Comparison of the titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test to determine the breakthrough titres to be used in the IDvet ELISA

Transcription

1 Comparison of the titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test to determine the breakthrough titres to be used in the IDvet ELISA Final Report No part of this report may be copied or reproduced without permission from GD Author: Dr. J.J. de Wit Contract partner: ID.vet Project manager: A. Kolkman Final approval by: Dr. J.J. de Wit Date: Funded by: ID.vet

2 Table of contents 1. SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS IDEXX batches used at GD Titres Sequential sera Decrease in MDA in time and calculations of the breakthrough titres DISCUSSION CONCLUSIONS AND RECOMMENDATIONS LITERATURE ANNEXES p. 2 of 14

3 1. Summary The aim of the study was to compare the titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test to determine the breakthrough titres to be used in the IDvet ELISA (IDEXX was considered as gold standard). The comparison of the titres of both ELISAs showed a high correlation between the titres of both tests This was also the case for the sequential sera of young, non-vaccinated broilers. The comparison showed that the breakthrough titres of the IDvet ELISA are somewhat lower than in IDEXX. Due to the high correlation in titres, it was shown that the Deventer formula can well be used by the IDvet ELISA using its own breakthrough titres and the same half-life times as those used by the IDEXX ELISA for the different types of chickens. 2. Introduction Gumboro disease is an important threat for the health of poultry. For this reason, a very high percentage of the chickens are vaccinated against infectious bursal disease virus. The vaccines are applied in the hatchery by injection (vector-vaccines and immune-complex vaccines) or in the field by a drinking water vaccination. The timing of these vaccinations is critical as the maternally derived antibodies (MDA) can neutralize the vaccine (Van den Berg & Meulemans, 1991; Tsukamoto et al., 1995; De Wit & van Loon, 1998; Alam et al., 2002; Hair-Bejo et al., 2004; McCarty et al., 2005; Moraes et al., 2005; Rautenschlein et al., 2005; Block et al., 2007). The amount of MDA that can be overcome by a vaccine is the so-called breakthrough titre of this vaccine. These titres are vaccine and ELISA specific. After measuring the level and variation of the MDA in the flock, the optimal day of vaccination can be determined using an appropriate formula (De Wit, 1998; Block et al., 2007). The aim of the study was to compare the titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test to determine the breakthrough titres to be used in the IDvet ELISA (IDEXX is gold standard). 3. Materials and Methods GD Animal Health collected 261 sera of chickens to be tested in the IDEXX IBDV antibody ELISA and the IDvet IBDV antibody ELISA. Each serum was tested in two different IDEXX batches by GD Animal Health (according to the ISO accredited protocol using two batches that were accepted according to the acceptance criteria of GD) and afterwards sent to IDvet to perform the IDvet ELISA on each serum in two different batches. GD Animal Health compared the results and titres to determine the breakthrough titres that can be used in the IDvet ELISA (IDEXX is gold standard). 99 sera were sequential sera from a group of slow-growing broilers to be able to compare the half-life time measured by both ELISAs. For the comparison, the mean titre in both batches of each ELISA were used. Separate comparisons were made for the sequential sera and for the sera that were positive in both ELISAs. ELISAs: ID Screen IBD Indirect, batches 950 and A04 IDEXX IBD Ab test, batches CL361 and LL477 p. 3 of 14

4 4. Results 4.1. IDEXX batches used at GD Two different batches of the IDEXX ELISA were used: CL361 and LL477. Both batches fulfilled the requirements of the batch controls before acceptance (accredited according to ISO 17025). The performance of these two batches appeared to be very similar to the other four batches that have been used by GD in 2015 and The average titre of the internal positive control samples that is used on every plate for the six batches was 12.3 (log2). Batch CL361 (first one) scored on average 12.3 and batch LL477 scored on average Titres All results of all sera are listed in tables 2a, 2b, 2c, 2d, and 2e (See Annexes). The correlation between the titres of both tests was determined using the normal titres and log2 titres. The correlation between the titres of both ELISAs was high. The correlation coefficient was highest with the normal titres (Figure 1 to 6). For the sequential sera, the correlation coefficients were similar for both methods. Therefore, these titres were used for the determination of the breakthrough titres. IDEXX titres were on average about 40% higher than the IDvet titres. Figure 1. Correlation of titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test using all 261 sera p. 4 of 14

