Design Verification IMTEC-TSH R -A C Intended Use... 2 Diagnostic Sensitivity and Diagnostic Specificity... 2 Interferences... 4 Imprecision...

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1 Design Verification IMTEC-TSH RECEPTOR-ANTIBODIES CONTENTS 1 Intended Use Diagnostic Sensitivity and Diagnostic Specificity Determination of Diagnostic Sensitivity and Diagnostic Specificity Evaluation Data Calculation of Diagnostic Sensitivity and Diagnostic Specificity Conclusion Analytical Sensitivity (Detection Limit) Interferences Imprecision Within-run Imprecision (Intra-Assay) Between-run Imprecision (Inter-Assay) Between-lot Imprecision (Inter-Lot) Linearity Standardisation Stability Conclusion... 6 Form: /6 valid of

2 1 Intended Use IMTEC-TSH Receptor-Antibodies is an enzyme immunoassay (ELISA) for the quantitative measurement of autoantibodies against TSH receptor in human serum. The assay is intended for in vitro diagnostic use only as an aid in the diagnosis of Graves disease. Thyroid dysfunction may have a variable clinical presentation. Laboratory tests help to determine thyroid function and to differentiate common causes for hypothyroidism and hyperthyroidism. Low or undetectable TSH levels are diagnostic for hyperthyroidism and may be caused by Graves Disease, toxic multinodular goiter (MNG), toxic adenoma and subacute thyroiditis. Graves Disease is the most common cause of hyperthyroidism, accounting for 60 to 80 percent of all cases. Hyperthyroidism in Graves disease is attributable to autoantibodies to the thyroid-stimulating hormone receptor (TSHR), and measurement of these TSHR autoantibodies can help in the diagnosis and management of the disease. Second generation TSHR antibody assays measure the ability to inhibit receptor binding of TSH and are a more sensitive and specific alternative to first generation assays. The IMTEC-TSH Receptor-Antibodies ELISA is a nonisotopic and automatable second generation assay. 1. Rees Smith B. et al., A new assay for thyroid receptor, Thyoid : Kamijo K.,TSH receptor antibody measurement [ ], Endocrine J 50, (2003) 2. Bolton et al., Measurement of thyroid stimulating hormone receptor autoantibodies by ELISA, Clin. Chem. 45, (1999) 2 Diagnostic Sensitivity and Diagnostic Specificity 2.1 Determination of Diagnostic Sensitivity and Diagnostic Specificity Diagnostic sensitivity and diagnostic specificity were calculated according to DIN : 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 Design Verification and Product Data for IMTEC-TSH Receptor-Antibodies 2/6

3 2.1.1 Evaluation Data A total of 154 apparently healthy donors, 35 Disease controls and 50 patients with Graves disease were assayed. The following table summarises the results. Control and Disease groups Total number Negative < 1.0 IU/L Equivocal IU/L Positive 1.5 IU/L Blood Donors Graves Disease Calculation of Diagnostic Sensitivity and Diagnostic Specificity Diagnostic Sensitivity (Graves Disease) = 49/50 = 98 % Diagnostic Specificity (Blood Donors) = 152/154 = 98.7 % Conclusion The data support a sensitivity of 98 % and a specificity > 95 % for the IMTEC-TSH Receptor-Antibodies ELISA. 2.2 Analytical Sensitivity (Detection Limit) The analytical sensitivity (detection limit [IU/L]) was determined using the negative control. The absolute mean was obtained from 32 determinations. The detection limit of 0.21 IU/L was calculated from the absolute mean plus two standard deviations. Design Verification and Product Data for IMTEC-TSH Receptor-Antibodies 3/6

4 3 Interferences No interferences was observed by spiking three low positive samples with bilirubin at 0.2 mg/ml, hemoglobin at 5 mg/ml, lipid at 30 mg/dl and TSH at 3.35 mu/ml. The following table lists the tested substances in their final concentration which has been set to a level above physiologically relevant concentration. Substance Bilirubin Hemoglobin Lipids TSH 0.2 mg/ml 5.0 mg/ml 30 mg/dl 3.35 mu/ml 4 Imprecision 4.1 Within-run Imprecision (Intra-Assay) For determination of the intra-assay imprecision two controls were assayed twenty-five times each in one assay. 1 2 IU/l IU/l Mean SD CV [%] The obtained data confirm an intra-assay reproducibility of CV %. 4.2 Between-run Imprecision (Inter-Assay) For determination of the inter-assay imprecision two controls were assayed in single determination on twenty different runs. 1 2 IU/l IU/l Mean SD CV [%] The obtained data confirm an inter-assay reproducibility of CV %. 4.3 Between-lot Imprecision (Inter-Lot) For determination of the inter-lot imprecision four controls were assayed twenty times each in twenty batches IU/l IU/l IU/l IU/l Mean SD CV [%] The obtained data confirm an inter-lot reproducibility of CV %. Design Verification and Product Data for IMTEC-TSH Receptor-Antibodies 4/6

5 5 Linearity For determination of the linearity one positive sample was diluted using a negative pool and assayed in the IMTEC- TSH Receptor-Antibodies ELISA. The assay shows linearity within the measuring range. Sample Dilution Observed Mean Expected Mean Recovery [O/E] 1 1 in % 1 in % 1 in % 1 in % 1 in % 1 in % 1 in % 1 in % 1 in % 1 in % 6 Standardisation The IMTEC-TSH Receptor-Antibodies ELISA is calibrated against the 1 st international reference preparation for thyroid stimulating antibodies (NIBSC 90/672). Batch NIBSC 90/672 standard Observed Mean Recovery [O/N] % % % % % % % % Design Verification and Product Data for IMTEC-TSH Receptor-Antibodies 5/6

6 7 Stability The stability of the IMTEC-TSH Receptor-Antibodies ELISA has been evaluated by real time studies of all test kit components. A function test was applied with the whole kit employing all standards and controls. LOT 2ATERK358A: Time/ Sample 0 months 6 months 15 months Absorbance U/L Absorbance U/L Absorbance U/L LP MP HP LOT 2ATERK359: Time /Sample 0 months 6 months 15 months Absorbance U/L Absorbance U/L Absorbance U/L LP MP HP LOT 2ATERK360B: Time /Sample 0 months 6 months 15 months Absorbance U/L Absorbance U/L Absorbance U/L LP MP HP Conclusion The stability data support the shelf life claim of 14 months after production. Design Verification and Product Data for IMTEC-TSH Receptor-Antibodies 6/6