Cut-off value approaches for MELISA-TEK Ruminant

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1 Laboratory report RAP Cut-off value approaches for MELISA-TEK Ruminant summary MELISA-TEK is validated at CCL for the analysis of various non-ruminant PAP samples, with or without added ruminant PAPs (processed at 133 C, 137 C, 141 C and 145 C). In report RAP results are shown, using a fixed decision level (OD = 0.017). New experiments (including an exploratory inter-laboratory ring trial) have indicated however that the variability within test plates and between kits can be larger than seen in those former results. With current test plates, samples have been tested again to look for a new cut off that is more robust and that is also applicable in case of high variability within a plate. This report shows seven different approaches for setting the cut-off value. All seven cut-offs are applied on new results of positive (spiked) samples. Based on these results two cut-off values are chosen and applied on all data used in RAP From all seven tested cut-off approaches approach number 4 seems to be the most robust one. This cut-off ratio value of 1.186, based on the mean (OD negative sample / OD blank) + 2 SD (OD negative sample / OD blank) gives good results for both current plates with larger standard deviations and for former plates with small standard deviations. The specificity of the method is 99%. For positive samples with 1% ruminant spikes a sensitivity of 100% is found in samples treated up to 137ºC. For positive samples with 2% spikes a sensitivity of 100% is found in samples treated up to 145ºC. See table 1. Table 1 shows the sensitivity of MELISA-TEK for ruminant- material spiked samples, processed at the various temperatures. processing n / N = number of correct samples / Number of samples tested temperature of ruminant spike (%) ruminant PAPs ºC 7 / 7 6 / 6 26 / / / 13 4 / 4 137ºC n.a. n.a. n.a. 12 / 12 8 / 8 4 / 4 141ºC n.a. n.a. n.a. 11 / / 11 4 / 4 145ºC 0 / 1 0 / 1 3 / 4 9 / 12 9 / 9 4 / 4 n.a. = not analysed CCL Nutricontrol en CCL Research zijn onderdeel van CCL B.V. Op alle diensten en leveringen zijn de algemene voorwaarden van CCL B.V. van toepassing waarin een beperking van aansprakelijkheid is opgenomen. Deze zijn gedeponeerd bij de KvK te Eindhoven. Het onderzoek is uitgevoerd in de periode tussen de datum van ontvangst monster op CCL B.V. en datum van rapportage. De met (Q) gemerkte analyses zijn erkend door RvA. De geschatte onzekerheid van de vermelde resultaten is opvraagbaar. Dit rapport mag niet anders dan in zijn geheel worden gekopieerd zonder toestemming van CCL B.V. Niet elke analyse is geaccrediteerd. In het accreditatiecertificaat L 053 is beschreven welke analyses geaccrediteerd zijn. Dit certificaat is opvraagbaar. Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrolpag. 1 / 27 BTW nr. NL B01, Handelsregister Eindhoven , NL: Rabobank Utrecht , D: ABN-AMRO Frankfurt Blz KoNr /001

2 introduction The performance of MELISA-TEK Ruminant kit in combination with the MELISA-TEK high Sensitivity Sample Extraction kit was tested before on various non-ruminant PAP samples, with or without added ruminant PAPs (processed at 133 C, 137 C, 141 C and 145 C). Results based on the cut-off (decision limit) OD have been shown in RAP This is why the cut-off 0.017, based on blank-subtracted average OD of all negative samples (0.003) plus 3 times a standard deviation (0.0045), is not always applicable. With current test plates samples have been tested again to look for a new cut off that is more robust and that is also applicable in case of high variability within a plate. This report shows seven different approaches for setting the cut-off value. All seven cut-offs are applied on new results of positive (spiked) samples. Based on these results two cut-off values are chosen and applied on all data used in RAP analysis method ANAL MELISA-TEK RUMINANT Species Kit (ELISA Technologies, Inc., Cat. No ) is a commercial sandwich ELISA that allows for detection of ruminant muscle tissue in extracts made from cooked meat or feed products (e.g. meat meals and meat and bone meals). It is based on the recognition of species specific troponin-i, a heat-stable muscle-specific protein, by specific monoclonal antibodies. 5 Grams of each sample is extracted singular according to the kit s manual and concentrated with the MELISA-TEK High Sensitivity Extraction Kit, (ELISA Technologies, Inc., Cat. No ), using funnels and concentrator tubes. The extract of a sample is analysed in duplicate on a plate. The intensity of the formed colour (OD=optical density) is measured with an ELISA reader at 450nm. samples The samples tested are a wide range of Processed Animal Proteins (PAPs). Some of these materials were processed by CCL in a dedicated sterilizer under strict conditions (133 C or 159 C for 20 min at 3 bar; both pre- and post-pressure cooked). Other reference materials (bovine, ovine, porcine and avian carcase and muscle material) were processed by PDM Ltd (Doncaster, UK) at 133 C, 137 C, 141 C and 145 C. Further, some pure commercially available products were tested. In total thirty-nine non-ruminant PAPs are tested: 13 porcine PAPs, 12 poultry PAPs, 9 porcine-poultry PAP mixtures, 1 porcine blood plasma, 1 dicalciumphosphate (DCP), 1 tricalciumphosfate (TCP) and 1 fish meal. Further also one negative feed sample with 5% porcine PAP is tested. The samples are shown in appendix I and II. Some positive samples are made by adding a known content (2%, 1%, 0.5%) of ruminant PAPs (carcase or muscle) to non-ruminant PAPs (porcine PAP and poultry PAP). The ruminant PAPs were treated at different temperatures (133 C, 141 C and 145 C). The number and composition of these positive samples are shown in appendix III. seven cut-off approaches A cut-off needs to be calculated which can be used as decision criterion at which a sample is considered as positive (containing ruminant PAP) or not. There are different approaches to set such cut-off values. In this report 7 different manners, which are frequently used for ELISA s, are shown. Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 2 / 27