5 Figure 2. Correlation of log2 titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test using all 261 sera IDvet IBDV log2 titre 15,0 14,0 13,0 12,0 11,0 10,0 9,0 8,0 7,0 Correlation IDEXX and IDvet IBDV 2log titres of all 261 sera y = 0,7624x + 2,2617 R² = 0,8751 6,0 6,0 8,0 10,0 12,0 14,0 IDEXX IBDV log2 titre Figure 3. Correlation of titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test using the 246 sera that were positive in both ELISAs p. 5 of 14

6 Figure 4. Correlation of log2 titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test using the 246 sera that were positive in both ELISAs 15,0 Correlation IDEXX and IDvet IBDV 2log titres (positive sera for both tests only) IDvet IBDV log2 titre 14,0 13,0 12,0 11,0 10,0 y = 0,7404x + 2,538 R² = 0,8432 9,0 8,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0 IDEXX IBDV log2 titre 4.3. Sequential sera Figure 5. Correlation of titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test using 99 sequential sera p. 6 of 14

7 Figure 6. Correlation of log2 titres of the ID Screen IBD Indirect ELISA and IDEXX IBD Ab test using 99 sequential sera with maternally derived antibodies 15,0 14,0 Correlation IDEXX and IDvet IBDV 2log titres sequential MDA sera IDvet IBDV log2 titre 13,0 12,0 11,0 10,0 9,0 8,0 y = 0,7824x + 2,2065 R² = 0,91 7,0 6,0 6,0 8,0 10,0 12,0 14,0 IDEXX IBDV log2 titre 4.4. Decrease in MDA in time and calculations of the breakthrough titres In figure 7 and 8 the decrease in MDA titres in time is shown for both ELISAs. As can be expected, the log2 titres show a linear decrease in antibodies, therefore, the log2 titres of the sequential sera were used to determine the breakthrough titres for the IDvet ELISA. Figure 7. Mean titres in time for the sequential sera of the slow growing broilers mean MDA titres Mean MDA titres in IDEXX and IDvet IBDV ELISA in sequential sera of slow-growing broilers age (in days) ID-vet mean IDEXX mean p. 7 of 14

8 Figure 8. Mean log2 titres in time for the sequential sera of the slow growing broilers. 14,00 Mean MDA log 2 titres in IDEXX and IDvet IBDV ELISA in sequential sera of slow-growing broilers mean MDA log2 titres 13,00 12,00 11,00 10,00 9,00 ID-vet mean IDEXX mean 8, age (in days) Table 1 shows the comparison of MDA titres in both ELISAs using the [IDvet = 0,7824 x IDEXX + 2,2065] (Figure 6) that can be used to determine the breakthrough titres of both ELISAs. Table 1. Comparison of MDA titres (breakthrough titres) Comparison MDA titres IDEXX IDvet (intermediate) (intermediate plus) (hot) p. 8 of 14

