Design Verification. Form:

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1 Design Verification SYPHILIS TPHA liquid CONTENTS 1 Function Sensitivity... 2 Preparation of Serum Control Panel... 2 Kit Controls Positive and Negative Traceability Specificity and Sensitivity... 4 Study Study Study Cross-reactivity, Interferences Stability... 6 Study for Shelf Life Extension Lot-to-lot Consistency, Reproducibility and Stability... 7 Form: /9 Rev. 009 valid of

2 1 Function SYPHILIS TPHA liquid has been designed as an indirect haemagglutination test for the qualitative and quantitative detection of antibodies to Treponema pallidum antigen in serum. The test comprises test and control cell suspensions, positive and negative controls and a special diluent. Microtiter plates, preferably with rigid U-wells, and micropipettes/-dilutors are required but not provided with the test kit. Avian erythrocytes have been coated with T. pallidum antigen. In the presence of specific antibodies to T. pallidum the test cells agglutinate due to an antigen-antibody reaction. If no specific antibodies to T. pallidum are present, the cells will sediment rapidly. A compact button at the bottom of the wells indicates a negative result cells, while an even layer of agglutinated cells, sometimes surrounded by a more a less distinct ring, indicate a positive result. With the control cells always a well-defined button should be seen. If there is an indication for agglutination with the control cells, this implies the presence of non-specific agglutinins in the sample. In this case the sample should be first absorbed with control cells and the test repeated with the pre-adsorbed sample. Each sample positive in the qualitative test should be tested quantitatively. For this the sample is diluted stepwise. The final dilution which produces an agglutination of test cells is defined as the titer of the sample. 2 Sensitivity Description of Control Materials For sensitivity check a standard material is employed (syphilis standard, biomérieux, cat. no ). This standard material is prepared according to the instructions of the manufacturer. A stepwise dilution of the standard is performed in the microtiter wells as described below: Well 1: 100 µl diluent + 25 µl sample = 125 µl mix and transfer to well 2 Well 2: 25 µl diluent + 25 µl from well 1 = 50 µl mix and transfer to well 3 add 75 µl control cell suspension Well 3: 25 µl diluent + 25 µl from well 2 = 50 µl mix and transfer to well 4 add 75 µl test cell suspension Well 4: 25 µl diluent + 25 µl from well 3 = 50 µl proceed as above... Well X: mix and transfer 25 µl from previous well add 75 µl test cell suspension add 75 µl test cell suspension Well Titer 1 : 80 1 : : 320 1: : 1,280 1 : 2,560 1 : 5,120 1 : 10,240 The test cells should produce a visible agglutination at that dilution which equals the stated titer of the standard ± 1 dilution step. Preparation of Serum Control Panel A panel of each 8 sera, negative for anti-t. pallidum, is selected and determined by an independent assay. The panel sera are aliquoted and kept deep-frozen. Before use, the panel aliquots are thawed, mixed and employed in the test to a final dilution of 1:160. The panel sera are tested with the test and control cell suspension. The above panel produced no agglutination with both the test and control cells. Design Verification and Product Data for SYPHILIS TPHA liquid 2/9 Rev. 009

3 Kit Controls Positive and Negative The kit controls are employed for function test of control and test cells. The positive control produced a visible agglutination with the test cells at a final titer of 1:2,560 (± 1 dilution step). No agglutination is observed with the control cells. The negative control produced no agglutination with both the test and control cells at a titer of 1:40 and 1:80, respectively. 3 Traceability SYPHILIS TPHA liquid has been standardized against the WHO 1st ISP (1958). This international standard preparation has been prepared from a pool of strongly reactive sera. One ampoule has been defined to contain 49 IU. The freeze-dried content of one ampoule is redissolved with 2 ml dist. water to make a concentration of 24.5 IU/ml. The WHO 1st ISP together and available control sera and an in-house master control have been applied to 2 different lots of Syphilis TPHA. The results are summarized in the following tables. Lot: H1110 Titer Syphilis TPHA Titer (without test cell dilution) WHO biomerieux (Serotrol) LTB In-house master 1:5120 1: :2,560 pos pos pos neg 1: :5,120 pos neg neg neg 1: :10,240 neg neg neg neg Lot: H1118 Titer Syphilis TPHA Titer (without test cell dilution) WHO biomerieux (Serotrol) LTB In-house master 1:5120 1:10,240 1:2,560 pos pos pos neg 1:20,480 1:5,120 pos neg neg neg 1:40,960 1:10,240 neg neg neg neg The LTB (La Techique Biologique/Fournier) preparation is also traceable to the WHO Reference Serum for Serodiagnostic Tests for Treponemal Infections Ref Conclusion: No significant difference could be observed between 2 independent lots SYPHILIS TPHA liquid. Therefore, the sensitivity of SYPHILIS TPHA liquid can be assigned to 24.5 / 20,480 IU/ml = 1.2 x 10-3 IU/ml (in regard to the WHO 1st ISP). Contributing to the concentration of the sample (without the additional dilution effect by the test cell suspension) this makes a sensitivity of 4.79 x 10-3 IU/ml. Syphilis International Standards 05/122 and 05/132 The WHO Reference Serum for Serodiagnostic tests for Treponemal Infections- Ref has been superseded by the International Standards 05/122 and 05/132. However the reference remains valid as both 05/122 and 05/132 standards are relative to the Syphilis Standard Ref Three QC passed batches of TPHA were tested with the Syphilis International Standard 05/122 (0.3 IU per ampoule) and the Syphilis International Standard 05/132 (3 IU per ampoule) to verify the test sensitivity (detection level) against these new standards. Materials: Current biomérieux standard batch used: TPHA Negative batch used: TPHA Positive batch used: Design Verification and Product Data for SYPHILIS TPHA liquid 3/9 Rev. 009

