Post on 19-Oct-2014
description
Enzyme Linked Immunosorbent Assay
ELISA
Enzyme-linked Immunosorbent Assay
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
ELISA Kits for avian Ab Detection
SYNBIOTICS
IDEXX
Definition of ELISA
A sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody. It is often used as a diagnostic test to determine exposure to a particular infectious agent (technique involving the reaction of the antigen or antibodies in vitro )
HISTORY
Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies.
Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.
ELISA objective
To detect the presence of an antigen or antibodies in a sample and to use it as a diagnostic tool in medicine.
To detect potential food allergens.
To be used in toxicology as a rapid presumptive screen for certain classes of drugs
Principle of ELISA
Principle of ELISA
Antibody is immobilized on micro-plate wells
Competition between in sample and labeled enzyme for antibody binding sites
The unbound material is washed out
Chromogenic substrate added to develop color
Resulting color is read
Component of ELISA
Antigen
Primary antibodies
Secondary antibodies
Enzyme
Substrate
Stop solution
• Equipments are widely available.
• No radiation hazards.
• Reagents are cheap with long shelf life.
• Adaptable to automation and high speed.
• Qualitative and quantitative.
• Reproducible.
• ELISA can be used on most types of
biological samples, such as plasma,
serum, urine, and cell extracts
• Sensitive assay
Types of ELISA
Direct method
In direct method
Sandwich method
Competitive method
Direct ELISA
Direct ELISA
The direct Enzyme-Linked Immunoabsorbent Assay (ELISA) is a method for detecting and measuring antigen concentration in a sample. Using a capture monoclonal antibody, the presence of a particular antigen in a sample is detected.
Advantage of direct ELISA
This type of ELISA has two main advantages:
It is faster, since fewer steps are required
It is less prone to error, since there are fewer steps and reagents
Indirect ELISA
Indirect ELISA is a two-step method that uses a primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation of the secondary antibody. But, this may give a nonspecific signal result because cross-reaction with the secondary antibody may occur
Antigen coated to a polystyrene multiwell plate is
detected in two stages or layers. First an unlabeled
primary antibody, which is specific for the
antigen, is applied. Next, an enzyme-labeled
secondary antibody is bound to the first antibody.
The secondary antibody is usually an anti-species antibody and is often polyclonal.
Advantage of indirect method
This method has several advantages: Increased sensitivity, since more than
one labeled antibody is bound per primary antibody
Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody
Cost savings, since fewer labeled antibodies are required
ELISA Results
Results should be recorded by reading the optical densities of the plates in a plate reader at the correct absorbance:
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
With the IDEXX kit S/P ratios of greater than 0.5 are considered positive
ELISA Results
Example:
Sample mean= 0.820
Negative control mean=0.053
Positive control mean=0.563
ELISA titer =(1.642xlog10 SP)+3.568
Values are relatively quantitative: a higher value indicates more antibody.
ELISA Laboratory
Materials Needed
ELISA plate
Record Sheet
Test samples
Dilution Tubes
Pipets and tips
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
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
ELISA Laboratory 2
1 2 3 4 5 6 7 8 9 10 11 12
A + - + 1 2 3 4 5 6 7 8 9
B - + -
C
D
E
F
G
H
Add 100μl of diluted test serum to the plate according to your record sheet
Incubate for 30 minutes
1 2 3 4 5 6 7 8 9 10 11 12
A + - + 1 2 3 4 5 6 7 8 9
B - + -
C
D
E
F
G
H
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
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
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
Calculation of Results
Average the 2 negative control wells
Average the 2 positive control wells
Average 2 wells for each sample
Calculation of Results
Example:
Sample mean= 0.820
Negative control mean=0.053
Positive control mean=0.563
S/P ratios of greater than 0.5 are considered positive
(Positive values will be different for each kit)
Sensitivity
ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody –antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results. As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive.
are negative controlsIf the results: positivegiving
Contamination of the substrate solution, enzyme-labelled antibody, control themselves.
Inadequate rinsing of plates.
Inadequate blocking of plates.
If no colour has developed for the positive controls or for the samples:
a. Check all reagents for dating and storage conditions.
b. Microwell plates not coated properly.
c. Reagents applied in wrong order or step omitted.
d. Enzyme conjugate defective or inhibited by contaminant.
If very little colour has developed for positive controls and the test samples:
a. Check the dilution of the enzyme labelled antibody.
b. The concentration of the substrate.
c. Wash buffer not adequately drained after every wash step.
d. Inadequate incubation times.
e. Enzyme conjugate defective or inhibited by contaminant, Substrate defective or contaminated,
f. Micro well plates poorly coated.
If colour has developed for the test samples but
not the positive controls:
Check the source of positive controls, their
expiry date and storage.
If the colour can be seen, but the absorbance is
not high as expected, check the wave length.