9 5. Discussion The correlation between titres of both ELISAs was high which makes it possible to produce a reliable estimation of the breakthrough titres to be used in the IDvet ELISA. These titres are somewhat lower in the IDvet ELISA compared to the IDEXX ELISA. Based on the experience of IDvet, comparable titres were expected. Based on the IDEXX results at GD, it would be recommended to use lower breakthrough titres for the IDvet ELISA (intermediate 100, intermediate plus around 400, and 500 for hot vaccines). However, when IDvet would conclude on multiple comparisons using more batches that IDvet titers and IDEXX titers are very similar, IDvet might still decide to use the same breakthrough titres as IDEXX. It is important to realize that the difference in the recommended day of vaccination for broilers will only be one day which is not a major problem between the two breakthrough titres for the same vaccine. 6. Conclusions and recommendations The comparison of the titres of the IDEXX IBDV antibody ELISA and the IDvet IBDV antibody ELISA showed a high correlation between the titres of both tests. This was also the case for the sequential sera of young, non-vaccinated broilers. The comparison showed that the breakthrough titres of the IDvet ELISA are somewhat lower than in IDEXX. Due to the high correlation in titres, it shows that the Deventer formula can well be used by the IDvet ELISA using its own breakthrough titres and the same half-life times as those used by the IDEXX ELISA for the different types of chickens. 7. Literature Alam, J., Rahman, M.M., Sil, B.K., Khan, M.S.R., Giasuddin & Sarker, M.S.K. (2002). Effect of maternally derived antibody on vaccination against infectious bursal disease (Gumboro) with live vaccine in broiler.. International Journal of Poultry Science, 1, Block, H., Meyer-Block, K., Rebeski, D.E., Scharr, H., de Wit, S., Rohn, K. & Rautenschlein, S. (2007). A field study on the significance of vaccination against infectious bursal disease virus (IBDV) at the optimal time point in broiler flocks with maternally derived IBDV antibodies. Avian Pathology, 36, De Wit, J.J. (1998). Gumboro disease: estimation of optimal time of vaccination by the Deventer formula. Polish Veterinary Journal, 3, De Wit, J.J. & van Loon, A.A. (1998). Gumboro vaccination. Tijdschrift voor Diergeneeskunde, 123, Hair-Bejo, M., Ng, M.K. & Ng, H.Y. (2004). Day old vaccination against infectious bursal disease in broiler chickens. International Journal of Poultry Science, 3, McCarty, J.E., Brown, T.P. & Giambrone, J.J. (2005). Delay of infectious bursal disease virus infection by in ovo vaccination of antibody positive chicken eggs. Journal of Applied Poultry Research, 14, Moraes, H.L.S., Salle, C.T.P., Nascimento, V.P., Salle, F.O., Rocha, A.C.G.T., Souza, G.F., Furian, T.Q. & Artencio, J.O. (2005). Infectious bursal disease: evaluation of maternal immunity and protection by vaccination of one-day old chicks against challenge with very virulent virus isolate. Brazilian Journal of Poultry Science, 7, Rautenschlein, S., Kraemer, C., Vanmarcke, J. & Montiel, E. (2005). Protective efficacy of intermediate and intermediate plus infectious bursal disease virus (IBDV) vaccines against very virulent IBDV in commercial broilers. Avian Dis, 49, Tsukamoto, K., Tanimura, N., Kakita, S., Ota, K., Mase, M., Imai, K. & Hihara, H. (1995). Efficacy of three live vaccines against highly virulent infectious bursal disease virus in chickens with or without maternal antibodies. Avian Diseases, 39, Van den Berg, T.P. & Meulemans, G. (1991). Acute infectious bursal disease in poultry: protection afforded by maternally derived antibodies and interference with live vaccination. Avian Pathology, 20, p. 9 of 14

