Diametra Anti CP IgG Elisa Kit

PRINCIPLE
Anti CP IgG test is based on the binding of antibodies present in calibrators, controls or prediluted patient samples to the syntetic Citrullinated Peptides (CP) coated on the inner surface of the wells. After a 30 minutes incubation the microplate is washed with a wash buffer to remove the non-reactive serum components. Then an anti-human-IgG horseradish peroxidase conjugated solution recognizes the IgG class antibodies bound to the immobilized antigens. After a 30 minutes incubation, any excess of enzyme conjugate not specifically bound is washed away withthe wash buffer. Finally a chromogenic substrate solution containingTMB is dispensed into the wells. After 15 minutes ofincubation the color development is stopped byadding the stop solution. The solutions color changesinto yellow. The amount of color is directlyproportional to the concentration of IgG antibodiespresent in the original sample.

CLINICAL SIGNIFICANCE 
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 this 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 citrullinated peptides (CP) autoantibodies are a hallmark of RA and are used in diagnostic assays, through the use of citrullinated sequences (such as: filaggrin and filaggrin derived cyclized peptides, citrullinated recombinant vimentin, fibrin, viral citrullinated proteins) as antigens. Dia.Metra Anti CP IgG assay is based on viral and human citrullinated peptidic sequences that are used as antigens; this combined use allows to detect different subgroups of anti CP antibodies in the same set of patients, leading to a better diagnostic investigation.

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use.
All reagents should be stored refrigerated at 2-8°Cin their original container. Any exceptions areclearly indicated. The reagents are stable until theexpiry date when stored and handled as indicated.
Allow all kit components and specimens to reachroom temperature (22-28°C) and mix well prior touse.
Do not interchange kit components from differentlots. The expiry date printed on box and vialslabels must be observed. Do not use any kitcomponent beyond their expiry date.

Preparation of the Sample
For determination of anti CP antibodies, human serum or plasma are the preferred sample matrixes. All serum and plasma samples have to be prediluted with sample diluent 1:100; for example 10 μL of sample may be diluted with 990 μL of sample diluent. Samples may be stored refrigerated at 2-8°C for at least 5 days. For longer storage of up to six months samples should be stored frozen at -20°C. To avoid repeated thawing and freezing the samples should be aliquoted. The Controls are ready to use.

RESULTS
Calibration curveFor the Anti CP IgG assay a 4-Parameter-Fit with linlog coordinates for optical density and concentrationis the preferred data reduction method. SmoothedSpline Approximation and log-log coordinates arealso suitable. However we recommend using a Lin-Log curve.First calculate the averaged optical densities for eachcalibrator well. Use lin-log graph paper and plot theaveraged optical density of each calibrator versus theconcentration. Draw the best fitting curveapproximating the path of all calibrator points. Thecalibrator points may also be connected with straightline segments. The concentration of unknowns maythen be estimated from the calibration curve byinterpolation.