Design Verification. Form: /5 Rev. 007 valid of

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1 Design Verification g 1 Determination of Diagnostic Sensitivity and Diagnostic Specificity... 2 Crossreactivity... 3 Potentially interfering substances Imprecision Standardisation Stability... 5 Real-time stability... 5 Open-vial stability... 5 Form: /5 valid of

2 1 Determination of Diagnostic Sensitivity and Diagnostic Specificity Sensitivity and specificity were calculated with following formulas: Diagnostic Sensitivity = Number of correctly positive determined samples Number of true positive + false negative determined samples Diagnostic Specificity = Number of correctly negative determined samples Number of true negative + false positive determined samples External Study: A total of 418 serum samples of predetermined status have been employed. 32 samples have been confirmed positive for CMV IgM and 386 samples have been confirmed negative. The Eurogenetics CMV IgM test has been employed as a reference method to confirm the sample status. Diagnostic sensitivity and specificity The result of the external study can be summarized in the following table: Competitor ELISA ELISA Antibody Test Diagnostic specificity = 99.2% Diagnostic sensitivity = 93.75% Predictive value = 90.9% Overall agreement = 98.8%. Design Verification and Product Data for CMV IgM ELISA 2/5

3 Internal Evaluation 232 samples have been tested with the ELISA in parallel with a commercially available ELISA. True positive and negative status was defined by the results from other commercially available tests. Equivocal results have not been considered for calculation of diagnostic sensitivity and specificity. Independent Tests ELISA Antibody Test Diagnostic specificity = 98.9% Diagnostic sensitivity = 98.15% Predictive value = 96.4% Overall agreement = 98.71%. Internal Re-Evaluation Recently, the test has been re-evaluated on the basis of true 88 positive and 116 negative samples (equivocals not considered). The results are summarized below. Competitor ELISA ELISA Antibody Test Diagnostic specificity = 100% Diagnostic sensitivity = 100% Predictive value = 100% Overall agreement = 100% The result from this re-evaluation confirms the results obtained from previous studies. Crossreactivity Each 20 sera with RF, ANA and from patients confirmed for infectious mononucleosis (I.M.) have been tested. No interference / crossreactivity could be observed with RF and ANA sera. 5 out of 20 I.M. sera produced weak positive results. These positive results may be related to a minor crossreactivity but can as well be explained by a reactivation of CMV IgM producing cells due to acute I.M. Potentially interfering substances Potentially interfering substances have been evaluated by spiking of negative [NC] and positive [PC] control with concentrated aqueous substances (analytic grade) and in parallel with matrix only. - Hemoglobin (5.0 mg/ml) - Bilirubin (0.4 mg/ml) Design Verification and Product Data for CMV IgM ELISA 3/5

4 - Triglyceride (25 mg/ml) No inference (positive control recovery > 95%) was observed for the indicated concentrations. 2 Imprecision Test Design The imprecision of the ELISA antibody test has been checked by employing positive and negative kit controls and three sera with predetermined high positive (HP), low positive (LP) and negative (N) status. Each sample has been tested five-fold. The samples were randomly distributed over the microtiter plate. The cut-off value (COV) has been established from a triple determination of both positive and negative kit controls. The mean of the values of both positive and negative controls (MNC, MPC) has been used for calculation of the COV: COV = MNC + (0.2* MPC) Results No. Pos. contr. Neg. contr. HP LP N Lot: 086, MNC = 0.044, MPC = 0.768, COV = No. Pos. contr. Neg. contr. HP LP N Lot: 088, MNC = 0.054, MPC = 0.812, COV = The results from the above imprecision study are summarized below. For lot 086: Mean abs SD CV% Status pos. neg. pos. pos. neg. For lot 088: Mean abs SD CV% Status pos. neg. pos. pos. neg. These results clearly demonstrate the good precision of the assay. 3 Standardisation The positive control of the HUMAN IgM ELISA test has been standardized by a reference laboratory against a validated CE marked test in routine use. Design Verification and Product Data for CMV IgM ELISA 4/5

5 4 Stability Real-time stability The stability of the ELISA antibody test was checked by real-time studies on four independent production lots. One kit of each lot has been tested at the date of manufacture and re-tested after the indicated periods. A function test was applied to each kit employing the positive and negative kit controls as well as panel sera of predetermined positive and negative status. Results Real time storage at C Lot CMV IgM ELISA control Real time storage (months) control control Low pos. patient sera High pos. patient sera H (-38.1%) passed passed H (-11.4%) passed passed H (-8.9%) passed passed The above data obtained with lots stored much longer than the specified 18 months shelf life confirm that an inevitable decrease of the positive control during storage is well below the specification. Also we demonstrate for this excessive storage time that patient sera were correctly recovered with their predetermined status. These stability data clearly support the shelf life claim of 18 months after production. Open-vial stability To test the open vial stability of the CMV IgM ELISA one lot (#16002) was stored for 65 days after opening. The kit components were tested at different time points. The criteria for the CMV IgM ELISA should be met after storage for 65 days. Acceptance criteria: Blank OD NC < OD PC OD samples > cut off samples < cut off Mean values Days OD sub BL NC PC pos pos neg neg neg neg The results are within the acceptance criteria. The data demonstrate the claimed open vial stability of the CMV IgM ELISA of 60 days. Design Verification and Product Data for CMV IgM ELISA 5/5