10 8. Annexes Table 2a. Results of ID Screen IBD Indirect and IDEXX IBD Ab ELISA on test panel, samples 1-45 IDvet ELISA performed by IDvet IDEXX by IDvet IDEXX by GD Batch 950 Batch A04 Batch LL477 First batch second batch DO S/P Titers DO S/P Titers DO S/P Titers Titers Titers Block Day C - 0,106 0,00 0 0, ,049 0,00 0 C + 0,518 0, , ,287 1, Animal 01 1,851 3, ,957 3, ,543 2, Animal 02 0,922 1, ,034 1, ,345 1, Animal 03 1,323 2, ,527 2, ,490 1, Animal 04 1,476 2, ,744 2, ,526 2, Animal 05 1,379 2, ,671 2, ,585 2, Animal 06 1,346 2, ,601 2, ,548 2, Animal 07 1,339 2, ,341 2, ,484 1, Animal 08 1,305 2, ,376 2, ,456 1, Animal 09 0,693 1, ,603 0, ,213 0, Animal 10 0,904 1, ,838 1, ,359 1, Animal 11 1,096 1, ,130 1, ,491 1, Animal 12 1,004 1, ,059 1, ,408 1, Animal 13 1,135 1, ,111 1, ,599 2, Animal 14 1,082 1, ,022 1, ,442 1, Animal 15 0,807 1, ,748 1, ,343 1, Animal 16 0,957 1, ,964 1, ,413 1, Animal 17 1,229 2, ,088 1, ,379 1, Animal 18 0,980 1, ,863 1, ,303 1, Animal 19 0,997 1, ,895 1, ,374 1, Animal 20 1,469 2, ,354 2, ,642 2, Animal 21 0,909 1, ,817 1, ,409 1, Animal 22 1,167 1, ,102 1, ,535 2, Animal 23 0,709 1, ,708 0, ,270 0, Animal 24 0,583 0, ,538 0, ,226 0, Animal Animal 26 0,782 1, ,843 1, ,275 0, Animal 27 0,720 1, ,775 1, ,223 0, Animal 28 0,832 1, ,920 1, ,233 0, Animal 29 0,746 1, ,810 1, ,223 0, Animal 30 0,707 1, ,836 1, ,261 0, Animal 31 0,617 0, ,686 0, ,208 0, Animal 32 0,360 0, ,469 0, ,129 0, Animal 33 0,847 1, ,927 1, ,265 0, Animal 34 0,679 1, ,766 1, ,207 0, Animal 35 0,716 1, ,885 1, ,265 0, Animal 36 0,945 1, ,089 1, ,349 1, Animal 37 0,507 0, ,605 0, ,166 0, Animal 38 0,597 0, ,749 1, ,210 0, Animal 39 0,893 1, ,052 1, ,343 1, Animal 40 0,901 1, ,067 1, ,309 1, Animal 41 0,766 1, ,835 1, ,262 0, Animal 42 0,877 1, ,869 1, ,283 0, Animal 43 0,658 0, ,707 0, ,227 0, Animal 44 0,475 0, ,518 0, ,145 0, Animal 45 0,481 0, ,628 0, ,158 0, p. 10 of 14

