Kits
Anti-CCP
Kits
Description
Reumathoid arthritis (RA) is one of the most common
autoimmune diseases (1-2% European population). The
most significant clinical symptom is an inflammation of the
synovial membranes which causes a painful swelling of the
articulations and the ankylosis.
In order to correctly diagnose RA it is necessary to exclude
other forms of arthritis: in such a diagnostic process, the
laboratory plays an important role in the determination of
Rheumatoid Factor (RF) antibodies of class IgM,
detectable in 60-80% of the patients with RA. The RF
antibodies are sensitive but not very specific markers; on
the contrary, Anti CCPs are characterized by a specificity
of over 90% in patients affected by RA ,and are detectable
in a very early asymptomatic stage in the approximately
70% of RA patients whereas only 2% of the control
subjects resulted positive.
Therefore, the presence of Anti CCP antibodies can be
used in the diagnosis of RA, particularly in the case of
erosive arthritis, in childhood in the case of juvenile RA.
The test also appears, to be useful in differentiating the
collagen pathologies with concomitant arthritis from the
RA. The Anti CCP antibodies test has an important
prognostic value in the monitoring of articular radiologically
detectable damage. The kit’s quantitative determination is
useful in the control and verification of the effects of
pharmacological therapy.
The Anti CCP antibody test, together with the
determination of RF, increases the ratio of sensitivity /
specificity. 20% of the RAs are RF-negative and 15/20% of
the RAs are positive only to RF. The simultaneous positive
result of a sample to RF and CCP has a positive predictive
value of about 100%.
The levels of Anti CCP antibodies are not necessarily
correlated to the evolutionary stage of the illness. The
advantage of the Anti CCP antibodies is that they are
detectable in the patient sera up to 10 years prior to the
appearance of symptoms. In addition, in cases of early
arthritis a positive test result, according to some studies is
related to the development of bone erosive lesions of the
articulations.
2. PRINCIPLE
Anti CCP test is based on the binding of the antibodies
present in the sample, to the cyclical citrullinated peptides
adsorbed on the microplate.
In the first step the antibodies present in the calibrators, in
the controls or in the prediluted patient samples are bound
to the internal surface of the wells. After 60 minutes of
incubation, the microplate is washed with a wash buffer to
remove the non-reacted serum components. Then a
solution of anti-human IgG conjugated with peroxidase
recognizes the antibodies of class IgG bound to the
immobilized antigens.
After 30 minutes of incubation the excess of enzyme
conjugate, that is not specifically bound, is removed by the
wash buffer. At this point, a substrate solution containing
chromogenic TMB is added to the microplate. After 15
minutes of incubation the colour development is stopped
by adding the stop solution. The solution turns yellow at
this point. The level of developed colour is directly
proportional to the concentration of the anti CCP IgG
antibodies present in the original sample.
The concentration of the anti CCP IgG antibodies in the
sample is calculated through a calibration curve.
autoimmune diseases (1-2% European population). The
most significant clinical symptom is an inflammation of the
synovial membranes which causes a painful swelling of the
articulations and the ankylosis.
In order to correctly diagnose RA it is necessary to exclude
other forms of arthritis: in such a diagnostic process, the
laboratory plays an important role in the determination of
Rheumatoid Factor (RF) antibodies of class IgM,
detectable in 60-80% of the patients with RA. The RF
antibodies are sensitive but not very specific markers; on
the contrary, Anti CCPs are characterized by a specificity
of over 90% in patients affected by RA ,and are detectable
in a very early asymptomatic stage in the approximately
70% of RA patients whereas only 2% of the control
subjects resulted positive.
Therefore, the presence of Anti CCP antibodies can be
used in the diagnosis of RA, particularly in the case of
erosive arthritis, in childhood in the case of juvenile RA.
The test also appears, to be useful in differentiating the
collagen pathologies with concomitant arthritis from the
RA. The Anti CCP antibodies test has an important
prognostic value in the monitoring of articular radiologically
detectable damage. The kit’s quantitative determination is
useful in the control and verification of the effects of
pharmacological therapy.
The Anti CCP antibody test, together with the
determination of RF, increases the ratio of sensitivity /
specificity. 20% of the RAs are RF-negative and 15/20% of
the RAs are positive only to RF. The simultaneous positive
result of a sample to RF and CCP has a positive predictive
value of about 100%.
The levels of Anti CCP antibodies are not necessarily
correlated to the evolutionary stage of the illness. The
advantage of the Anti CCP antibodies is that they are
detectable in the patient sera up to 10 years prior to the
appearance of symptoms. In addition, in cases of early
arthritis a positive test result, according to some studies is
related to the development of bone erosive lesions of the
articulations.
2. PRINCIPLE
Anti CCP test is based on the binding of the antibodies
present in the sample, to the cyclical citrullinated peptides
adsorbed on the microplate.
In the first step the antibodies present in the calibrators, in
the controls or in the prediluted patient samples are bound
to the internal surface of the wells. After 60 minutes of
incubation, the microplate is washed with a wash buffer to
remove the non-reacted serum components. Then a
solution of anti-human IgG conjugated with peroxidase
recognizes the antibodies of class IgG bound to the
immobilized antigens.
After 30 minutes of incubation the excess of enzyme
conjugate, that is not specifically bound, is removed by the
wash buffer. At this point, a substrate solution containing
chromogenic TMB is added to the microplate. After 15
minutes of incubation the colour development is stopped
by adding the stop solution. The solution turns yellow at
this point. The level of developed colour is directly
proportional to the concentration of the anti CCP IgG
antibodies present in the original sample.
The concentration of the anti CCP IgG antibodies in the
sample is calculated through a calibration curve.