Changes

*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

**'''Heterogenous'''- 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 indentification of disease agents, e.g. type and subtype

*Detection and identification of disease agents, e.g. type and subtype

*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

*Quantification of specific antibody isotypes, e.g. IgM/IgG ratio

==Techniques==

==Techniques==

*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-labelled antigen/antibody

*The addition of enzyme-labeled 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 labelled antigen and an 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.

*As their name implies, competitive assays measure the competition between a pre-titrated (fixed amount) of labeled antigen and an unknown quantity of sample antigen in their affinity to an antibody. The process can be reversed to measure the competition between labeled and unlabeled 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 labelled ligand

**However they are more demanding with regard to the accuracy of the reagents and the purity of the labeled 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

# Sample is added- specific antigen binds to antibody

# Sample is added- specific antigen binds to antibody

# Wash

# Wash

# Enzyme-labelled specific antibody is added- attaches to bound antigen

# Enzyme-labeled specific antibody is added- attaches to bound antigen

# Wash

# Wash

# Enzyme substrate is added

# Enzyme substrate is added

# Antigen is added- attaches to specific antibody  

# Antigen is added- attaches to specific antibody  

# Wash

# Wash

# Enzyme-labelled antibody is added

# Enzyme-labeled antibody is added

# Wash

# Wash

# Enzyme substrate is added

# Enzyme substrate is added

# Sample is added- any specific antibody present in sample binds to antigen

# Sample is added- any specific antibody present in sample binds to antigen

# Wash

# Wash

# Enzyme-labelled antiglobulin is added- attaches to antibody

# Enzyme-labeled antiglobulin is added- attaches to antibody

# Wash

# Wash

# Enzyme substrate is added

# Enzyme substrate is added

# Antigen is adsorbed onto solid phase

# Antigen is adsorbed onto solid phase

# Wash

# Wash

# Enzyme-labelled antibody (pre-titrated- optimal colour development) and sample are added

# Enzyme-labeled antibody (pre-titrated- optimal colour development) and sample are added

# Wash

# Wash

# Enzyme substrate is added

# Enzyme substrate is added