EIA

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• EIAs
• (Enzyme Immunoassays) for Food Safety

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EIAs (Enzyme Immunoassays) for Food Safety

• EIAs 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.
•  

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EIAs for Food Safety
Competitive (Inhibition) EIAs For detection of
low molecular weight components (residues and
contaminants)
Non-Competitive (Sandwich) EIAs For detection
of high molecular weight components (proteins)
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Format of the Competitive EIA

• Residue X Target molecule
• Horseradish Peroxidase (HRP) Enzyme

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Target Molecules for the Competitive EIAs

• Drugs and steroids
• Antibiotics
• Mycotoxins

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Format of the Sandwich EIA

• Protein Y Target molecule
• Horseradish Peroxidase (HRP) Enzyme

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Target Molecules for the Sandwich EIAs

• Food allergens (proteins)
• Proteins involved in adulteration

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Production of Immunogens and Antibodies
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Antibody Purification
Rabbit Polyclonal or Mouse Monoclonal Antibodies
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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)

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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)

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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

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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

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Storage of Samples
Take care of the following points during storage
of the samples

• The samples should be stored at the appropriate
  temperature (-20C, 4C, 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

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Sample Preparation 1
Depending on the nature of the material the
sample is either liquid (in all gradations) or
solid
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
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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

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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)

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Performing the EIA

• Example the chloramphenicol (CAP) kit

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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

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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 4C 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 20-25C
• Add the stop solution
• Shake the EIA plate for maximal 5 sec and measure
  the absorption at 450 nm

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Performing the CAP EIA 3

• 1. The several reagents are added in order A, B,
  C

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Performing the CAP EIA 4

• The plate is incubated for 60 min at 4C
• 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

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Performing the CAP EIA 5

• Sample with CAP

Sample without CAP
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Performing the CAP EIA 6

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

5. The plate is incubated for 30 min at 20-25C
During incubation the colourless chromogen is
converted by the HRP enzyme into a blue reaction
product
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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
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Performing the CAP EIA 8

• The colour development is stopped by adding 0.5 M
  of sulfuric acid (H2SO4). In this acidic
  environment the blue colour changes into yellow

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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
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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

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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

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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 (lt60C)
• Use glass tubes when applying organic solvents.
  Some agents, e.g. aminoglycosides, adhere to
  glass. In such cases use siliconised glass (see
  manual)

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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

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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

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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

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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

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Pitfalls 8 Temperature

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

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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

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Pitfalls 10 Substrate/Chromogen (H2O2/TMB)

• TMB crystallises at 2-8C
• 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

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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

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Pitfalls 12 Validation of the results
OD maximal signal gt 0.8 OD blanc lt 0.2

• Its 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 lt 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

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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

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For further information you may contact our local
distribution partners

• For details, please visit our website
• www.eurodiagnostica.com