RIDASCREEN Aflatoxin M1
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1 R-Biopharm AG Product information Reliable Extensively tested, excellent performance Sensitive Low limit of detection (5 ppt for milk) Easy All reagents ready to use Automatable Options available R-Biopharm for the safety of your analysis.
2 is a competitive enzyme immunoassay for the quantitative analysis of aflatoxin M1 in milk and milk powder. Application notes for cheese and butter samples are available on request. Background Aflatoxins are carcinogenic and highly toxic metabolites of the fungi species Aspergillus flavus and Aspergillus parasiticus. Aflatoxin M1 is produced as a metabolite of aflatoxin B1. It is secreted with the milk after feeding of aflatoxin B1 containing feed to lactating cows. As aflatoxin M1 is relatively stable towards the pasteurizing process, not only a comprehensive routine check of the raw materials to be processed is required but also of the final products. Brief information Technology: Format: Sample preparation: Incubation time: Limit of detection (LOD): Detection range: ELISA Microtiter plate with 96 wells (12 strips with 8 wells each) Milk: degrease / Milk powder: reconstitute and degrease 1 h 15 min Milk 5 ppt / Milk powder 50 ppt 5-80 ppt (ng/kg) Recovery: Milk 100 % / Milk powder 116 % (ESM reference material 111 %) Specificity: Aflatoxin M1 100% / Aflatoxin M2 < 10 % Benefits Sensitive: Low limit of detection (LOD) Easy: Fast: Precise: Ready-to-use test kit components, simple and efficient sample preparation Only 1 h 15 min test duration Reliable and reproducible results Test principle The basis of the test is the antigen-antibody reaction. The wells of the microtiter strips are coated with capture antibodies directed against anti-aflatoxin M1 antibodies. Anti-aflatoxin M1 antibodies are added which are bound by the immobilized capture antibodies. After incubation and washing step, standards or sample solutions are added. After a further incubation with a subsequent washing step, the aflatoxin M1 enzyme conjugate is added. Free and enzyme conjugated aflatoxin M1 compete for the antibody binding sites (competitive enzyme immunoassay). Any unbound enzyme conjugate is then removed in a washing step. Substrate/chromogen is added to the wells and incubated. Bound enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm and the absorption is inversely proportional to the aflatoxin M1 concentration in the sample. 2
3 Test procedure Place the required number of wells in the frame. 1 Add 100 µl of antibody solution 15 min Incubation at room temperature 2 Wash 3 times with washing buffer Add 100 µl of standard solution or prepared sample 30 min Incubation at room temperature in the dark 3 Wash 3 times with washing buffer Add 100 µl of enzyme conjugate 15 min Incubation at room temperature in the dark 4 Wash 3 times with washing buffer Add 100 µl substrate/chromogen 15 min Incubation at room temperature in the dark 5 Add 100 µl of stop solution Photometrical measurement at 450 nm 3
4 Limit of detection (LOD) Milk Negative samples Lot 1 Lot 2 Lot 3 B/B 0 c [ppt] B/B 0 c [ppt] B/B 0 c [ppt] n = <Std <Std <Std <Std Mean SD LOD 4.4 SD = standard deviation LOD = limit of detection (mean + 3 x SD) 4
5 Recovery Milk Spiked samples Lot 1 Lot 2 Lot 3 c [ppt] Recovery [%] c [ppt] Recovery [%] c [ppt] Recovery [%] 10 ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt Mean Mean (total) 100 LOD = limit of detection 5
6 Limit of detection (LOD) Milk powder (referring to reconstituted milk) Negative samples Lot 1 Lot 2 Lot 3 B/B 0 c [ppt] B/B 0 c [ppt] B/B 0 c [ppt] n = <Std <Std <Std <Std Mean SD LOD 49 SD = standard deviation LOD = limit of detection (mean + 3 x SD) 6
7 Recovery Milk powder (referring to reconstituted milk) Spiked samples Lot 1 Lot 2 Lot 3 c [ppt] Recovery [%] c [ppt] Recovery [%] c [ppt] Recovery [%] 100 ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt ppt Mean Mean (total) 116 LOD = limit of detection 7
8 Recovery Milk powder (referring to reconstituted milk) ERM reference Target value Lot 1 Lot 2 Lot 3 material [ppt] c [ppt] Recovery [%] c [ppt] Recovery [%] c [ppt] Recovery [%] ERM 282 ERM 282 ERM 282 ERM 282 ERM 282 ERM 282 ERM ERM ERM ERM ERM ERM ERM ERM ERM ERM ERM ERM Mean Mean (total) 111 LOD = limit of detection 8
9 Repeatability and reproducability Milk powder (referring to reconstituted milk) Participant Lot Spiked sample [250ppt] c [ppt] c [ppt] c [ppt] c [ppt] Sample Sample Sample Sample Sample Sample Sample Sample Standard deviation of repeatability (sr) 16.9 Repeatability (r) 47.3 Standard deviation of reproducability (sr) 22.7 Reproducablility (R) 66.1 Remark corresponds with specifications of ISO Norm 14675:2003(E). Data calculated from in-house QC testing over several lots and months with different technicians using one stable sample. Collaborative data not available until the time point of finilization of this document. 9
10 Related products Product Description No. of Tests / Amount Code No. Aflatoxin ELISA Microtiter Plates RIDASCREEN FAST Aflatoxin M1 Enzyme immunoassay for quantitative analysis of aflatoxin M1 in milk and milk powder Detection limit: < 125 ppt 48 determinations Incubation time: 15 min R5812 Immunoaffinity Columns AFLAPREP M Immunoaffinity columns for sample clean-up prior to the analysis of aflatoxin M1 using HPLC 25 columns (1 ml format) RBRP04 AFLAPREP M WIDE Immunoaffinity columns for sample clean-up prior to the analysis of aflatoxin M1 using HPLC 10 columns (3 ml format) 50 columns (3 ml format) RBRP124 RBRP124B Standard liquid AFLATOXINM1 Standard Aflatoxin M1 standard solution at a concentration of 1000 ng/ml in acetonitrile 6 ml RBRP42 Trilogy Liquid Standard Aflatoxin M1 Aflatoxin M1; 0.5 µg/ml acetonitrile 2 ml TSL-143 Standard dried Trilogy Dried Standard Aflatoxin M1 Aflatoxin M1 1 µg/ml after reconstitution TS-130 For further information or orders please contact R-Biopharm AG: International Sales: Phone: +49 (0) Fax: +49 (0) sales@r-biopharm.de Order Department: Phone: +49 (0) Fax: +49 (0) orders@r-biopharm.de R-Biopharm AG An der neuen Bergstraße 17, Darmstadt, Germany info@r-biopharm.de
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