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4.1.4.2 Work package II: Cross reactivity ............................................................................................................................................................ 4-414.1.4.2.1 Cross reactivity in solvent ................................................................................................................................................................... 4-414.1.4.2.1.1 Study design ............................................................................................................................................................................. 4-414.1.4.2.1.2 Data handling and statistical analyses ........................................................................................................................................ 4-414.1.4.2.1.2.1 Elisa kits .................................................................................................................................................................................................... 4-414.1.4.2.1.2.2 LFD............................................................................................................................................................................................................ 4-414.1.4.2.2 Cross reactivity in matrix ................................................................................................................................................................... 4-414.1.4.2.2.1 Study design ............................................................................................................................................................................. 4-414.1.4.2.2.2 Data handling and statistical analyses ........................................................................................................................................ 4-424.1.4.3 Work package III: Matrix effect .............................................................................................................................................................. 4-424.1.4.3.1 Study design ...................................................................................................................................................................................... 4-424.1.4.3.2 Data handling and statistical analyses ................................................................................................................................................ 4-424.1.4.3.2.1.1 Elisa kits .................................................................................................................................................................................................... 4-424.1.4.3.2.1.2 LFD............................................................................................................................................................................................................ 4-444.1.4.4 Work package IV: Accuracy ................................................................................................................................................................... 4-444.1.4.4.1 Study design ...................................................................................................................................................................................... 4-444.1.4.4.2 Data handling and statistical analyses ................................................................................................................................................ 4-444.1.4.5 Work package V: Blank sample analysis................................................................................................................................................. 4-444.1.4.5.1 Study design ...................................................................................................................................................................................... 4-444.1.4.5.2 Data handling and statistical analyses ................................................................................................................................................ 4-444.1.5 Extraction protocols .................................................................................................................................................................................... 4-454.2 ROSA OCHRATOXIN GRAIN by CHARM ............................................................................................................ 4-464.2.1 Concentration accuracy of standards......................................................................................................................................................... 4-464.2.2 Cross-reactivity ............................................................................................................................................................................................. 4-464.2.2.1 Solvent .................................................................................................................................................................................................... 4-464.2.2.2 Matrix ................................................................................................................................................................................................... 4-474.2.3 Matrix effect ................................................................................................................................................................................................. 4-484.2.4 Accuracy ....................................................................................................................................................................................................... 4-494.2.5 Blank sample analysis .................................................................................................................................................................................. 4-494.2.6 Conclusions .................................................................................................................................................................................................. 4-504.3 RIDASCREEN® Ochratoxin A 30/15 by R-Biopharm ............................................................................................ 4-514.3.1 Concentration accuracy of standards......................................................................................................................................................... 4-514.3.2 Cross-reactivity ............................................................................................................................................................................................. 4-514.3.2.1 Solvent .................................................................................................................................................................................................... 4-514.3.2.2 Matrix ................................................................................................................................................................................................... 4-524.3.3 Matrix effect ................................................................................................................................................................................................. 4-534.3.4 Accuracy ....................................................................................................................................................................................................... 4-534.3.5 Blank sample analysis .................................................................................................................................................................................. 4-544.3.6 Conclusions .................................................................................................................................................................................................. 4-544.4 AgraQuant® Ochratoxin by Romerlabs ........................................................................................................................ 4-554.4.1 Concentration accuracy of standards......................................................................................................................................................... 4-554.4.2 Cross-reactivity ............................................................................................................................................................................................. 4-554.4.2.1 Solvent .................................................................................................................................................................................................... 4-554.4.2.2 Matrix ................................................................................................................................................................................................... 4-564.4.3 Matrix effect ................................................................................................................................................................................................. 4-574.4.4 Accuracy ....................................................................................................................................................................................................... 4-574.4.5 Blank sample analysis .................................................................................................................................................................................. 4-584.4.6 Conclusions .................................................................................................................................................................................................. 4-584.5 OCHRATOXIN-A ELISA by Europroxima ............................................................................................................... 4-594.5.1 Concentration accuracy of standards......................................................................................................................................................... 4-594.5.2 Cross-reactivity ............................................................................................................................................................................................. 4-594.5.2.1 Solvent .................................................................................................................................................................................................... 4-594.5.2.2 Matrix ................................................................................................................................................................................................... 4-604.5.3 Matrix effect ................................................................................................................................................................................................. 4-604.5.4 Accuracy ....................................................................................................................................................................................................... 4-614.5.5 Blank sample analysis .................................................................................................................................................................................. 4-624.5.6 Conclusions .................................................................................................................................................................................................. 4-624.6 Conclusions of the experimental evaluation .................................................................................................................. 4-635. Conclusion and future prospects ............................................................................... 5-65