3 According to Commission Decision 2002/657/EC it is sufficient to use 95% reliability (2 times the standard deviation) instead of 99%, for screening methods with an established permitted limit. A report of Det Norske Veritas Ltd. (DNV) concluded that up to 5% ruminant contamination of non-ruminant PAP, together with up to 1% contamination of ruminant diets with non ruminant PAP, did not lead to any higher risk of BSE in the EU cattle herd. As a result of the DNV report, EFPRA recommends that a 2% threshold limit for the presence of ruminant protein in monopap is accepted as providing a safe level of protection. Therefore, a 2% threshold limit for the presence of ruminant PAPs in non-ruminant PAPs is used and the cut-off values are based on 95% reliability. Seven different approaches to set a cut-off are: 1 mean OD bl + 2 SD OD bl 2 mean (OD ns - OD bl) + 2 SD (OD ns - OD bl) 3 mean (OD ns - OD nc) + 2 SD (OD ns - OD nc) 4 mean (OD ns / OD bl) + 2 SD (OD ns / OD bl) 5 mean (OD ns / OD nc) + 2 SD (OD ns / OD nc) 6 mean (OD ns / OD hpc) + 2 SD (OD ns / OD hpc) 7 mean (OD ns / OD lpc) + 2 SD (OD ns / OD lpc) bl = blank, ns = negative sample, nc = negative control, hpc = high positive control, lpc = low positive control, SD = standard deviation Approach number 4 is based on report Ruggedness study of immunoassays for processed animal proteins detection in feed of Joint Research centre (GE/R/FSQ/04/2006/09/04), (JRC, 2006). setting cut-off values Forty non-ruminant samples are analysed with MELISA-TEK TM Ruminant. Three different ELISA kits are used. The results and data analysis are shown in appendix II. Based on these data seven different cut-off values are determined as mentioned above. The first plate tested shows a large standard deviation between blanks (0.020) and negative controls (0.018). Normally this plate would be rejected according to the criteria of ANAL (standard deviation between blanks not more than 0.010). However in this study these results are included to gain a cut-off value which is based on great variability between and within plates. Table 2 shows the cut-off values that are calculated. In red the cut-off values based on 95% reliability are given, which will be used further on. Table 2. Cut-off values based on 7 different approaches OD bl OD ns - OD bl OD ns - OD nc OD ns / OD bl OD ns / OD nc OD ns / OD hpc OD ns / OD lpc Cut-off approach: mean SD SD cut-off SD cut-off SD = standard deviation All cut-off values are applied on the forty negative samples in appendix II. Only one false positive result is shown. This false positive result is due to a large variability between duplicates and would be analysed again in case of a commercial sample. Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 3 / 27

4 choice of cut-off approach With two different ELISA plates some positive samples are tested. The results are analysed and all seven different approaches are applied on these results and shown in appendix III. Sample avian meal with 0.5% bovine muscle spike 133 C is tested as control sample. The other samples are more difficult (porcine) samples, spiked with 1 or 2% bovine carcase material processed at 141 C or 145 C. In the first plate the blank is tested 8 times and shows a very large standard deviation. Unless this large standard deviation the samples with 2% spike are tested positive with all seven different cut-off approaches. Based on the tested positive samples, cut-off approach 3, 4 and 5 give the best results. Chosen is to apply only approach 4 and 5 on all data from RAP , as these approaches resemble the one mentioned in report Ruggedness study of immunoassays for processed animal proteins detection in feed of Joint Research centre (JRC, 2006). cut-off approach number 4 From all negative and positive samples reported in RAP the values [ / OD blank] are calculated and compared to the cut-off value 1.186, as mentioned above. The raw data is shown in appendix IV and V and VIII. Figure 1a and b show the results of the negative samples and positive samples with 1% spike and 2a and b show the results of the negative samples and positive samples with 2% spike. Figure b is a close-up view from figure a. It can be seen that 107 of the 108 negative samples have a value below the cut-off. Some of the positive samples with 1% spike also have a value below the cut-off, while all positive samples with 2% have a value above the cut-off. This means the sensitivity for samples with 1% spike is 94% and for samples with 2% spike the sensitivity is 100%. All samples with 1% spike which have a value close to or below the cut-off value, are samples with a spike of carcase material. Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 4 / 27

