Difference between revisions of "ELISA testing"
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==Introduction== | ==Introduction== | ||
[[Image:ELISA.jpg|thumb|right|150px|Double Antibody Sandwich ELISA]] | [[Image:ELISA.jpg|thumb|right|150px|Double Antibody Sandwich ELISA]] | ||
| − | The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual | + | The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual is positive for a specified pathogen, so is often the first test used in disease diagnosis. Utilizing the principle of antigen-antibody interaction, the test allows easy visualisation of results and, since its introduction in 1971, has quickly replaced radioimmunoassays for diagnostic purposes. |
*There are two basic types of ELISA: | *There are two basic types of ELISA: | ||
**'''Homogenous'''- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin | **'''Homogenous'''- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin | ||
| − | **''' | + | **'''Hetereogenous'''- various reagents added sequentially, primarily used to detect microbial antigens and antibodies |
This article describes the '''heterogenous''' type | This article describes the '''heterogenous''' type | ||
==Applications== | ==Applications== | ||
| − | *Detection and | + | *Detection and indentification of disease agents, e.g. type and subtype |
| + | *Quantification of disease agent, e.g. estimation of parasite load | ||
*Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies | *Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies | ||
| − | * | + | *Quanitification of specific antibody isotypes, e.g. IgM/IgG ratio |
==Techniques== | ==Techniques== | ||
| Line 20: | Line 21: | ||
*The addition of the chosen sample and reagents | *The addition of the chosen sample and reagents | ||
*Incubation and washing | *Incubation and washing | ||
| − | *The addition of enzyme- | + | *The addition of enzyme-labelled antigen/antibody |
*The addition of a specific substrate | *The addition of a specific substrate | ||
'''Competitive VS Non-competitive''' | '''Competitive VS Non-competitive''' | ||
| − | *As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of | + | *As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labelled antigen and unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labelled and unlabelled antibody. |
*Competitive techniques: | *Competitive techniques: | ||
**Easier to quantify | **Easier to quantify | ||
**Less likely to be influenced by contaminants | **Less likely to be influenced by contaminants | ||
| − | **However they are more demanding with regard to the accuracy of the reagents and the purity of the | + | **However they are more demanding with regard to the accuracy of the reagents and the purity of the labelled ligand |
*Non-competitive assays: | *Non-competitive assays: | ||
**Errors in dispensing reagents have little effect on the result | **Errors in dispensing reagents have little effect on the result | ||
| Line 35: | Line 36: | ||
'''Solid VS Fluid-phase''' | '''Solid VS Fluid-phase''' | ||
| − | |||
An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested: | An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested: | ||
| Line 42: | Line 42: | ||
*Solid-phase (''surface of protein-binding material, e.g. plastic'') | *Solid-phase (''surface of protein-binding material, e.g. plastic'') | ||
**Easier to perform and more sensitive | **Easier to perform and more sensitive | ||
| − | |||
| − | |||
| − | |||
| − | |||
===Non-competitive ELISA=== | ===Non-competitive ELISA=== | ||
| − | |||
'''Double Antibody Sandwich''' (for antigen detection) | '''Double Antibody Sandwich''' (for antigen detection) | ||
# Antibody is adsorbed onto solid phase | # Antibody is adsorbed onto solid phase | ||
# Wash | # Wash | ||
| − | # Sample | + | # Sample is added- specific antigen binds to antibody |
# Wash | # Wash | ||
| − | # Enzyme- | + | # Enzyme-labelled specific antibody is added- attaches to bound antigen |
# Wash | # Wash | ||
| − | # Enzyme substrate is added | + | # Enzyme substrate is added |
| − | ''visualised product = amount of antigen in the | + | ''visualised product = amount of antigen in the sample'' |
'''Antibody Class Capture Assay''' (for antibody detection) | '''Antibody Class Capture Assay''' (for antibody detection) | ||
# Class-specific antiglobulin is adsorbed onto solid phase | # Class-specific antiglobulin is adsorbed onto solid phase | ||
# Wash | # Wash | ||
| − | # Sample | + | # Sample is added- class-specific antibody in the sample binds to antiglobulin |
# Wash | # Wash | ||
| − | # Antigen is added - attaches to specific antibody | + | # Antigen is added- attaches to specific antibody |
# Wash | # Wash | ||
| − | # Enzyme- | + | # Enzyme-labelled antibody is added |
# Wash | # Wash | ||
# Enzyme substrate is added | # Enzyme substrate is added | ||
| − | ''visualised product = amount of specific antibody in | + | ''visualised product = amount of specific antibody in sample'' |
'''Indirect Method''' (for antibody detection) | '''Indirect Method''' (for antibody detection) | ||
| − | # | + | # Antigen is adsorbed onto solid phase |
# Wash | # Wash | ||
| − | # | + | # Sample is added- any specific antibody present in sample binds to antigen |
# Wash | # Wash | ||
| − | # Enzyme- | + | # Enzyme-labelled antiglobulin is added- attaches to antibody |
# Wash | # Wash | ||
# Enzyme substrate is added | # Enzyme substrate is added | ||
| − | ''visualised product = amount of antibody in the | + | ''visualised product = amount of antibody in the sample'' |
