1. EIA’s (Enzyme Immunoassays) for Food Safety

2. EIA’s (Enzyme Immunoassays) for Food Safety EIA’s are tests that use either an enzyme-bound antibody or enzyme-bound analyte to detect a particular antigen. The enzyme catalyzes a colour reaction when exposed to a substrate. Such tests can be used for semi-quantitative analysis of contaminants and residues in different biological matrices.

3. EIA’s for Food Safety Competitive (Inhibition) EIA’s: For detection of low molecular weight components (residues and contaminants) Non-Competitive (Sandwich) EIA’s: For detection of high molecular weight components (proteins)

4. Format of the Competitive EIA Residue X = Target molecule Horseradish Peroxidase (HRP) = Enzyme Sheep anti-rabbit antibody Residue X (standard/sample) Microtitre plate well Rabbit antibody to residue X Residue X - HRP conjugate

5. Target Molecules for the Competitive EIA’s Drugs and steroids Antibiotics Mycotoxins

6. Format of the Sandwich EIA Biotinylated rabbit anti- protein Y Streptavidin coated microtitre plate well Streptavidin coated microtitre plate well Protein Y (standard/sample) Rabbit anti-protein Y- HRP conjugate Rabbit anti-protein Y- HRP conjugate Protein Y = Target molecule Horseradish Peroxidase (HRP) = Enzyme

7. Target Molecules for the Sandwich EIAs Food allergens (proteins) Proteins involved in adulteration

8. Production of Immunogens and Antibodies Residue X Coupling to a carrier protein (BSA, BTG) Coupling to a carrier protein (BSA, BTG) Protein Y Antibody production Immunisation of mice or rabbits

9. Antibody Purification Rabbit Polyclonal or Mouse Monoclonal Antibodies Ammonium Sulfate Precipitation Antibody Fraction Immunoaffinity Chromatography Conjugation to HRP or biotin Conjugation to HRP or biotin Competitive Assay Sandwich Assay

10. Conjugate in the Competitive-EIA The conjugate in the competitive-EIA consists of residue X that has been labelled with the enzyme horseradish peroxidase (HRP) Residue X + + HRP Residue X - HRP conjugate

11. Conjugate in the Sandwich-EIA The conjugate in the sandwich-EIA consists of an antibody (polyclonal or monoclonal) to protein Y that has been labelled with the enzyme horseradish peroxidase (HRP) + + HRP Anti-protein Y- HRP conjugate Antibody to protein Y Antibody to protein Y

12. Different Steps in the Analysis of Samples Collection of samples and transport Storage of samples Sample preparation Performing the EIA Calculation and interpretation of the results

13. Collection of Samples and Transport Take a representative amount of the product in question Prevent cross contamination of the samples Label the samples Record the shelf-life for the sample product Treat all samples as being potentially infective or toxic Make sure that the samples are transported at the appropriate temperature Be aware of possible microbial contamination in the samples The basis for obtaining good laboratory results is an adequate collection of the samples and transport under controlled conditions

14. Storage of Samples The samples should be stored at the appropriate temperature (-20°C, +4°C, room temperature) The use of a sample registration system is recommended Divide the samples in aliquots Repeated freezing and thawing of the samples should be avoided Take care of the following points during storage of the samples

15. Sample Preparation 1 Liquid Urine Milk Serum, Plasma Fruit juice Egg Honey Bile Saliva Solid Tissue (muscle, organs, sea food) Feed Silage Food (products) Retina Choroids Fat Hair Depending on the nature of the material the sample is either liquid (in all gradations) or solid

16. Sample Preparation 2 The sample should be a representative part of the total batch The sample should be as homogeneous as possible Be aware of matrix effects, i.e. high background values The sample should have the appropriate pH Be aware of the dilution factor Sample preparation demands attention to the following critical points

17. Sample Preparation 3 Methods used in sample preparation Dilution (aqueous buffer, organic solvent) pH adjustment Centrifugation Filtration Defatting Proteolytic treatment / Hydrolysis Liquid extraction (aqueous, organic) Solid phase extraction (SPE)

18. Performing the EIA Example: the chloramphenicol (CAP) kit Structure of chloramphenicol (CAP) detecting CAP in e.g. shrimps

19. Performing the CAP EIA 1 Planning Determine the number of samples and required amounts of reagents Determine the sample matrix Make a time frame Construct a pipetting scheme Place all kit components at room temperature prior to use Organise the appropriate equipment Prepare reagents exactly as described in the manual Carefully follow instructions as described in the manual Put samples in a pipetting bloc to facilitate the use of an 8 channel pipette Number the EIA strips to be used

20. Performing the CAP EIA 2 Assay Protocol Pipette the samples and standards into the relevant wells Add the CAP - HRP conjugate solution Add the anti - CAP antibody solution Shake the EIA plate for maximal 5 seconds Incubate at +4°C for 60 min (see manual) Wash the plate three times manually or in an automated washer Add the substrate solution Incubate for 30 min at 20-25°C Add the stop solution Shake the EIA plate for maximal 5 sec and measure the absorption at 450 nm

21. Performing the CAP EIA 3 1. The several reagents are added in order A, B, C Sheep anti-rabbit antibodies Microtitre plate well CAP standards, samples A Rabbit anti - CAP antibodies C CAP - HRP Conjugate B

22. 2.The plate is incubated for 60 min at 4˚C 3.The wells are then washed with washing buffer Two situations can now be considered: The sample contains a certain amount of CAP The sample contains no CAP Performing the CAP EIA 4