5 Negative samples and samples with 1% spike / OD blank cut off negative samples 133 spike 137 C spike 141 C spike 145 C spike USA spike Figure 1a. Values [ / OD blank] of negative and positive samples (1% spike) with regard to cut-off value Negative samples and samples with 1% spike (close-up) 2.50 / OD blank carcase spike cut off negative samples 133 spike 137 C spike 141 C spike 145 C spike USA spike Figure 1b. Close-up values [ / OD blank] of negative and positive samples (1% spike) with regard to cut-off value Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 5 / 27

6 Negative samples and samples with 2% spike / OD blank cut off negative samples 133 C spike 137 C spike 141 C spike 145 C spike USA spike Figure 2a. Values [ / OD blank] of negative and positive samples (2% spike) with regard to cut-off value Negative samples and samples with 2% spike (close up) 2.50 / OD blank cut off negative samples 133 C spike 137 C spike 141 C spike 145 C spike USA spike Figure 2b. Close-up values [ / OD blank] of negative and positive samples (2% spike) with regard to cut-off value Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 6 / 27

7 cut-off approach number 5 From all negative and positive samples reported in RAP the values [ / OD negative control] are calculated and compared to the cut-off value 1.236, as mentioned above. The raw data is shown in appendix VI and VII. Figure 3a and b show the results of the negative samples and positive samples with 1% spike and 4a and b show the results of the negative samples and positive samples with 2% spike. Figure b is a close-up view from figure a. It can be seen that with cut-off approach number 5 all negative samples have a value below the cut-off, which means the specificity is 100%. Some positive samples with 1% spike and some positive samples with 2% spike also have a value below the cut-off, which give false negative results. The sensitivity of samples with 1% spike is 88% and for samples with 2% spike the sensitivity is 91%. All positive samples with [OD sample / OD negative control] value < are samples with a spike of carcase material. Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 7 / 27

8 Negative samples and samples with 1% spike / OD negative control cut off negative samples 133 C spike 137 C spike 141 C spike 145 C spike USA spike Figure 3a. Values [ / OD negative sample] of negative and positive samples (1% spike) with regard to cut-off value Negative samples and samples with 1% spike (close-up) 2.50 / OD negative control carcase spike 0.00 cut off negative samples 133 C spike 137 C spike 141 C spike 145 C spike USA spike Figure 3b. Close-up values [ / OD negative sample] of negative and positive samples (1% spike) with regard to cut-off value Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 8 / 27

9 Negative samples and samples with 2% spike / OD negative control cut off negative samples 133 C spike 137 C spike 141 C spike 145 C spike USA spike Figure 4a. Values [ / OD negative sample] of negative and positive samples (2% spike) with regard to cut-off value Negative samples and samples with 2% spike (close-up) 2.50 / OD negative control carcase spike cut off negative samples 133 C spike 137 C spike 141 C spike 145 C spike USA spike Figure 4b. Close-up values [ / OD negative sample] of negative and positive samples (2% spike) with regard to cut-off value Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 9 / 27

10 Conclusions From all seven tested cut-off approaches approach number 4 seems to be the most robust one. This cut-off value of 1.186, based on the mean (OD negative sample / OD blank) + 2 SD (OD negative sample / OD blank) gives good results for both current plates with larger standard deviations and for former plates with small standard deviations. The specificity of the method is 99%. For positive samples with 1% ruminant spikes a sensitivity of 100% is found in samples treated up to 137ºC. For positive samples with 2% spikes a sensitivity of 100% is found in samples treated up to 145ºC. Veghel, 12 th March 2010 mailing list: M. Alm P. Derks C. van Vuure S. Woodgate Agreed by author CCL Nutricontrol Agreed by reviewer CCL Research Name: J. Vaessen Name: A. van Doremalen Date: Date: Signature: Signature: Agreed by group manager CCL Nutricontrol Name:R. Margry Date: Signature: Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 10 / 27

11 Appendix I. Composition of 39 negative samples tested with MELISA-TEK TM Ruminant CCL1 Pork soft pre-pressure cooking 133 C CCL2 Pork soft pre-pressure cooking 159 C CCL32 Pork soft defatted post-pressure cooking 133 C CCL33 Pork soft defatted post-pressure cooking 159 C CCL5 Pork bones pre-pressure cooking 133 C Porcine Poultry / avian PAP Porcine and poultry PAP Others CCL6 Pork bones pre-pressure cooking 159 C CCL34 Pork bones defatted post-pressure cooking 133 C CCL35 Pork bones defatted post-pressure cooking 159 C SW19 Porcine carcase 133 C SW45 Porcine muscle 133 C SW47 Porcine muscle 141 C ER23 Porcine protein 40 no (MBM 40%) ER22 Porcine protein 58 no:3150 (meat meal 58%) CCL13 Chicken soft pre-pressure cooking 133 C CCL14 Chicken soft pre-pressure cooking 159 C CCL50 Chicken soft defatted post-pressure cooking 133 C CCL25 Chicken bones pre-pressure cooking 133 C CCL10 Chicken bones pre-pressure cooking 159 C CCL53 Chicken bones defatted post-pressure cooking 159 C MM40 Chicken soft and bones 133 C (CCL13 + CCL25) ER30 Poultry meal standard SW5 Avian carcase 133 C SW24 Avian carcase 145 C ER16 Avian meal CCL40 Feather meal (Sonac Son) CCL29 Porcine PAP 98% + Chicken PAP 2% (pre-pressure 133 C) CCL31 Porcine PAP 99.5% + Chicken PAP 0.5% (pre-pressure 133 C) CCL48 Porcine PAP 95% + Chicken PAP 5% (post-pressure 133 C) CCL47 Porcine PAP 98% + Chicken PAP 2% (post-pressure 133 C) CCL49 Porcine PAP 99.5% + Chicken PAP 0.5% (post-pressure 133 C) CCL26 Chicken PAP 98% + Porcine PAP 2% (pre-pressure 133 C) CCL45 Chicken PAP 95% + Porcine PAP 5% (post-pressure 133 C) CCL44 Chicken PAP 98% + Porcine PAP 2% (post-pressure 133 C) CCL46 Chicken PAP 99.5% + Porcine PAP 0.5% (post-pressure 133 C) CCL42 Fish meal (IFFO) CCL54 Porcine DCP (Rousselot) CCL55 Porcine TCP (Smits Vuren) CCL37 Porcine Spray dried plasma (Harimex) MM41 Chicken feed 95% + Porcine PAP (CCL1 +CCL5) Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 11 / 27

