Enzyme-Linked%20Immunosorbent%20Assay%20(ELISA)

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Enzyme-Linked Immunosorbent Assay (ELISA)

• Mary Lea Killian
• USDA APHIS VS
• National Veterinary Services Laboratories
• Ames, Iowa

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Enzyme-linked Immunosorbent Assay
http//microvet.arizona.edu/Courses/MIC419/ToolBox
/elisa.html
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ELISA Kits for Antibody Detection

• Commercial ELISA test kits are available to
  detect
• Avian influenza virus antibody in chicken serum
• Newcastle disease virus antibody in chicken serum
• Newcastle disease virus antibody in turkey serum

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ELISA Kits for AIV Ab Detection
IDEXX
SYNBIOTICS
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ELISA Results

• Results should be recorded by reading the optical
  densities of the plates in a plate reader at the
  correct absorbance IDEXX 650nm
• Each manufacturer supplies computer software
  specific for their test which calculates which
  samples are negative and the titers of positive
  samples.

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ELISA Results

• The status of a sample are evaluated by the
  sample to positive ratio (S/P ratio)
• Sample mean - negative control mean
• positive control mean - negative control mean
• (mean of optical absorbance)

With the IDEXX kit S/P ratios of greater than
0.5 are considered positive
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ELISA Results

• Example
• Sample mean 0.820
• Negative control mean0.053
• Positive control mean0.563

Values are relatively quantitative a higher
value indicates more antibody.
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Valid ranges for the positive and negative
controls

• Negative control 0.150 or less
• The difference between the positive and negative
  control means must be greater than 0.075
• Example if negative control 0.100, the
  positive control must be 0.176 or greater.

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ELISA Laboratory

• Materials Needed
• AIV ELISA plate
• Record Sheet
• Test samples (same set as AGID)
• Dilution Tubes
• Pipets and tips

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Materials Needed

• The materials for your kit
• ELISA plate
• Positive control
• Negative control
• Dilution Buffer (already in dilution tubes)
• Conjugate (secondary antibody)
• TMB Substrate
• Stop solution

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ELISA Laboratory 1

• Label dilution tubes
• Add 1ml of diluent to dilution tubes (done)
• Add 2µl of test serum to a dilution tube
• Do NOT dilute controls

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ELISA Laboratory 2

• Add 100 µl negative control to wells A1 and A2
• Add 100 µl positive control to wells A3 and A4

1 2 3 4 5 6 7 8 9 10 11 12
A - - 1 1 2 2 3 3 4 4
B 5 5 6 6 7 7 8 8 9 9 10 10
C
D
E
F
G
H
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Add 100µl of diluted test serum to the plate
according to your record sheet
1 2 3 4 5 6 7 8 9 10 11 12
A - - 1 1 2 2 3 3 4 4
B 5 5 6 6 7 7 8 8 9 9 10 10
C
D
E
F
G
H

• Incubate for 30 minutes

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ELISA Laboratory 3

• Wash with 350 µl distilled water (three times)
• Add 100 µl of conjugate to test wells on your
  plate
• Incubate for 30 minutes

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ELISA Laboratory 4

• Wash with distilled water (3 times)
• Add 100 µl of TMB substrate to each well
• Incubate for 15 minutes
• Add 100 µl of stop solution to each well
• Read results

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Interpretation of Results

• Negative control 0.150 or less
• The difference between the positive and negative
  control means must be greater than 0.075
• Example if negative control mean 0.100, the
  positive control mean must be 0.176 or greater

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Calculation of Results

• Average the 2 negative control wells
• Average the 2 positive control wells
• Average 2 wells for each sample

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Calculation of Results

• Example
• Sample mean 0.820
• Negative control mean0.053
• Positive control mean0.563

S/P ratios of greater than 0.5 are considered
positive (Positive values will be different for
each kit)