4 : Standard/Control Expected Titre TPHA Test Cells batch TPHA Diluent batch TPHA Test Cells batch TPHA Diluent batch TPHA Test Cells batch TPHA Diluent batch Syphilis International standard 05/122 N/A 1/320 1/320 1/320 Syphilis International standard 05/132 N/A 1/ / /10240 Current biomérieux standard 1/ / / /10240 TPHA Positive Control 1/2560 +/- 1 double dilution 1/2560 1/2560 1/2560 TPHA Negative Control Negative Negative Negative Negative Interpretation Syphilis International Standard 05/122 has a concentration of 0.3IU/ml relative to 05/132 the 1st IS for human syphilitic plasma IgG and IgM. SYPHILIS TPHA liquid detects up to 9,375 x 10-4 IU/ml as defined by Syphilis International Standard 05/122 = 1/320 dilution of 0.3IU/ml Syphilis International Standard 05/132 has a concentration of 3IU/ml relative to HS, the 1st IS for human syphilitic antibodies (WHO Reference Serum for Serodiagnostic tests for Treponemal Infections- Ref ). SYPHILIS TPHA liquid detects up to x 10-4 IU/ml as defined by Syphilis International Standard 05/132 = 1/10240 dilution of 3IU/ml. 4 Specificity and Sensitivity Description of Method and Specimen The diagnostic specificity and sensitivity of SYPHILIS TPHA liquid has been determined by three external studies. SYPHILIS TPHA liquid was compared against a similar test system approved by the German Paul-Ehrlich-Institut or by a confirmatory test (FTA-ABS). Fresh patient sera have been employed in the studies. If direct measurement was not possible, sera have been kept deep-frozen at <-20 C. Sera from confirmed Syphilis positive and -negative persons, potentially cross-reacting sera and confirmed negative sera were employed. Positive sera have been classified, whereever possible in primary/secondary syphilis infection, untreated and treated syphilis. Additionally, in one laboratory a panel of 50 selected sera was quantitatively determined with SYPHILIS TPHA liquid and a TPHA test employed in the routine of this laboratory. The results of the individual studies are summarised below. Study 1 Competitive Syphilis TPHA test SYPHILIS TPHA liquid From the above table a diagnostic sensitivity of 95.6% can be calculated while the diagnostic specificity comes up to 100%. The overall agreement is 97.8%. Study 2 FTA-ABS confirmatory test SYPHILIS TPHA liquid Compared to the confirmatory test FTA-ABS, SYPHILIS TPHA liquid yielded a diagnostic sensitivity of 92.7% and a diagnostic specificity of 100%. The overall agreement is 92.9%. Only one potentially cross-reacting serum showed a weak positive result with SYPHILIS TPHA liquid and a negative one with the comparison test. The FTA-ABS test also gave a positive result with this sample. No other indications of cross-reactivities could be obtained from the above studies. Design Verification and Product Data for SYPHILIS TPHA liquid 4/9 Rev. 009

5 Study 3 FTA-ABS confirmatory test SYPHILIS TPHA liquid Of the 3 false negative results, two sera were from patients with primary or secondary syphilis, 1 serum was from a patient with untreated syphilis. In all three cases the results with the FTA-ABS confirmatory test were low. The above results yield an overall diagnostic sensitivity of 98.5% and specificity of 100%. In respect to primary / secondary syphilis infection or untreated syphilis the sensitivities were slightly lower, giving 93.3% and 97.4%, respectively. With samples from patients with treated syphilis the sensitivity was 100%. Quantitative determination No. TPHA in-house SYPHILIS TPHA liquid 1 20,480 20, ,120 2, ,280 1, ,120 5, ,280 1, ,280 1, ,280 1, ,120 5, ,560 2, No. TPHA in-house SYPHILIS TPHA liquid 26 1,280 1, >1,280 1, >20,480 >20, >1,280 1, , ,120 5, >1,280 2, >1,280 1, ,120 5, , The quantitative determination showed a very good agreement between the existing in-house method and SYPHILIS TPHA liquid. 5 Cross-reactivity, Interferences Selected serums from patients with Lyme disease (Borrelia antibodies), leptospirosis, EBV, HBV, HSV and SLE have been employed to confirm the specificity of the assay. None of the tested specimens reacted positive. Thus, a crossreaction with these diseases could be excluded. By testing serums containing rheumatoid factors an interference with RF positive specimens could be excluded for concentrations up to 1,074 IU/ml. Potentially interfering substances, such as bilirubin, hemoglobin and triglycerides have been added to the TPHA positive control and to the TPHA negative control. Design Verification and Product Data for SYPHILIS TPHA liquid 5/9 Rev. 009