12 Appendix II. Results and data analysis of 40 negative samples tested with MELISA-TEK TM Plate 1 MRM /78 Plate 2 MRM /79 Plate 3 MRM /78 Controls Negative samples Controls Negative samples Controls Negative samples cut-off approach: OD sm - OD sm - OD sm / OD OD sm / OD OD sm / OD OD sm / OD nr sample % OD sm OD sm SD mean OD bl OD nc bl nc hpc lpc blank blank low controle 0, high controle negative controle 10% pork negative controle 10% pork ER16 0.5% SW ER16 0.5% SW CCL CCL CCL CCL CCL CCL CCL CCL CCL CCL CCL CCL OD sm - OD sm - OD sm / OD OD sm / OD OD sm / OD OD sm / OD nr sample % OD sm OD sm SD mean OD bl OD nc bl nc hpc lpc blank blank low controle 0, high controle negative controle 10% pork negative controle 10% pork ER16 0.5% SW ER16 0.5% SW CCL CCL CCL SW OD sm - OD sm - OD sm / OD OD sm / OD OD sm / OD OD sm / OD nr sample % OD sm OD sm SD mean OD bl OD nc bl nc hpc lpc blank blank low controle 0, high controle negative controle 10% pork negative controle 10% pork ER16 0.5% SW ER16 0.5% SW MM MM SW CCL SW SW SW SW CCL CCL CCL CCL CCL CCL CCL CCL CCL ER ER ER ER CCL CCL CCL bl = blank, sm = sample, nc = negative control, cut-off approach: hpc = high positive control, lpc = low positive control, mean SD = standard deviation SD SD cut off SD cut off Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 12 / 27

13 Appendix III. Results and data analysis of positive samples tested with MELISA-TEK TM Plate: MRM /78 Plate: MRM /80 controls positive samples controls positive samples cut-off approach: OD sm - OD sm - OD sm / OD sm / OD sm / OD sm / sample/controls % spike OD sm OD sm SD mean OD bl OD nc OD bl OD nc OD hpc OD lpc bl bl bl bl lpc hpc nc 10% pork Avian meal (ER 16) 0.5 bovine muscle 133 C (SW7) Avian meal (ER 16) 0.5 bovine muscle 133 C (SW7) Avian meal (ER 16) 0.5 bovine muscle 133 C (SW7) Avian meal (ER 16) 0.5 bovine muscle 133 C (SW7) Porcine meal (ER23) 2 bovine carcase 145 C (SW14) Porcine meal (ER23) 2 bovine carcase 145 C (SW14) Porcine meal (ER23) 1 bovine carcase 145 C (SW14) Porcine meal (ER23) 1 bovine carcase 145 C (SW14) Porcine meal (ER23) 2 bovine muscle 133 C (SW7) Porcine meal (ER23) 2 bovine muscle 133 C (SW7) OD sm - OD bl OD sm - OD nc OD sm / OD bl OD sm / OD nc OD sm / OD hpc OD sm / OD lpc sample/controls % spike OD sm OD sm SD mean bl bl nc 10% pork nc 10% pork lpc hpc Avian meal (ER 16) 0.5 bovine muscle 133 C (SW7) Avian meal (ER 16) 0.5 bovine muscle 133 C (SW7) Porcine meal (ER23) 1 bovine carcase 145 C (SW14) Porcine meal (ER23) 2 bovine carcase 145 C (SW14) Porcine meal (ER22) 1 bovine carcase 145 C (SW14) Porcine meal (ER22) 2 bovine carcase 145 C (SW14) Porcine carcase 141 C (SW21) 1 bovine carcase 145 C (SW14) Porcine carcase 141 C (SW21) 2 bovine carcase 145 C (SW14) Porcine carcase 141 C (SW21) 1 bovine carcase 141 C (SW13) Porcine carcase 141 C (SW21) 2 bovine carcase 141 C (SW13) bl = blank, sm = sample, nc = negative control, cut-off approach: hpc = high positive control, lpc = low positive control, mean SD = standard deviation SD SD cut off SD cut off Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 13 / 27

