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New page: ELISA ==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 i...
ELISA
==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
===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''
==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
===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''