11 Table 2b. Results of ID Screen IBD Indirect and IDEXX IBD Ab ELISA on test panel, samples IDvet ELISA performed by IDvet IDEXX by IDvet IDEXX by GD Batch 950 Batch A04 Batch LL477 First batch second batch DO S/P Titers DO S/P Titers DO S/P Titers Titers Titers Block Day C - 0,106 0,00 0 0, ,049 0,00 0 C + 0,518 0, , ,287 1, Animal 46 0,413 0, ,509 0, ,114 0, Animal 47 0,436 0, ,515 0, ,145 0, Animal 48 0,520 0, ,653 0, ,169 0, Animal 49 0,541 0, ,402 0, ,127 0, Animal 50 0,518 0, ,370 0, ,146 0, Animal 51 0,525 0, ,408 0, ,152 0, Animal 52 0,607 0, ,559 0, ,182 0, Animal 53 0,684 1, ,594 0, ,186 0, Animal 54 0,661 0, ,552 0, ,195 0, Animal 55 0,741 1, ,639 0, ,205 0, Animal 56 0,512 0, ,457 0, ,170 0, Animal 57 0,662 0, ,782 1, ,264 0, Animal 58 0,455 0, ,562 0, ,157 0, Animal 59 0,209 0, ,214 0, ,088 0, Animal 60 0,670 1, ,788 1, ,291 1, Animal 61 0,498 0, ,547 0, ,173 0, Animal 62 0,268 0, ,277 0, ,081 0, Animal 63 0,390 0, ,418 0, ,157 0, Animal 64 0,307 0, ,333 0, ,107 0, Animal 65 0,436 0, ,325 0, ,126 0, Animal 66 0,507 0, ,397 0, ,158 0, Animal 67 0,396 0, ,392 0, ,149 0, Animal 68 0,428 0, ,434 0, ,148 0, Animal 69 0,468 0, ,408 0, ,161 0, Animal 70 0,368 0, ,326 0, ,131 0, Animal 71 0,374 0, ,333 0, ,119 0, Animal 72 0,323 0, ,305 0, ,107 0, Animal 73 0,349 0, ,245 0, ,103 0, Animal 74 0,408 0, ,331 0, ,114 0, Animal 75 0,300 0, ,246 0, ,096 0, Animal 76 0,397 0, ,356 0, ,132 0, Animal 77 0,251 0, ,229 0, ,074 0, Animal 78 0,392 0, ,396 0, ,138 0, Animal 79 0,359 0, ,349 0, ,122 0, Animal 80 0,354 0, ,340 0, ,129 0, Animal 81 0,511 0, ,411 0, ,116 0, Animal 82 0,423 0, ,352 0, ,098 0, Animal 83 0,253 0, ,219 0, ,069 0, Animal 84 0,322 0, ,277 0, ,083 0, Animal 85 0,374 0, ,349 0, ,112 0, Animal 86 0,371 0, ,404 0, ,120 0, Animal 87 0,292 0, ,296 0, ,082 0, Animal 88 0,281 0, ,292 0, ,091 0, Animal 89 0,323 0, ,272 0, ,088 0, Animal 90 0,208 0, ,240 0, ,087 0, Animal 91 0,142 0, ,168 0, ,065 0, Animal 92 0,314 0, ,326 0, ,166 0, Animal 93 0,232 0, ,281 0, ,122 0, Animal 94 0,205 0, ,228 0, ,101 0, Animal 95 0,162 0, ,157 0, ,069 0, Animal 96 0,204 0, ,211 0, ,078 0, Animal 97 0,222 0, ,245 0, ,112 0, Animal 98 0,157 0, ,197 0, ,074 0, Animal 99 0,245 0, ,290 0, ,112 0, Animal 100 0,131 0, ,155 0, ,058 0, p. 11 of 14

12 Table 2c. Results of ID Screen IBD Indirect and IDEXX IBD Ab ELISA on test panel, samples IDvet ELISA performed by IDvet IDEXX by IDvet IDEXX by GD Batch 950 Batch A04 Batch LL477 First batch second batch DO S/P Titers DO S/P Titers DO S/P Titers Titers Titers Block Day C - 0,106 0,00 0 0, ,049 0,00 0 C + 0,518 0, , ,287 1, B7 1,002 1, ,956 1, ,484 1, B8 0,831 1, ,797 1, ,457 1, B9 1,821 3, ,781 2, ,876 3, B10 2,012 3, ,846 2, ,782 3, A1 0,560 0, ,751 1, ,320 1, A2 1,383 2, ,764 2, ,677 2, A3 1,435 2, ,528 2, ,613 2, A4 1,071 1, ,266 1, ,657 2, A5 0,920 1, ,235 1, ,711 2, A6 0,900 1, ,340 2, ,565 2, A7 1,091 1, ,249 1, ,716 2, A8 1,270 2, ,308 1, ,514 1, A9 1,907 3, ,932 3, ,799 3, A10 0,535 0, ,650 0, ,454 1, B1 0,552 0, ,726 1, ,507 1, B2 0,686 1, ,926 1, ,588 2, B3 1,054 1, ,303 1, ,799 3, B4 0,799 1, ,894 1, ,527 2, B5 0,633 0, ,719 0, ,367 1, B6 1,785 2, ,761 2, ,963 3, A1 0,877 1, ,750 1, ,360 1, A2 0,817 1, ,757 1, ,447 1, A3 0,920 1, ,907 1, ,550 2, A4 0,895 1, ,950 1, ,544 2, A5 0,768 1, ,774 1, ,487 1, A6 1,206 1, ,194 1, ,643 2, A7 1,530 2, ,461 2, ,798 3, A8 1,088 1, ,921 1, ,615 2, A9 0,731 1, ,750 1, ,427 1, A10 1,135 1, ,286 1, ,650 2, A11 0,782 1, ,948 1, ,492 1, A12 0,428 0, ,518 0, ,693 2, B1 1,381 2, ,447 2, ,697 2, B2 1,144 1, ,273 1, ,657 2, B3 1,038 1, ,238 1, ,595 2, B4 2,372 4, ,931 4, ,132 4, B5 0,717 1, ,669 0, ,272 0, B6 1,936 3, ,859 2, ,026 4, B7 1,377 2, ,369 2, ,662 2, B8 1,799 3, ,065 3, ,873 3, B9 0,882 1, ,949 1, ,428 1, B10 0,878 1, ,880 1, ,412 1, B11 2,093 3, ,984 3, ,858 3, B12 1,067 1, ,982 1, ,459 1, C1 0,930 1, ,903 1, ,419 1, C2 0,708 1, ,706 0, ,320 1, C3 1,125 1, ,206 1, ,517 1, C4 0,688 1, ,707 0, ,324 1, C5 0,639 0, ,667 0, ,331 1, C6 0,829 1, ,797 1, ,414 1, C7 1,135 1, ,132 1, ,505 1, C8 1,402 2, ,324 2, ,610 2, C9 1,044 1, ,107 1, ,436 1, C10 1,066 1, ,762 1, ,490 1, p. 12 of 14