6 Substance Concentration up to Result with negative control Result with positive control Bilirubin 40 mg/dl negative positive Hemoglobin 500 mg/dl negative positive Triglycerides 1,000 mg/dl negative positive Conclusion: SYPHILIS TPHA liquid showed no cross-reactivity with selected serum specimens and no interferences with RF positive specimens, with bilirubin, hemoglobin and triglycerides within the tested concentration range. 6 Stability The stability of SYPHILIS TPHA liquid has been demonstrated in real-time stability studies and was checked by temperature stress studies on each produced lot. A function tests on the finalized kit has been performed using standard materials and kit controls: SYPHILIS TPHA liquid, #060; Date of manufacturing: ; Expiry: :80 1:160 1:320 1:640 1:1,280 1:2,560 1:5,120 1:10,240 1:20,480 control 1 control 2 control 3 pos ± control 4 pos ± control 5 standard standard standard standard standard SYPHILIS TPHA liquid #062; Date of manufacturing: ; Expiry: :80 1:160 1:320 1:640 1:1,280 1:2,560 1:5,120 1:10,240 1:20,48 0 control 1 control 2 control 3 pos control 4 pos ± control 5 standard standard standard standard standard Note: 1 initial result; 2 after 4 months; 3 after 13 months; 4 after 16 months; 5 after 19 months The above results from 2 independent production batches stored for 19 months and checked at suitable intervals clearly demonstrate the stability of the SYPHILIS TPHA liquid and support the claimed stability of 18 months. Design Verification and Product Data for SYPHILIS TPHA LIQUID 6/9 Rev. 006

7 Study for Shelf Life Extension In order to evaluate an extension of the shelf life, 5 different lots of SYPHILIS TPHA liquid have been tested between 23 and 28 months from the date of manufacturing. A function test has been performed using the positive and negative kit controls. The negative control cells showed with all lots negative test results. The results with the positive control are summarized in the following table. Lot Expiry Month 1:80 1:160 1:320 1:640 1:1,280 1:2,560 1:5,120 1:10,240 H pos pos pos pos pos pos pos. ± H pos pos pos pos pos pos pos H pos pos pos pos pos pos ± H pos pos pos pos pos pos ± H pos pos pos pos pos pos ± 0 The above mentioned results demonstrate excellent product stability and clearly support an extension of the shelf life to 22 months from the date of manufacturing. 7 Lot-to-lot Consistency, Reproducibility and Stability The quality control results from 6 independent lots have been compared to identify lot-to-lot consistency and reproducibility. Additional results from temperature stress tests performed during quality control are taken for stability evaluation. The overall results are summarized in the following tables. Kit:H1119 Initial Stress (7d@ 37 C) Exp.: biomérieux biomérieux Titer #73701 H1119 #73701 H1119 1:10,240 equiv. neg neg neg Kit: H1116 Initial Stress (7d@ 37 C) Exp.: biomérieux biomérieux #73701 H1116 #73701 H1116 1:10,240 equiv. neg equiv. neg Design Verification and Product Data for SYPHILIS TPHA LIQUID 7/9 Rev. 006

8 Kit:H1118 Initial Stress 37 C) Exp.: biomérieux biomérieux #73701 H1118 #73701 H1118 1:10,240 pos equiv. pos equiv. Kit: H1115 Initial Stress 37 C) Exp.: biomérieux biomérieux #72191 H1115 #72191 H1115 1:10,240 equiv. equiv. equiv. equiv. Kit: H1105 Initial Stress 37 C) Exp.: biomérieux biomérieux #72881 H1105 #72881 H1105 1:10,240 equiv. equiv. equiv. equiv. Kit: H1107 Initial Stress 37 C) Exp.: biomérieux biomérieux #72191 H1107 #72191 H1107 1:10,240 equiv. equiv. equiv. equiv. Design Verification and Product Data for SYPHILIS TPHA LIQUID 8/9 Rev. 006

9 Conclusion: The above results from 6 independent production lots confirm the high degree of lot-to-lot consistency and reproducibility. All batches showed within ± one titer step identical sensitivity with the kit control and an independent commercially available control serum. The stability is further confirmed by the routine temperature stress results. Design Verification and Product Data for SYPHILIS TPHA LIQUID 9/9 Rev. 006