===Competitive ELISA=== | ===Competitive ELISA=== | ||
| Line 85: | Line 80: | ||
# Antigen is adsorbed onto solid phase | # Antigen is adsorbed onto solid phase | ||
# Wash | # Wash | ||
| − | # Enzyme- | + | # Enzyme-labelled antibody (pre-titrated- optimal colour development) and sample are added |
# Wash | # Wash | ||
# Enzyme substrate is added | # Enzyme substrate is added | ||
| − | ''visualised product = amount of | + | ''visualised product = amount of enzyme-labelled antibody'' |
'''Direct Antigen Competition''' (for antigen detection) | '''Direct Antigen Competition''' (for antigen detection) | ||
# Antigen is adsorbed onto solid phase | # Antigen is adsorbed onto solid phase | ||
# Wash | # Wash | ||
| − | # Antigen in | + | # Antigen in sample is incubated with enzyme-labelled antibody (again pre-titrated): this is directed against the antigen on the solid phase |
# Wash | # Wash | ||
# Enzyme substrate is added | # Enzyme substrate is added | ||
''visualised product = amount of enzyme-labelled antigen'' | ''visualised product = amount of enzyme-labelled antigen'' | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
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Revision as of 10:57, 22 August 2008
Introduction
The Enzyme Linked Immunosorbent Assay (ELISA) is an immunoassay commonly used to detect the presence of an antigen or antibody in a sample. It is a powerful tool in clinical immunology and can be used to determine whether an individual is positive for a specified pathogen, so is often the first test used in disease diagnosis. Utilizing the principle of antigen-antibody interaction, the test allows easy visualisation of results and, since its introduction in 1971, has quickly replaced radioimmunoassays for diagnostic purposes.
- There are two basic types of ELISA:
- Homogenous- completed in one step, all reagents added simultaneously. Primarily used to detect small molecules such as digoxin and gentamicin
- Hetereogenous- various reagents added sequentially, primarily used to detect microbial antigens and antibodies
This article describes the heterogenous type
Applications
- Detection and indentification of disease agents, e.g. type and subtype
- Quantification of disease agent, e.g. estimation of parasite load
- Identification of specific antibodies, e.g. used in serodiagnosis for epidemiological studies
- Quanitification of specific antibody isotypes, e.g. IgM/IgG ratio
Techniques
There are many methods of ELISA, but each assay involves these basic steps:
- The adsorption of antibody/antigen to solid phase (the medium)
- The addition of the chosen sample and reagents
- Incubation and washing
- The addition of enzyme-labelled antigen/antibody
- The addition of a specific substrate
Competitive VS Non-competitive
- As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labelled antigen and unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labelled and unlabelled antibody.
- Competitive techniques:
- Easier to quantify
- Less likely to be influenced by contaminants
- However they are more demanding with regard to the accuracy of the reagents and the purity of the labelled ligand
- Non-competitive assays:
- Errors in dispensing reagents have little effect on the result
- Therefore they are easier to control and yield accurate results
- However they are easily influenced by cross reactions and non-specific binding
Solid VS Fluid-phase
An important consideration is the medium in which the assay is carried out, largely dependent on what is being tested:
- Fluid-phase (in solution)
- Main advantage is that the behaviour of molecules in solution is easier to predict
- Solid-phase (surface of protein-binding material, e.g. plastic)
- Easier to perform and more sensitive
Non-competitive ELISA
Double Antibody Sandwich (for antigen detection)
- Antibody is adsorbed onto solid phase
- Wash
- Sample is added- specific antigen binds to antibody
- Wash
- Enzyme-labelled specific antibody is added- attaches to bound antigen
- Wash
- Enzyme substrate is added
visualised product = amount of antigen in the sample
Antibody Class Capture Assay (for antibody detection)
- Class-specific antiglobulin is adsorbed onto solid phase
- Wash
- Sample is added- class-specific antibody in the sample binds to antiglobulin
- Wash
- Antigen is added- attaches to specific antibody
- Wash
- Enzyme-labelled antibody is added
- Wash
- Enzyme substrate is added
visualised product = amount of specific antibody in sample
Indirect Method (for antibody detection)
- Antigen is adsorbed onto solid phase
- Wash
- Sample is added- any specific antibody present in sample binds to antigen
- Wash
- Enzyme-labelled antiglobulin is added- attaches to antibody
- Wash
- Enzyme substrate is added
visualised product = amount of antibody in the sample
Competitive ELISA
Direct Antibody Competition (for antibody detection)
- Antigen is adsorbed onto solid phase
- Wash
- Enzyme-labelled antibody (pre-titrated- optimal colour development) and sample are added
- Wash
- Enzyme substrate is added
visualised product = amount of enzyme-labelled antibody
Direct Antigen Competition (for antigen detection)
- Antigen is adsorbed onto solid phase
- Wash
- Antigen in sample is incubated with enzyme-labelled antibody (again pre-titrated): this is directed against the antigen on the solid phase
- Wash
- Enzyme substrate is added
visualised product = amount of enzyme-labelled antigen