14 Appendix IV. Cut-off approach 4 applied on negative samples RAP NEGATIVE SAMPLES OD sample duplo mean OD SD OD sm / OD bl sample % code blank low controle 0, high controle negative controle 10% pork pork soft pre-pressure 133 C 100 CCL pork soft pre-pressure 159 C 100 CCL pork soft post-pressure 133 C 100 CCL pork soft post-pressure 159 C 100 CCL pork soft defatted post-pressure 133 C 100 CCL pork soft defatted post-pressure 159 C 100 CCL pork bones pre-pressure 133 C 100 CCL pork bones pre-pressure 159 C 100 CCL pork bones post-pressure 133 C 100 CCL pork bones post-pressure 159 C 100 CCL pork bones defatted post-pressure 133 C 100 CCL pork bones defatted post-pressure 159 C 100 CCL chicken soft pre-pressure 133 C 100 CCL chicken soft pre-pressure 159 C 100 CCL chicken soft post-pressure 133 C 100 CCL chicken soft post-pressure 159 C 100 CCL chicken soft defatted post-pressure 133 C 100 CCL chicken bones pre-pressure 133 C 100 CCL chicken bones pre-pressure 159 C 100 CCL chicken bones post-pressure 133 C 100 CCL chicken bones post-pressure 159 C 100 CCL chicken bones defatted post-pressure 133 C 100 CCL chicken bones defatted post-pressure 159 C 100 CCL Avian carcase 133 C 100 SW blank low controle 0, high controle negative controle 10% pork Chicken soft +bones pre-pressure cooking 133 C MM Pork soft +bones pre-pressure cooking 133 C MM Porcine carcase 133 C SW Porcine carcase 141 C SW Porcine carcase 145 C SW Avian carcase 133 C SW Avian carcase 145 C SW Porcine muscle 133 C SW Porcine muscle 141 C SW Porcine muscle 145 C SW pork 49.75%+49.75% + Chicken 0.25%+0.25% (soft/bones pre-pressure 133 C) CCL Chicken 47.5%+47.5% + Pork 2.5%+2.5% (soft/bones post-pressure 133 C) CCL Chicken 49%+49% + Pork 1%+1% (soft/bones post-pressure 133 C) CCL Chicken 49.75%+49.75% + Pork 0.25%+0.25% (soft/bones post-pressure 133 C) CCL Pork 47.5%+47.5% + Chicken 2.5%+2.5% (soft/bones post-pressure 133 C) CCL Pork 49%+49% + Chicken 1%+1% (soft/bones post-pressure 133 C) CCL Pork 49.75%+49.75% + Chicken 0.25%+0.25% (soft/bones post-pressure 133 C) CCL blank low controle 0.05% high controle 1% negative controle 10% pork pork soft pre-pressure 133 C 100 CCL pork soft pre-pressure 159 C 100 CCL pork soft post-pressure 133 C 100 CCL pork soft post-pressure 159 C 100 CCL pork soft defatted post-pressure 133 C 100 CCL pork soft defatted post-pressure 159 C 100 CCL pork bones pre-pressure 133 C 100 CCL pork bones pre-pressure 159 C 100 CCL pork bones post-pressure 133 C 100 CCL pork bones post-pressure 159 C 100 CCL pork bones defatted post-pressure 133 C 100 CCL pork bones defatted post-pressure 159 C 100 CCL chicken soft pre-pressure 133 C 100 CCL Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 14 / 27

