Design Verification T IgG ELISA C Determination of Diagnostic Sensitivity and Diagnostic Specificity... 2 Crossreactivity... 3

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1 Design Verification TOXO IgG ELISA CONTENTS 1 Determination of Diagnostic Sensitivity and Diagnostic Specificity External Study: Internal Evaluation Crossreactivity Potentially Interfering Substances Imprecision Within-run imprecision (Intra-Assay) Standardisation Stability Real Time Stability Stability Working Wash Solution... 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 1.1 External Study: 120 serum samples from blood donors and 79 paediatric serum samples have been tested. The results with the HUMAN Toxo. IgG ELISA have been compared with a commercially available EIA (Abbott). Equivocal or doubtful results have been retested and confirmed by clinical anamnesis and independent test method (EIKEN, EIA). Diagnostic sensitivity and diagnostic specificity The result of the external study can be summarized in the following table: Competitor ELISA Negative Human Toxo IgG ELISA Antibody Test 74 1 Negative This means according to the above calculation: Diagnostic specificity = 99.2% Diagnostic sensitivity = 96.1% Predictive value = 98.7% Overall agreement = 98.0% Design Verification and Product Data for TOXO IgG 2/5

3 1.2 Internal Evaluation 100 positive and 99 negative patient samples have been tested with the HUMAN TOXO IgG ELISA in parallel with a commercially available ELISA. The result of the internal studies are summarized in the following table: Competitor Test Negative Human TOXO IgG ELISA Antibody Test Negative 0 98 This means according to the above calculation: Diagnostic specificity = 99.0% Diagnostic sensitivity = 100.0% Predictive value = 99.0 % Overall agreement = 99.5%. The results are in good agreement with the results from the external studies. 2 Crossreactivity 62 RF, anti-nuclear antibodies (ANA) and infectious mononucleosis (IM) positive samples have been checked in order to check potential interferences. No crossreactivity could be observed. 3 Potentially Interfering Substances Description of control materials Potentially interfering substances have been evaluated by spiking serum of positive and negative controls with concentrated aqueous or ethanolic stock solutions, freshly prepared from pure substances (analytic grade) or from medical grade infusion solutions (lipids). The following table lists the tested substances in their final concentration. The final test concentration has been set to a level above physiological relevant concentration. Bilirubin Hemoglobin Triglycerides Substance Concentration 0.4 mg/ml 5.0 mg/ml 25.0 mg/ml For initial absorbances of positive and negative TOXO IgG sera no interference was observed by the concentrations of the tested substances up to the concentrations listed above. 4 Imprecision 4.1 Within-run imprecision (Intra-Assay) The within-run imprecision of the HUMAN TOXO IgG ELISA antibody test has been checked by employing positive, negative and Cut-off 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 (MCC) has been established from a triple determination of both positive and negative kit controls. The mean absorbance values of MNC (mean negative control), of MCC (mean cut-off control) and of MPCL (mean positive control low), MPCM (mean positive control medium) and MPCH (mean positive control high) are calculated according following example: Design Verification and Product Data for TOXO IgG 3/5

4 A 450 (D1) + A 450 (E1) MCC = 2 No. Pos. contr. Cut-off contr. Neg. contr. HP MP N Mean SD CV% Status pos. neg. pos. pos. neg. Lot: 101, MNC = 0.002, MCC = 0.249, MPC low = 0.996, MPC med = 2.144, MPC high = No. Pos. contr. Cut-off contr. Neg. contr. HP MP N Mean SD CV% Status pos. neg. pos. pos. neg. Lot: 102, MNC = 0.047, MCC = 0.219, MPC low = 1.070, MPC med = 2.053, MPC high = The results demonstrate the good presision of the assay. 5 Standardisation The positive (low, medium and high) controls and the cut-off control of the HUMAN TOXO IgG ELISA test has been standardized against the 1 st ISP WHO and adjusted to the following levels: high positive control = 40 IU/ml (±10%) medium positive control = 20 IU/ml (±10%) low positive control = 6 IU/ml (±15%) cut-off control = 1 IU/ml (±20%). The positive and cut-off controls are employed for quantitative determination of anti-toxoplasma gondii IgG antibodies. For this purpose the absorbance values of the controls are plotted vs. their respective antibody concentrations (point-to-point connection). The resulting calibration curve is used to calculate the IgG content in unknown samples from their respective absorbance values. Design Verification and Product Data for TOXO IgG 4/5

5 6 Stability 6.1 Real Time Stability The stability of the HUMAN TOXO IgG 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. Real time storage at C Lot TOXO IgG ELISA control absorbance (lot-release) Real time storage (months since lotrelease) control absorbance specification > 0.75 Pos. patient sera (Index) H (-31.0%) passed passed H (-16.6%) passed passed H (-20.5%) passed passed H (-40.3%) 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. Conclusion: These stability data clearly support the shelf life claim of 18 months after production. 6.2 Stability Working Wash Solution The working wash solution has been stored 60 days at 25 C. Reference LOT# BA01 Dilution of OD OD LOT # KT01 LOT# % OD LOT# % OD % 1: : : : : : : : Conclusion: The recovery shows a working wash solution stability of 60 days stored at 25 C. Design Verification and Product Data for TOXO IgG 5/5