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The enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) was developed
independently and simultaneously by the research group of Peter Perlmann and Eva Engvall at
Stockholm University in Sweden and by the research group of Anton Schuurs and Bauke van
Weemen in The Netherlands.
Source: http://clinchem.aaccjnls.org/content/51/12/2415.short
PRINCIPLE
In ELISA, the presence of antigen or antibody is measured by the antigen-antibody interactions , an antigen or
antibody are labeled with enzyme, and enzyme activity is measured colorimetrically.
The enzyme activity is measured using a substrate that changes colour when modified by the enzyme. Light
absorption of the product is measured and converted to numeric values.
Source: http://ruo.mbl.co.jp/bio/e/support/method/elisa.html
PROCEDURE
- Antigen solution are added to individual wells of a microtiter plate.
- Incubate the plate for 2 hours at 37°C or overnight at 4°C.
- Remove the coating solution and wash the the wells with PBS buffer.
- Block the remaining protein-binding sites in the coated wells by adding blocking buffer.
- Incubate for 1 hour at Room Temperature.
- Wash the plate with PBS buffer.
- Add diluted enzyme linked-antibody to each well.
- Incubate the plate at 37℃ for an hour with gentle shaking.
- Wash the plate with PBS buffer.
- Dispense the substrate solution per well with a multichannel pipe.
- Incubate the plate at 37℃ in dark for 15-30mins.
- After sufficient color development, add stop solution to the wells (if necessary).
- Read the absorbance (optical density at 450nm) of each well with a plate reader.
Source: https://www.avivasysbio.com/protocols-procedures/elisa
TYPES OF ELISA
Direct
Indirect
Sandwich
Competitive
Direct ELISA Procedure
When the antigen level in the sample is high, the level of antibody-bound enzyme-labeled antigen is lower
and the color is lighter. Conversely, when it is low, the level of antibody-bound enzyme-labeled antigen is higher
and the color, darker.
Source: http://ruo.mbl.co.jp/bio/e/support/method/elisa.html
Advantages disadvantages
Direct ELISA Simple protocol No signal amplification, since only a
time-saving primary antibody is used and a
secondary antibody is not needed
Signal amplification, since one or more Complex protocol compared with direct
Indirect ELISA secondary antibodies can be used to ELISA
bind to the primary antibody.
Competitive ELISA Best for the detection of small antigens, Relatively complex protocol.
even when they are present in low Needs the use of inhibitor.
concentrations.
ACKNOWLEDGEMENT