15 Appendix IV. Cut-off approach 4 applied on negative samples RAP (continuation) chicken soft pre-pressure 159 C 100 CCL chicken soft post-pressure 133 C 100 CCL chicken soft post-pressure 159 C 100 CCL chicken soft defatted post-pressure 133 C 100 CCL chicken soft defatted post-pressure 159 C CCL chicken bones pre-pressure 133 C 100 CCL chicken bones pre-pressure 159 C 100 CCL chicken bones post-pressure 133 C 100 CCL chicken bones post-pressure 159 C 100 CCL chicken bones defatted post-pressure 133 C 100 CCL chicken bones defatted post-pressure 159 C 100 CCL Pork 49.75%+49.75% + Chicken 0.25%+0.25% CCL Chicken 47.5%+47.5% + Pork 2.5%+2.5% CCL Chicken 49%+49% + Pork 1%+1% CCL Chicken 49.75%+49.75% + Pork 0.25%+0.25% CCL Pork 47.5%+47.5% + Chicken 2.5%+2.5% CCL Pork 49%+49% + Chicken 1%+1% CCL Pork 49.75%+49.75% + Chicken 0.25%+0.25% CCL Porcine Spray dried plasma (Harimex) CCL Spray dried Porcine bloodmeal (DAKA) CCL Porcine haemoglobine (PDM) CCL Feather meal (Sonac Son) CCL Feather meal (Saria Isse) CCL Fish meal (IFFO) CCL Porcine DCP (Rousselot) CCL Porcine TCP (Smits Vuren) CCL blank low controle high controle negative controle 10% pork fish meal (ER19) porcine protein 40 no.3025 (ER23) porcine protein 58 no (ER22) poultry meal standard (ER30) avian meal (ER16) Daka Porcine Protein 54, 1 CL Chicken 47.5%+47.5% + Pork 2.5%+2.5% (soft/bones pre-pressure 133 C) CCL Chicken 49%+49% + Pork 1%+1% (soft/bones pre-pressure 133 C) CCL Chicken 49.75%+49.75% + Pork 0.25%+0.25% (soft/bones pre-pressure 133 C) CCL Pork 47.5%+47.5% + Chicken 2.5%+2.5% (soft/bones pre-pressure 133 C) CCL Pork 49%+49% + Chicken 1%+1% (soft/bones pre-pressure 133 C) CCL blank (extractie buffer) low controle 0, high controle negative controle 10% pork Porcine carcase 133 C SW Porcine carcase 137 C SW Porcine carcase 141 C SW Porcine carcase 145 C SW Avian carcase 133 C SW Avian carcase 137 C SW Avian carcase 145 C SW Porcine muscle 133 C SW Porcine muscle 137 C SW Porcine muscle 141 C SW Porcine muscle 145 C SW Avian muscle 133 C SW Avian muscle 137 C SW Avian muscle 141 C SW Avian muscle 145 C SW Chicken soft +bones pre-pressure cooking 133 C MM Pork soft +bones pre-pressure cooking 133 C MM total number 108 FP 1 Based on the present cut off % FP 1 Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 15 / 27

16 Appendix V. Cut-off approach 4 applied on positive samples >0.5% spike RAP POSITIVE SAMPLES > 0.5% sample % % OD sample duplo mean OD SD blank low controle high controle negative controle 0 pork bovine carcase 133 C (SW7) 2 fish meal (ER19) bovine carcase 133 C (SW7) 1 fish meal (ER19) bovine carcase 145 C (SW14) 2 fish meal (ER19) bovine carcase 145 C (SW14) 1 fish meal (ER19) bovine muscle 145 C (SW28) 1 fish meal (ER19) ovine muscle 145 C (SW52) 1 fish meal (ER19) ovine carcase 145 C (SW18) 1 fish meal (ER19) bovine muscle + bovine carcase 145 C (SW28+SW14) (1:1) 1 fish meal (ER19) ovine muscle + ovine carcase 145 C (SW52+SW18) (1:1) 1 fish meal (ER19) bovine carcase 145 C (SW14) 1 porcine PAP (ER23) bovine carcase 145 C (SW14) 1 porcine PAP (ER22) ruminant spike RR1 A 1 RP1 (20% CCL1, 80% CCL5) B ruminant spike RR1 A 1 porcine protein 58 no: 3150 (ER22) blank low controle 0, high controle negative controle 10% pork ruminant spike RR1A 1 porcine protein 40 no (ER23) P ruminant spike RR1A 1 aquafeed (PAB1.8) P bovine carcase 133 SW7 1 porcine carcase 141 C SW bovine carcase 133 SW7 2 porcine carcase 141 C SW bovine muscle 133 C SW25 1 porcine carcase 141 C SW bovine muscle 133 C SW25 2 porcine carcase 141 C SW bovine carcase 133 SW7 1 porcine muscle 141 C SW bovine carcase 133 SW7 2 porcine muscle 141 C SW bovine muscle 133 C SW25 1 porcine muscle 141 C SW bovine muscle 133 C SW25 2 porcine muscle 141 C SW bovine carcase 137 C SW8 1 porcine carcase 141 C SW bovine carcase 137 C SW8 2 porcine carcase 141 C SW bovine muscle 137 C SW26 1 porcine carcase 141 C SW bovine muscle 137 C SW26 2 porcine carcase 141 C SW bovine carcase 137 C SW8 1 porcine muscle 141 C SW bovine carcase 137 C SW8 2 porcine muscle 141 C SW bovine muscle 137 C SW26 1 porcine muscle 141 C SW bovine muscle 137 C SW26 2 porcine muscle 141 C SW bovine carcase + bovine muscle 133 C (SW7+SW25) (1:1) 1 chicken MM ovine carcase + ovine muscle 133 C (SW15+SW49) (1:1) 1 chicken MM bovine carcase + bovine muscle 133 C (SW7+SW25) (1:1) 1 porcine MM ovine carcase + ovine muscle 133 C (SW15+SW49) (1:1) 1 porcine MM bovine carcase + bovine muscle 133 C (SW7+SW25) (1:1) 5 pig feed + chicken PAP (5%) MM bovine carcase + bovine muscle 133 C (SW7+SW25) (1:1) 1 pig feed + chicken PAP (5%) MM bovine carcase + bovine muscle 133 C (SW7+SW25) (1:1) 5 chicken feed + porcine PAP (5%) MM bovine carcase + bovine muscle 133 C (SW7+SW25) (1:1) 1 chicken feed + porcine PAP (5%) MM blank low controle high controle negative controle 0 10% pork ruminant spike RR1 A 1 porcine protein 58 no: 3150 (ER22) ruminant spike RR1 A 1 RC1 (20% CCL13, 80% CCL25) C ruminant spike RR1 A 1 avian meal (ER16) ruminant spike RR1 A 1 poultry meal standard (ER30) ruminant spike RR1 A 1 feather meal (ER9) ruminant spike RR1 A 1 hydrolysed feather meal (ER29) ruminant spike RR1 A 1 fish meal (ER19) ruminant spike RR1 A 1 A2.2 Europa Eel (FM5) DQ/06/ USA 1 Daka protein 54,1; CL DQ/06/ USA 1 Daka Porcine Protein 40,2; CL DQ/06/ USA 1 Daka Porcine Protein 58,3; CL DQ/06/ USA 1 Pig Protein Powder 1327; CL DQ/06/ USA 1 fish meal (ER19) DQ/06/ USA 1 Poultry Meal Standard (ER30) DQ/06/ USA 1 Avian meal (ER16) DQ/06/ USA 1 RP1 (20% CCL1, 80% CCL5)B DQ/06/ USA 1 RC1 (20% CCL13, 80% CCL25)C DQ/06/ USA 1 Porcine musle 141 C (SW47) blank (extractie buffer) low controle 0, high controle negative controle 10% pork Bovine 133 C 1 Avian 133 C SW Bovine carcase 133 C 2 feathermeal NS Bovine carcase 133 C 1 feathermeal NS bovine muscle 133 C SW25 2 feathermeal NS bovine muscle 133 C SW25 1 feathermeal NS ovine carcase 133 C 2 feathermeal NS ovine carcase 133 C 1 feathermeal NS OD sm / OD bl Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 16 / 27