23. Performing the CAP EIA 5 Sample with CAP CAP-HRP conjugate as well as free CAP can bind to the anti-CAP antibodies Sample without CAP Only the CAP-HRP conjugate is available for binding to the anti- CAP antibodies

24. Performing the CAP EIA 6 4.The substrate/chromogen (H 2 O 2 /TMB) is added H 2 O 2 /TMB 5.The plate is incubated for 30 min at 20-25˚C During incubation the colourless chromogen is converted by the HRP enzyme into a blue reaction product

25. Performing the CAP EIA 7 The blue colour is inversely proportional to the amount of bound CAP. The more CAP present in the sample, the less colour is developed H 2 O 2 /TMB

26. Performing the CAP EIA 8 6.The colour development is stopped by adding 0.5 M of sulfuric acid (H 2 SO 4 ). In this acidic environment the blue colour changes into yellow

27. Performing the CAP EIA 9 7.The absorption of each well (OD) is measured at 450 nm 8.The amount of CAP in the samples is calculated according to the calculation program ‘simple fit’

28. Pitfalls 1 General Before performing the Euro-Diagnostica Food Safety EIA read the complete Instruction Manual Take care of proper sampling and storage of the samples Apply the appropriate sample treatment Treat all unknown samples as being potentially infective or toxic Turbid samples should be centrifuged or filtrated Do not use kit components that have past the expiry date Do not intermix kit components with different lot numbers

29. Pitfalls 2 General Use distilled water for preparation of the reagents Take care that all reagents are prepared and all equipment (including columns) are ready for use Completely dissolve all reagents: check for crystallisation or contaminations Apply appropriate washing procedures Avoid contact of the pipette tips with the coating of the wells All safety precautions, as valid in your laboratory and reflected in the instruction manual should be strictly followed

30. Pitfalls 3 Evaporation Evaporation is an essential process during the extraction Use a solid heating block, and adjustable taps, to regulate the nitrogen flow Avoid cross-contamination Organic solvents disturb the EIA. Take care to evaporate all solvent but avoid overheating of the residue (<60°C) Use glass tubes when applying organic solvents. Some agents, e.g. aminoglycosides, adhere to glass. In such cases use siliconised glass (see manual)

31. Pitfalls 4 Evaporation Use control samples to estimate the recovery of the entire process Take care that the dry residue is completely dissolved before applying to the EIA When columns or tubes are re-used take care for proper cleaning to avoid contamination Rinse with 100% methanol and wash with distilled water

32. Pitfalls 5 Matrix The nature of the matrix has a strong influence on the results in the EIA Dilution and/or extraction of samples reduce the matrix effect The pH value, salt concentration and protein contents of the samples influence the optical density value Optimal results are obtained when the standard line is made in the sample matrix

33. Pitfalls 6 Pipetting Prepare all reagents and equipment before starting the assay Too long pipetting time causes variation in incubation time, which influences the final results Use pipette formats in relation to the volumes to be used Use suitable pipette tips Do not damage the coating surface with pipette tips Use new pipette tips for each reagent

34. Pitfalls 7 Temperatures during the EIA performance Take care that the temperature is homogeneous For control use calibrated thermometers Incubate in a humid atmosphere To avoid evaporation from the wells use a closed incubation tray Minimize the edge effect e.g. by placing an empty strip besides the strip containing the standards or samples

35. Pitfalls 8 Temperature Incubation at 37°C ± 2°C Use an incubator with or without ventilation Do not place the EIA plate close to the heating element Incubation at room temperature Optimal 22°C ± 4°C. For higher or lower incubation temperatures it may be necessary to monitor the incubation times Incubation at +4°C The regular refrigerator temperature is 2-8°C The preferable temperature is 4-8°C Do not place the EIA plates close to the freezing element

36. Pitfalls 9 Wash procedure Take care of proper rinsing without causing damage to the coating When using an automatic washer, take care that all needles are open Tap out all rinsing buffer. Note that phosphates in the rinsing buffer disturb the enzyme reaction Take care that the wells do not dry out before the substrate solution is added

37. Pitfalls 10 Substrate/Chromogen (H 2 O 2 /TMB) TMB crystallises at 2-8°C TMB should be at room temperature before use and all crystals should be completely dissolved Blue colouring of the substrate in the pipetting tray is caused by contamination or light radiation. Do not use such substrate (contact Euro-Diagnostica) Incubate TMB in the dark. Any direct action of light should be avoided TMB should not come in contact with glass. Therefore, use plastic trays and vials

38. Pitfalls 11 Reading OD values The reader should be validated, and ready-for-use according to the instruction manual Check the presence of an appropriate filter (450 nm) Clean the bottom surface of the wells before reading Avoid air bubbles in the wells. Remove bubbles with a clean pipette tip Read the absorbance values immediately, within 30 min

39. Pitfalls 12 Validation of the results It’s important that the c.v. between the duplicates is not higher than 10% to 20%, depending on the concentration read from the standard curve Note that when OD values < 0.8 the curve will become very flat. In that case, the interpretation is difficult and the results are not acceptable For in-house quality control it is advised to analyse one positive and one negative sample in each series OD maximal signal > 0.8 OD blanc < 0.2

40. Pitfalls 13 Curve fitting Several curve fitting methods can be applied Euro-Diagnostica prefers a ‘3 parameter-fit’ model Check the calculation factors (result of sample treatment) If there are any suspected values, always check the raw OD values

41. For further information you may contact our local distribution partners For details, please visit our website: www.eurodiagnostica.com