DRG Cytomegalie Virus (CMV) IgM Elisa Kit

PRINCIPLE OF THE TEST 
The DRG Cytomegalie Virus (CMV) IgM ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA). Patient samples are diluted with Sample Diluent and additionally incubated with IgG-RF-Sorbent to eliminate competitive inhibition from specific IgG. This pretreatment avoids false negative results. Microtiter wells as a solid phase are coated with inactivated grade 2 Cytomegalovirus (CMV) antigen (strain AD-169). Diluted patient specimens and ready-for-use controls are pipetted into these wells. During incubation Cytomegalie Virus (CMV)-specific antibodies of positive specimens and controls are bound to the immobilized antigens. After a washing step to remove unbound sample and control material horseradish peroxidase conjugated anti-human IgM antibodies are dispensed into the wells. During a second incubation this anti-IgM conjugate binds specifically to IgM antibodies resulting in the formation of enzyme-linked immune complexes. After a second washing step to remove unbound conjugate the immune complexes formed (in case of positive results) are detected by incubation with TMB substrate and development of a blue color. The blue color turns into yellow by stopping the enzymatic indicator reaction with sulfuric acid. The intensity of this color is directly proportional to the amount of Cytomegalie Virus (CMV)-specific IgM antibody in the patient specimen. Absorbance at 450 nm is read using an ELISA microtiter plate reader.

SPECIMEN COLLECTION AND PREPARATION 
Serum or plasma (EDTA-, heparin- or citrate plasma) can be used in this assay. Do not use haemolytic, icteric or lipaemic specimens. Please note: Samples containing sodium azide should not be used in the assay. 
Specimen Collection 
Serum: Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.
Plasma: Whole blood should be collected into centrifuge tubes containing anti-coagulant (e.g. Sarstedt Monovette with the appropriate plasma preparation) and centrifuged immediately after collection.

Calculation of quantitative Results 
In order to obtain quantitative results in DU/mL (DU = DRG Units) plot the (mean) absorbance values of the Neg. Control and the 3 Standards 1, 2 and 3 on (linear/linear) graph paper in a system of coordinates against their corresponding concentrations (0, 50, 200 and 400 DU/mL) and draw a standard calibration curve (absorbance values on the vertical y-axis, concentrations on the horizontal x-axis). 
Read results from this standard curve employing the (mean) absorbance values of each patient specimen and control. All suitable computer programs available can be used for automated result reading and calculation. The following mathematical functions can be used: 4 PL (4 Parameter Logistics) curve fit, Linear regression or Point to Point calculation of the standard curve. We use DRG regression program for windows (4 parameter Rodbart regression). If other regression software is used, the obtained values have to be validated by the user.

QUALITY CONTROL
It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. After checking the above mentioned items without finding any error contact your distributor or DRG directly.