL. Prevalence and clinical significance of anti-cyclic citrullinated peptide antibodies in
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