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Evaluation of immunoassay kits for determination of ochratoxin A in cerealsinterest were subsequently constructed in the extracts. Besides these modifications the same protocol was used as in4.1.4.2.1.4.1.4.2.2.2 Data handling and statistical analysesThe same protocol was used as described in 4.1.4.2.1.2.4.1.4.3 Work package III: Matrix effect4.1.4.3.1 Study designTo assess the matrix effect a calibration curve of OTA is constructed in pure extraction solvent, in extract of amatrix (wheat, winter barley and oats) and in extract diluted 5 times with pure extraction solvent (see Table 41). Forall kits, the same sample of wheat, winter barley and oats was used. The extracts were pooled to minimize matrixinhomogenity.Table 41: Example of dilution scheme for matrix affect (calibration curve with 4 points)Control Water+std (9:1)Wheat Undiluted Extract+Std (9:1)Diluted Extract +std (9:1)Winter barley Undiluted Extract+Std (9:1)Diluted Extract +std (9:1)Oats Undiluted Extract+Std (9:1)Diluted Extract +std (9:1)Concentration 1 Concentration 2 Concentration 3 Concentration 4It is very well known in the art that mycotoxin identification and quantification can be hampered by matrix effects.Immunoassays are no exception to this. One common way to overcome the detrimental effects of matrixcompounds is to dilute the sample, implying the assumption that less matrix means less matrix effect. Diluting hassome drawbacks: it might increases significantly the LOD and LOQ as the component of interest is also diluted.Moreover it introduces an extra manipulation step (pipetting) whose error is afterwards multiplied by the dilutionfactor. Matrix effects are considered to be significant when higher than 10% compared to the solvent 69 .Matrix effect may be defined as the sum of the effects of all the components in a system with the exception of theanalyte to be measured 70 (see also Equation 11).4.1.4.3.2 Data handling and statistical analyses4.1.4.3.2.1.1 Elisa kitsDose-response curves were constructed by plotting the theoretical concentration against the relative absorbance(B/Bo).Two methods can be used to assess the matrix effect.69 Lee NA, Wang S, Allan RD, Kennedy IR. J Agric Food Chem. 2004 May 19; 52(10):2746-55.70 Wood WG, Matrix effects in immunoassays, Scand. J. Clin. Lab. Invest. Suppl., 1991, 205, 1054-42 Experimental evaluation | CODA-CERVA
Evaluation of immunoassay kits for determination of ochratoxin A in cerealsC50% reductionFrom this calibration curve the concentration at which 50% extinction occurs is calculated. Using the concentrationsof 50% reduction of absorbance, the matrix effect in % can be calculated as follows:Equation 10 CR = IC50[Diluted matrix] / IC50[Undiluted matrix] * 100With:C50% [Undiluted matrix]: concentration of OTA in undiluted matrixC50%[Diluted matrix]: concentration of OTA in matrix 5 times diluted with extraction solventPercentage matrix effect can be calculated as:Equation 11% Control Color ReductionUsing the absorbance values the color reduction was calculated as follows 71 .Table 42: Absorbance of the calibration curve in extraction solvent (A), undiluted matrix (B) and diluted matrix (C)StandardsControl A0 A1 A2 A3 A4 A5Undiluted B0 B1 B2 B3 B4 B5Diluted C0 C1 C2 C3 C4 C5 For undiluted extracts:Equation 12%CRmatrix assisted: [(1-B0/A0)+(1-B1/A1)+(1-B2/A2)+(1-B3/A3)+(1-B4/A4)+(1-B5/A5)]*100/6 For diluted extracts:Equation 13%CRmatrix assisted: [(1-C0/A0)+(1-C1/A1)+(1-C2/A2)+(1-C3/A3)+(1-C4/A4)+(1- C5/A5)]*100/6 Difference:Equation 14Diff=[(B0/A0-C0/A0)+(B1/A1-C1/A1)+(B2/A2-C2/A2)+(B3/A3-C3/A3)+(B4/A4-C4/A4)+(B5/A5-C5/A5)]*100/6 % Control Color Reduction:Equation 15%Control Color Reduction = (Acontrol-Diff)/(Acontrol/100)71Sabina Cairoli, Anna Arnoldi, and Silvia Pagani, J. Agric. Food Chem., 1996, 44 (12), pp 3849–3854CODA-CERVA | Experimental evaluation 4-43
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CODA-CERVAEvaluation of immunoassay
Page 5: We would like to thank the kit manu
Page 8 and 9: 4.1.4.2 Work package II: Cross reac
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
Page 21: Evaluation of immunoassay kits for
Page 27: 3. Administrative evaluation
Page 37 and 38: Measured value (ng/g)Evaluation of
Page 51: 4. Experimental evaluation
Page 60 and 61: ()Evaluation of immunoassay kits fo
Page 62 and 63: Observed Concentration (ng/g)Observ
Page 79: 5. Conclusion and futureprospects
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