17 Appendix V. Cut-off approach 4 applied on positive samples >0.5% spike RAP (continuation) blank low controle 0, high controle negative controle 10% pork bovine carcase 133 C (SW7) 1 porcine PAP ER bovine carcase 133 C (SW7) 2 porcine PAP ER bovine carcase 133 C (SW7) 1 porcine PAP ER bovine carcase 133 C (SW7) 2 porcine PAP ER bovine muscle 133 C (SW25) 1 porcine PAP ER bovine muscle 133 C (SW25) 2 porcine PAP ER bovine muscle 133 C (SW25) 1 porcine PAP ER bovine muscle 133 C (SW25) 2 porcine PAP ER bovine carcase 137 C (SW8) 1 porcine PAP ER bovine carcase 137 C (SW8) 2 porcine PAP ER bovine carcase 137 C (SW8) 1 porcine PAP ER bovine carcase 137 C (SW8) 2 porcine PAP ER bovine muscle 137 C (SW26) 1 porcine PAP ER bovine muscle 137 C (SW26) 2 porcine PAP ER bovine muscle 137 C (SW26) 1 porcine PAP ER bovine muscle 137 C (SW26) 2 porcine PAP ER bovine carcase 141 C (SW13) 1 porcine PAP ER bovine carcase 141 C (SW13) 2 porcine PAP ER bovine carcase 141 C (SW13) 1 porcine PAP ER bovine carcase 141 C (SW13) 2 porcine PAP ER bovine muscle 141 C (SW27) 1 porcine PAP ER bovine muscle 141 C (SW27) 2 porcine PAP ER bovine muscle 141 C (SW27) 1 porcine PAP ER bovine muscle 141 C (SW27) 2 porcine PAP ER bovine muscle 145 C (SW28) 1 porcine PAP ER bovine muscle 145 C (SW28) 2 porcine PAP ER bovine muscle 145 C (SW28) 1 porcine PAP ER bovine muscle 145 C (SW28) 2 porcine PAP ER blank low controle 0, high controle negative controle 10% pork Ovine muscle 133 C 2 feathermeal NS Ovine muscle 133 C 1 feathermeal NS DQ/06/ USA 2 porcine muscle 141 C (SW47) bovine carcase 145 C (SW14) 2 ER bovine carcase 145 C (SW14) 2 ER bovine carcase 141 C (SW13) 1 porcine carcase 141 C (SW21) bovine carcase 141 C (SW13) 2 porcine carcase 141 C (SW21) bovine carcase 141 C (SW13) 1 porcine muscle 141 C (SW47) bovine carcase 141 C (SW13) 2 porcine muscle 141 C (SW47) bovine muscle 141 C (SW27) 1 porcine muscle 141 C (SW47) bovine muscle 141 C (SW27) 2 porcine muscle 141 C (SW47) bovine carcase 145 C (SW14) 1 porcine carcase 141 C (SW21) bovine carcase 145 C (SW14) 2 porcine carcase 141 C (SW21) bovine muscle 145 C (SW28) 1 porcine carcase 141 C (SW21) bovine muscle 145 C (SW28) 2 porcine carcase 141 C (SW21) bovine carcase 145 C (SW14) 1 porcine muscle 141 C (SW47) bovine carcase 145 C (SW14) 2 porcine muscle 141 C (SW47) bovine muscle 145 C (SW28) 1 porcine muscle 141 C (SW47) bovine muscle 145 C (SW28) 2 porcine muscle 141 C (SW47) bovine carcase 141 C (SW13) 1 porcine carcase 133 C (SW19) bovine carcase 141 C (SW13) 2 porcine carcase 133 C (SW19) bovine muscle 141 C (SW27) 1 porcine carcase 133 C (SW19) bovine muscle 141 C (SW27) 2 porcine carcase 133 C (SW19) bovine carcase 141 C (SW13) 1 porcine muscle 133 C (SW45) bovine carcase 141 C (SW13) 2 porcine muscle 133 C (SW45) bovine muscle 141 C (SW27) 1 porcine muscle 133 C (SW45) bovine muscle 141 C (SW27) 2 porcine muscle 133 C (SW45) bovine carcase 133 C (SW7) 1 porcine carcase 133 C (SW19) bovine carcase 133 C (SW7) 1 porcine muscle 133 C (SW45) bovine muscle 133 C (SW25) 1 porcine carcase 133 C (SW19) bovine muscle 133 C (SW25) 1 porcine muscle 133 C (SW45) bovine carcase 137 C (SW8) 1 porcine carcase 133 C (SW19) bovine carcase 137 C (SW8) 1 porcine muscle 133 C (SW45) bovine muscle 137 C (SW26) 1 porcine carcase 133 C (SW19) bovine muscle 137 C (SW26) 1 porcine muscle 133 C (SW45) bovine muscle 141 C (SW27) 1 porcine carcase 141 C (SW21) blank low controle 0, high controle negative controle 10% pork DQ/06/ USA 2 Pig Protein Powder 1327; CL Based on the present cut off % total TP FN % FN Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 17 / 27