List of FiguresFigure 1: Partnership ................................................................................................................................................................................................................. 1-1Figure 2: Different forms of ochratoxin and analogues ........................................................................................................................................................ 1-4Figure 3: Open form of OTA .................................................................................................................................................................................................. 1-4Figure 4: Descriptive comparison of data in relative values obtained via Charm ............................................................................................................ 3-22Figure 5: Linear fit of measured OTA concentration versus reference value for Charm ............................................................................................... 3-23Figure 6: Descriptive comparison of data in relative values obtained via R-Biopharm .................................................................................................. 3-24Figure 7: Linear fit of measured OTA concentration versus reference value for R-Biopharm...................................................................................... 3-24Figure 8: Standard curve of OTA, OTB, OTα, Citrinin, Zearalenone, AFLA B1, Coumarin, 4-HydroxyCoumarin and L-phenylalanine (n=1) .. 4-46Figure 9: Standard curves of ochratoxin A (ng/g) in wheat ............................................................................................................................................... 4-48Figure 10: Standard curves of ochratoxin A (ng/g) in winter barley versus control (proprietary buffer) ..................................................................... 4-48Figure 11: Standard curves of ochratoxin A (ng/g) in oats ................................................................................................................................................ 4-48Figure 12: Standard curve of OTA, OTB, OTα, Citrinin, Zearalenone, AFLA B1, Coumarin, 4-HydroxyCoumarin and L-phenylalanine (n=2) 4-52Figure 13: Standard curve of OTA, OTB, OTα, Citrinin, Zearalenone, AFLA B1, Coumarin, 4-HydroxyCoumarin and L-phenylalanine (n=2) 4-56Figure 14: Standard curve of OTA, OTB, OTα, Citrinin, Zearalenone, AFLA B1, Coumarin, 4-HydroxyCoumarin and L-phenylalanine (n=2) 4-60List of TablesTable 1: Parameters for quantitative and qualitative kits ...................................................................................................................................................... 1-2Table 2: Different structures of ochratoxine .......................................................................................................................................................................... 1-4Table 3: Overview of contacted manufacturers and their kits ........................................................................................................................................... 2-11Table 4: Overview of kits involved in the administrative evaluation ................................................................................................................................. 2-12Table 5: Overview of kits involved in the experimental evaluation ................................................................................................................................... 2-12Table 6: Parameters for quantitative kits .............................................................................................................................................................................. 3-15Table 7: Quality assurance ...................................................................................................................................................................................................... 3-15Table 8: Guidelines used for performing the validation ..................................................................................................................................................... 3-16Table 9: Brief definition of various validation parameters .................................................................................................................................................. 3-16Table 10: Calculation of parameters ...................................................................................................................................................................................... 3-16Table 11: Extend of agreement based on Lin‟s concordance coefficient ......................................................................................................................... 3-17Table 12: Evaluation of the calibration curves ..................................................................................................................................................................... 3-17Table 13: Reference Matrices ................................................................................................................................................................................................. 3-17Table 14: Applicable matrices ................................................................................................................................................................................................ 3-18Table 15: LOD and LOQ for different matrices ................................................................................................................................................................. 3-18Table 16: Calculation of LOD and LOQ ............................................................................................................................................................................. 3-19Table 17: Recovery .................................................................................................................................................................................................................. 3-19Table 18: Accuracy and measurement uncertainties ............................................................................................................................................................ 3-21Table 19: Summary of the ordinary linear regression for Charm ....................................................................................................................................... 3-23Table 20: Summary of the ordinary linear regression for R-Biopharm ............................................................................................................................. 3-25Table 21: Intra-run repeatability ............................................................................................................................................................................................ 3-26Table 22: Inter-run repeatability ............................................................................................................................................................................................ 3-27Table 23: Reproducibility ........................................................................................................................................................................................................ 3-28Table 24: Cross-reactivity in% ............................................................................................................................................................................................... 3-29Table 25: Materials, time requirements and estimated costs............................................................................................................................................... 3-29Table 26: Parameters for quantitative kits ............................................................................................................................................................................ 3-30Table 27: References ............................................................................................................................................................................................................... 3-30Table 28: Standards provided for verifying the test ............................................................................................................................................................. 3-30Table 29: Guidelines used for performing the validation ................................................................................................................................................... 3-31Table 30: Standards ................................................................................................................................................................................................................. 3-32Table 31: Unreliability region ................................................................................................................................................................................................. 3-32Table 32: False positives/negatives & true positives/negatives rates................................................................................................................................ 3-32Table 33: Calculation of false positives/negatives & true positives/negatives ................................................................................................................ 3-33Table 34: Contingency table ................................................................................................................................................................................................... 3-33Table 35: Calculation of Relative Accuracy, Relative Sensitivity and Relative Specificity ............................................................................................... 3-33Table 36: Relative accuracy, relative sensitivity and relative specificity ............................................................................................................................. 3-33Table 37: Cross-reactivity in% ............................................................................................................................................................................................... 3-34Table 38: Materials, time requirements and estimated costs............................................................................................................................................... 3-34Table 39: Summary of kits ...................................................................................................................................................................................................... 4-39Table 40: Standard solutions .................................................................................................................................................................................................. 4-39Table 41: Example of dilution scheme for matrix affect (calibration curve with 4 points) ............................................................................................ 4-42Table 42: Absorbance of the calibration curve in extraction solvent (A), undiluted matrix (B) and diluted matrix (C) ............................................. 4-43Table 43: Extraction protocols .............................................................................................................................................................................................. 4-45Table 44: Cross-reactivity in solvent ..................................................................................................................................................................................... 4-47
Page 1: CODA-CERVAEvaluation of immunoassay
Page 5: We would like to thank the kit manu
Page 10 and 11: Table 45: Cross-reactivity in matri
Page 13: 1. Foreword & scope &background
Page 16 and 17: Evaluation of immunoassay kits for
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Page 37 and 38: Measured value (ng/g)Evaluation of
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Evaluation of immunoassay kits for
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()Evaluation of immunoassay kits fo
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Observed Concentration (ng/g)Observ
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5. Conclusion and futureprospects
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