PowerPoint Presentation

							EIA’s
(Enzyme Immunoassays) for Food Safety
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.
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)
Format of the Competitive EIA
• Residue X = Target molecule
• Horseradish Peroxidase (HRP) = Enzyme



                            Residue X - HRP conjugate

                            Residue X (standard/sample)

                            Rabbit antibody to residue X

                            Sheep anti-rabbit antibody

                            Microtitre plate well
Target Molecules for the Competitive EIA’s

• Drugs and steroids

• Antibiotics

• Mycotoxins
Format of the Sandwich EIA
• Protein Y = Target molecule
• Horseradish Peroxidase (HRP) = Enzyme




                           Rabbit anti-protein Y-
                           HRP conjugate

                            Protein Y (standard/sample)

                            Biotinylated rabbit anti-
                            protein Y

                            Streptavidin coated
                            microtitre plate well
Target Molecules for the Sandwich EIAs

• Food allergens (proteins)

• Proteins involved in adulteration
Production of Immunogens and Antibodies
         Residue X




 Coupling to a carrier protein      Protein Y
        (BSA, BTG)




           Immunisation of mice or rabbits




                                    Antibody production
  Antibody Purification
Rabbit Polyclonal or Mouse Monoclonal Antibodies



        Ammonium Sulfate Precipitation


        Immunoaffinity Chromatography


               Antibody Fraction         Competitive Assay



                  Conjugation
                to HRP or biotin         Sandwich Assay
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
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)




               +


 Antibody          HRP               Anti-protein Y- HRP
to protein Y                              conjugate
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
Collection of Samples and Transport
The basis for obtaining good laboratory results is an
adequate collection of the samples and transport under
controlled conditions

• 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
Storage of Samples
Take care of the following points during storage of the
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
Sample Preparation 1
Depending on the nature of the material the sample
is either liquid (in all gradations) or solid

Liquid                Solid
Urine                 Tissue (muscle, organs, sea food)
Milk                  Feed
Serum, Plasma         Silage
Fruit juice           Food (products)
Egg                   Retina
Honey                 Choroids
Bile                  Fat
Saliva                Hair
Sample Preparation 2
Sample preparation demands attention to the following
critical points

• 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 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)
Performing the EIA
Example: the chloramphenicol (CAP) kit


                                           OH   H
                                                N       CHCl2
                                                    C
                                              CH2   O
                                O2N
                                              OH
                            Structure of chloramphenicol (CAP)




detecting CAP in e.g. shrimps
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
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
Performing the CAP EIA 3
1. The several reagents are added in order A, B, C


                    Rabbit anti - CAP antibodies
       C

       B            CAP - HRP Conjugate



                    CAP standards, samples
       A

                           Sheep anti-rabbit antibodies
                           Microtitre plate well
Performing the CAP EIA 4
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 5
  Sample with CAP          Sample without CAP




CAP-HRP conjugate as      Only the CAP-HRP
 well as free CAP can   conjugate is available
 bind to the anti-CAP   for binding to the anti-
      antibodies            CAP antibodies
Performing the CAP EIA 6

4.   The substrate/chromogen (H2O2/TMB) is added

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

                       H2O2/TMB
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




                          H2O2 /TMB
Performing the CAP EIA 8
6. The colour development is stopped by adding 0.5 M of
   sulfuric acid (H2SO4). In this acidic environment the blue
   colour changes into yellow
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’
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
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
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)
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
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
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
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
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
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
Pitfalls 10 Substrate/Chromogen (H2O2/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
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
Pitfalls 12 Validation of the results
OD maximal signal > 0.8
OD blanc < 0.2


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