Application Note AgraQuant Melamine Sensitive ELISA COKAQ9400

Milk (dilution factor 5) 1. Pipette 5 mL of raw milk sample into a clean test tube. 2. Centrifuge milk sample with speed of 1500 g at 10C for 10 minutes (Note: if one does not have temperature controlled centrifuge, he/she can use chilled milk sample i.e., stored at 4C, and centrifuge with speed of 1500 g at room temperature (235C) for 10 minutes). 3. Pipette 0.2 mL of milk serum below the fat layer into a clean test tube. 4. Add 0.8 mL of assay diluent into the milk serum and mix. The sample is ready for testing. Milk powder (dilution factor 25) 1. Weigh 1 g of milk powder into a clean test tube. 2. Add 5 mL of 50C water to dissolve the milk powder. 3. Centrifuge the dissolved milk powder sample with speed of 1500 g at 10C for 10 minutes (Note: if one does not have temperature controlled centrifuge, he/she can centrifuge sample with speed of 1500 g at room temperature (235C) for 10 minutes). 4. Pipette 0.2 mL of milk serum below the fat layer into a clean test tube. 5. Add 0.8 mL of assay diluent into the milk serum and mix. The sample is ready for testing.

yogurt (dilution factor 5) 1. Weigh 1 g of yogurt sample into a clean test tube. 2. Dilute 1:5 with assay diluents (i.e. add 4 mL of assay diluents to 1 g of yogurt sample) and mix well. 3. Adjust pH of the mixed solution to fall within 6 8 by using alkaline. 4. Centrifuge yogurt sample with speed of 1500 g at 10C for 10 minutes (Note: if one does not have temperature controlled centrifuge, he/she can centrifuge samples with speed of 1500 g at room temperature (235C) for 10 minutes). 5. The supernatant in the mid layer is ready for testing.

yogurt drink (dilution factor 5) 1. Pipette 5 mL of yogurt drink sample into a clean test tube. 2. Centrifuge yogurt drink sample with speed of 1500 g at 10C for 10 minutes (Note: if one does not have temperature controlled centrifuge, he/she can use chilled yogurt drink sample i.e., stored at 4C, and centrifuge with speed of 1500 g at room temperature (235C) for 10 minutes). 3. Pipette 0.2 mL of yogurt drink below the fat layer into a clean test tube. 4. Add 0.8 mL of assay diluent into the yogurt drink serum and mix. The sample is ready for testing.

Egg (dilution factor 5) 1. Weigh 1 g of egg sample into a clean test tube. 2. Dilute 1:5 with assay diluents (i.e. add 4 mL of assay diluents to 1 g of egg sample) and vortex to mix the content well. 3. Centrifuge the diluted egg sample with speed of 1500 g at room temperature (23+/-5C) for 10 minutes. 4. The supernatant is ready for testing.

Egg powder (dilution factor 25) 1. Weigh 1 g of egg powder into a clean test tube. 2. Add 5 mL of 50C water to dissolve the egg powder. 3. Centrifuge the dissolved egg powder sample with speed of 1500 g at room temperature (235C) for 10 minutes. 4. Pipette 0.2 mL of serum below the fat layer into a clean test tube. 5. Add 0.8 mL of assay diluent into the serum and mix. The sample is ready for testing.

Biscuits (dilution factor 25) 1. Blend biscuit sample to make it homogenous. 2. Weigh 1 g of blended biscuits sample into a clean test tube. 3. Add 5 mL of distilled water into the biscuit sample, and vortex for 1 minute. 4. Centrifuge the content with speed of 1500 g for 10 minutes at room temperature (23+/-5C). 5. Pipette 0.2 mL of sample from the middle layer and add it into a clean tube. 6. Add 0.8 mL of assay diluents into the sample and mix the content well. 7. The sample is ready for testing. Ice cream (dilution factor 5) 1. Weigh 1 g of ice cream sample into a clean test tube. 2. Dilute 1:5 with assay diluents (i.e. add 4 mL of assay diluents to 1 g of ice cream sample) and vortex to mix the content well. 3. Centrifuge the diluted ice cream sample with speed of 1500 g at room temperature (23+/-5C) for 10 minutes. 4. The supernatant is ready for testing.

Matrix Milk Milk powder Yogurt Yogurt drink Egg Egg powder biscuits Ice cream

Dilution factor 5 25 5 5 5 25 25 5

Quantitation range 0.1 0.5 0.1 0.1 0.1 0.5 0.5 0.1 5 mg/kg 25 mg/kg 5 mg/kg 5 mg/kg 5 mg/kg 25 mg/kg 25 mg/kg 5 mg/kg

Spreadsheet for Milk-S Milk powder-S Milk-S Milk-S Milk-S Milk powder-S Milk powder-S Milk-S