13 Table 2d. Results of ID Screen IBD Indirect and IDEXX IBD Ab ELISA on test panel, samples IDvet ELISA performed by IDvet IDEXX by IDvet IDEXX by GD Batch 950 Batch A04 Batch LL477 First batch second batch DO S/P Titers DO S/P Titers DO S/P Titers Titers Titers Block Day C - 0,106 0,00 0 0, ,049 0,00 0 C + 0,518 0, , ,287 1, C11 0,770 1, ,647 0, ,487 1, C12 0,991 1, ,882 1, ,584 2, D1 0,709 1, ,658 0, ,353 1, D2 1,011 1, ,914 1, ,565 2, D3 0,982 1, ,896 1, ,503 1, D4 1,078 1, ,908 1, ,535 2, D5 0,851 1, ,779 1, ,391 1, D6 0,671 1, ,643 0, ,329 1, D7 0,595 0, ,589 0, ,301 1, D8 0,287 0, ,369 0, ,181 0, D9 0,567 0, ,595 0, ,294 1, D10 0,855 1, ,857 1, ,440 1, D11 1,041 1, ,014 1, ,484 1, D12 0,894 1, ,756 1, ,414 1, E1 2,456 4, ,002 3, ,920 3, E2 2,488 4, ,122 3, ,068 4, E3 2,513 4, ,222 3, ,116 4, E4 1,828 3, ,363 2, ,795 3, E5 1,196 1, ,883 1, ,913 3, E6 1,877 3, ,438 2, ,831 3, E7 1,414 2, ,067 1, ,525 2, E8 2,866 4, ,783 4, ,047 4, E9 2,349 4, ,770 4, ,917 3, E10 2,168 3, ,178 3, ,856 3, E11 0,908 1, ,919 1, ,351 1, E12 2,289 3, ,740 4, ,031 4, F1 0,485 0, ,552 0, ,180 0, F2 1,131 1, ,318 1, ,508 1, F3 2,269 3, ,799 4, ,811 3, F4 0,507 0, ,578 0, ,220 0, F5 1,290 2, ,387 2, ,479 1, F6 1,034 1, ,037 1, ,306 1, F7 0,880 1, ,789 1, ,304 1, F8 0,919 1, ,033 1, ,363 1, F9 1,383 2, ,322 2, ,531 2, F10 0,542 0, ,652 0, ,232 0, A1 0,477 0, ,347 0, ,206 0, A2 0,454 0, ,394 0, ,151 0, A3 0,594 0, ,513 0, ,209 0, A5 0,366 0, ,296 0, ,139 0, A6 0,437 0, ,404 0, ,184 0, A7 0,472 0, ,492 0, ,207 0, A8 0,732 1, ,588 0, ,326 1, A10 0,382 0, ,372 0, ,190 0, B4 0,294 0, ,294 0, ,141 0, B6 0,430 0, ,402 0, ,215 0, B7 0,464 0, ,469 0, ,190 0, B8 0,449 0, ,578 0, ,223 0, B9 0,419 0, ,530 0, ,173 0, B10 0,313 0, ,341 0, ,151 0, p. 13 of 14

