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ELISA

ENZYME-LINKED IMMUNOSORBENT ASSAY


 What is ELISA
 HISTORY
 PRINCIPLE
 PROCEDURE
 TYPES OF ELISA
What is ELISA
It is a test to detect and measure the presence of either antigen
or antibody in a sample .
HISTORY

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

•Antigen is coated onto the wells and incubation.


•Blocking agent is used to block the other binding sites
•The plates are washed with PBS-T to remove unbound
molecules.
•The enzyme conjugated antibody is added and incubated.
•The wells are washed again with PBS remove unbound
molecules.
•Substrate is added which detects the presence of the
enzyme.
Source: http://ruo.mbl.co.jp/bio/e/support/method/elisa.html
Indirect ELISA Procedure

•Antigen is coated onto the wells and incubation


•The plate is washed with PBS to remove unbound antigens.
•Blocking agent is used to block the other protein binding sites.
•Primary sample antibody is added to the plate and incubated with the
antigen.
•The plates are washed with PBS to remove unbound molecules.
•The secondary enzyme conjugated antibody is added and incubated.
•Wells are washed with PBS to remove unbound molecules.
•Substrate is added which detects the presence of the enzyme.
Source: http://ruo.mbl.co.jp/bio/e/support/method/elisa.html
Sandwich ELISA Procedure
•A known quantity of unlabeled antibodies are added to the wells and incubated.
•The antigen containing sample is then added to the plate.
•The plates are washed to remove unbound molecules.
•The primary antibodies are then added and incubated with the antigens.
•The plate is washed to remove unbound primary antibodies.
•Secondary antibody (the enzyme conjugated antibody) is added and incubated.
•The plates are washed so the unbound enzyme linked antibodies are removed.
•The substrates are added and incubated.
•Stop solution is added(if necessary) which terminates the reaction.
• The wells are then read at 450nm.
Source: http://ruo.mbl.co.jp/bio/e/support/method/elisa.html
Competitive ELISA Procedure
• An antibody specific for a target protein is coated onto the wells and incubation.
• The plates are washed to remove unbound molecules.
• Samples containing the target protein are added.
• The plates are washed to remove unbound molecules.
• A known amount of enzyme-labeled target protein are incubated with it.
• The plates are washed to remove unbound molecules.
• The substrates are added and incubated.
• After the reaction, the activity of the microplate well-bound enzyme is measured.

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.

Sandwich ELISA High specificity The antigen of interest must be large


enough so that two different antibodies
can bind to it at different epitopes

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

I wish to express my gratitude to Dr. Anindita Debnath for providing me an opportunity


to do this presentation.

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