18 Appendix VI. Cut-off approach 5 applied on negative samples RAP NEGATIVE SAMPLES OD sample duplo mean OD SD OD sm / OD nc sample % code blank low controle 0, high controle negative controle 10% pork pork soft pre-pressure 133 C 100 CCL pork soft pre-pressure 159 C 100 CCL pork soft post-pressure 133 C 100 CCL pork soft post-pressure 159 C 100 CCL pork soft defatted post-pressure 133 C 100 CCL pork soft defatted post-pressure 159 C 100 CCL pork bones pre-pressure 133 C 100 CCL pork bones pre-pressure 159 C 100 CCL pork bones post-pressure 133 C 100 CCL pork bones post-pressure 159 C 100 CCL pork bones defatted post-pressure 133 C 100 CCL pork bones defatted post-pressure 159 C 100 CCL chicken soft pre-pressure 133 C 100 CCL chicken soft pre-pressure 159 C 100 CCL chicken soft post-pressure 133 C 100 CCL chicken soft post-pressure 159 C 100 CCL chicken soft defatted post-pressure 133 C 100 CCL chicken bones pre-pressure 133 C 100 CCL chicken bones pre-pressure 159 C 100 CCL chicken bones post-pressure 133 C 100 CCL chicken bones post-pressure 159 C 100 CCL chicken bones defatted post-pressure 133 C 100 CCL chicken bones defatted post-pressure 159 C 100 CCL Avian carcase 133 C 100 SW blank low controle 0, high controle negative controle 10% pork Chicken soft +bones pre-pressure cooking 133 C MM Pork soft +bones pre-pressure cooking 133 C MM Porcine carcase 133 C SW Porcine carcase 141 C SW Porcine carcase 145 C SW Avian carcase 133 C SW Avian carcase 145 C SW Porcine muscle 133 C SW Porcine muscle 141 C SW Porcine muscle 145 C SW pork 49.75%+49.75% + Chicken 0.25%+0.25% (soft/bones pre-pressure 133 C) CCL Chicken 47.5%+47.5% + Pork 2.5%+2.5% (soft/bones post-pressure 133 C) CCL Chicken 49%+49% + Pork 1%+1% (soft/bones post-pressure 133 C) CCL Chicken 49.75%+49.75% + Pork 0.25%+0.25% (soft/bones post-pressure 133 C) CCL Pork 47.5%+47.5% + Chicken 2.5%+2.5% (soft/bones post-pressure 133 C) CCL Pork 49%+49% + Chicken 1%+1% (soft/bones post-pressure 133 C) CCL Pork 49.75%+49.75% + Chicken 0.25%+0.25% (soft/bones post-pressure 133 C) CCL blank low controle 0.05% high controle 1% negative controle 10% pork pork soft pre-pressure 133 C 100 CCL pork soft pre-pressure 159 C 100 CCL pork soft post-pressure 133 C 100 CCL pork soft post-pressure 159 C 100 CCL pork soft defatted post-pressure 133 C 100 CCL pork soft defatted post-pressure 159 C 100 CCL pork bones pre-pressure 133 C 100 CCL pork bones pre-pressure 159 C 100 CCL pork bones post-pressure 133 C 100 CCL pork bones post-pressure 159 C 100 CCL pork bones defatted post-pressure 133 C 100 CCL pork bones defatted post-pressure 159 C 100 CCL chicken soft pre-pressure 133 C 100 CCL Specifity_selectivity_MELISA-TEK_validation_CCL_Nutricontrol pag. 18 / 27