14 Table 2e. Results of ID Screen IBD Indirect and IDEXX IBD Ab ELISA on test panel, samples IDvet ELISA performed by IDvet IDEXX by IDvet IDEXX by GD Batch 950 Batch A04 Batch LL477 First batch second batch DO S/P Titers DO S/P Titers DO S/P Titers Titers Titers Block Day C - 0,106 0,00 0 0, ,049 0,00 0 C + 0,518 0, , ,287 1, A1 0,691 1, ,664 0, ,323 1, A2 0,525 0, ,493 0, ,281 0, A3 0,441 0, ,464 0, ,281 0, A4 0,532 0, ,578 0, ,294 1, A5 0,477 0, ,506 0, ,279 0, A6 0,607 0, ,630 0, ,369 1, A7 1,580 2, ,669 2, ,891 3, A8 0,565 0, ,486 0, ,325 1, A9 0,473 0, ,431 0, ,253 0, A10 0,360 0, ,408 0, ,346 1, B1 0,522 0, ,586 0, ,378 1, B2 0,605 0, ,649 0, ,473 1, B3 0,387 0, ,471 0, ,296 1, B4 0,439 0, ,484 0, ,295 1, B6 0,546 0, ,526 0, ,417 1, B7 0,454 0, ,448 0, ,251 0, B8 0,385 0, ,466 0, ,270 0, B9 0,487 0, ,577 0, ,344 1, B10 6 C1 0,358 0, ,414 0, ,283 0, C2 0,509 0, ,529 0, ,451 1, C3 0,660 0, ,690 0, ,483 1, C4 0,584 0, ,524 0, ,436 1, C5 0,989 1, ,107 1, ,570 2, C6 0,782 1, ,852 1, ,426 1, C7 0,655 0, ,599 0, ,406 1, C8 0,504 0, ,571 0, ,250 0, C9 0,719 1, ,704 0, ,510 1, C10 1,124 1, ,133 1, ,661 2, D1 0,340 0, ,341 0, ,205 0, D2 0,578 0, ,502 0, ,298 1, D3 1,701 2, ,780 2, ,762 3, D4 0,586 0, ,666 0, ,321 1, D5 0,442 0, ,540 0, ,271 0, D6 0,423 0, ,510 0, ,216 0, D7 0,558 0, ,692 0, ,379 1, D8 0,588 0, ,666 0, ,301 1, D9 0,645 0, ,570 0, ,320 1, D10 0,643 0, ,643 0, ,322 1, E1 0,507 0, ,622 0, ,331 1, E2 0,329 0, ,418 0, ,214 0, E3 0,363 0, ,499 0, ,268 0, E4 0,304 0, ,441 0, ,255 0, E5 0,522 0, ,700 0, ,377 1, E6 0,397 0, ,500 0, ,272 0, E7 0,363 0, ,479 0, ,269 0, E8 0,714 1, ,798 1, ,410 1, E9 0,770 1, ,848 1, ,448 1, E10 0,524 0, ,515 0, ,299 1, F1 0,536 0, ,545 0, ,463 1, F3 0,480 0, ,510 0, ,402 1, F4 0,487 0, ,475 0, ,355 1, F5 0,590 0, ,619 0, ,474 1, F6 0,582 0, ,560 0, ,395 1, F7 0,436 0, ,479 0, ,217 0, F8 0,349 0, ,413 0, ,215 0, F9 0,415 0, ,500 0, ,303 1, F10 0,649 0, ,702 0, ,481 1, p. 14 of 14