VALIDATION REPORT Colistin ELISA Beijerinckweg 18 NL-6827 BN Arnhem The Netherlands Phone: +31 26 363 0 364 info@europroxima.com
1. Introduction Colistin A and B are polypeptide antibiotics that have been used in medicine and veterinary for over 50 years. Due to the development of bacterial resistance to colistin European Medicines Agency has recenlty proposed to reduce colistin use in animals and restrict its application only in cases of infections for which no other effective treatments are available [1]. Colistin ELISA can be used to screen for the presence of colistin residues in milk, meat, eggs, liver, fish and feed at the levels below the maximum residue limits (MRLs) set in the European Union. MRLs established by Commission Regulation 37/2010 for colistin in tissues of all food producing animals are as follows [2]: Target tissue Muscle Fat Liver Kidney Milk Eggs MRL (μg/kg) 150 150 150 200 50 300 2. Kit characteristics: see manual The reactivity pattern of the antibody. Cross- reactions: Colistin A and B 100% Polymyxin B1 and B2 100% Bacitracin <0.1% 3. Scope of validation The validation study was carried out with the Colistin ELISA (5151COL).The study included: - Determination of the limit of detection (LOD) for milk, eggs, chicken, beef, pork, liver, fish and feed. - Determination of the detection capability (CCβ) for milk, eggs, chicken, beef, pork, liver, fish and feed. - Determination of the % recovery for milk, eggs, chicken, beef, pork, liver, fish and feed. - Determination of inter- and intra- assay coefficients of variation. 4. Sample material Non contaminated milk (cow s raw milk, cow s whole milk, cow s semi-skimmed milk, goat s milk, sheep s milk, buffalo s milk), eggs, chicken, beef, pork, beef liver, fish (salmon, cod, pollock), feed (cattle, swine, poultry). 5. Sample treatment Sample preparation was performed according the manual 5151COL[1]01.17, chapter 8.1 and 8.2. 6. Results For determination of LOD, CCβ and recovery, samples were spiked with colistin A/B purchased from Sigma, product number C4461 2
Table 1. Results of the validation study. Matrix Milk a Eggs b Chicken b Pork b Beef b Liver b Fish c Feed c LOD [ppb] 4.3 22.0 11.7 7.6 14.6 21.3 12.5 24.1 CCβ [ppb] 10 30 30 30 30 30 30 30 LOD: limit of detection, mean of blank samples + 3 x SD CCβ: detection capability a n=36; b n=40; c n=20 Table 2. Recovery [%] of colistin in different matrices. Spiking level [ppb] Milk Spiking level [ppb] Eggs 10 96.9 ± 9.1 30 96.1 ± 8.8 25 92.0 ± 16.2 150 103.1 ± 6.3 50 101.8 ± 14.4 300 100.1 ± 4.5 100 92.9 ± 13.1 450 100.1 ± 2.2 Spiking level [ppb] Chicken Pork Beef Liver Fish (n=20) Feed (n=20) 30 104.1 ± 3.5 107.6 ± 5.2 104.8 ± 5.2 114.5 ± 5.3 103.4 ± 3.4 101.2 ± 5.0 75 95.5 ± 4.2 104.5 ± 5.2 101.3 ± 6.6 108.8 ± 6.2 106.1 ± 5.6 100.0 ± 4.1 150 98.2 ± 3.2 100.0 ± 5.0 102.9 ± 5.5 107.4 ± 3.7 102.0 ± 4.5 102.7 ± 3.7 300 101.1 ± 2.6 100.2 ± 5.5 100.5 ± 4.7 110.5 ± 3.0 103.2 ± 3.1 101.7 ± 1.9 Table 3. Inter-assay coefficient of variation for 0.5 ng/ml colistin standard. Sample Mean OD ± SD* at 450nm CV (%) 0.5 ng/ml 1.077 ± 0.075 7.0 standard *Mean of 10 results in duplicate. Table 4. Intra-assay coefficient of variation for buffer spiked at 0.5 ng/ml with colistin. 3 Sample Buffer spiked at 0.5ng/mL *Mean of 40 samples Mean OD ± SD* at 450nm CV (%) 1.474 ± 0.093 6.3
Fig. 1. Determination of CCβ. 4
7. Literature [1] European Medicines Agency. Countries should reduce use of colistin in animals to decrease the risk of antimicrobial resistance. 27 July 2016 EMA/480583/2016. [2] Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Official Journal of the European Union L 15/1. 5