1. Lab # 6
ELISA
Enzyme – Linked Immuno
Sorbent Assay

2. Definition :
The ELISA can be used both qualitatively and quantitatively to measure antigen – antibody binding . Depending on what variation you use , it will detect antigen (hormones, enzymes , microbial antigens , illicit drugs ) or antibody ( anti – HIV in the screening test for HIV infection ) in body fluids or tissue culture supernatant.

3. ELYSA tools :
Purified antigen ( if you want to detect or quantify antibody )
Purified antibody ( if you want to detect or quantify antigen).
Standard solutions ( positive and negative controls)
Sample to be tested .
Microtiter dishes : plastic trays with small wells in which the assay is done.
Wash fluid (buffer)
Enzyme – labeled antibody and enzyme substrate.
ELISA reader ( spectrophotometer ) for quantitative measurements.

4. Direct (Sandwich ELISA)
Indirect
To detect antibody
Direct (Sandwich ELISA)
To detect antigen

6. How to Interpret the results :
The amount of colored product is proportional to the amount of enzyme- linked antibody that binds , which is directly related to the amount of antibody that was present to bind antigen or antigen that was present to bind antibody . If known amounts of antigen or antibody are added, a standard curve can be constructed which will allow the amount of unknown antigen or antibody to be determined .

7. Special proprieties of ELISA :
Versatility .
Sensitivity ( ability to detect small amount of antigen or antibody )
Specify ( ability to discriminate between closely related but antigenically different molecules .
Ease of automation.