Enzyme-linked immunosorbent assay (ELISA) Sarah Cheah March 6 th , 2020
Enzyme-linked
immunosorbent assay (ELISA)
Sarah Cheah
March 6th, 2020
Outline
❖ Introduction
❖ History of ELISA
❖ General Principles of ELISA
❖ Application of ELISA
❖ Current development on ELISA
What is ELISA
▪ A type of immunoassay used to detect and measure certain target molecule in a
mixture solution
▪ Qualitative (detect presence) or/and Quantitative (what is the level of molecule
present)
▪ Based on antibody-antigen reaction, they bind together to form complex
▪ The complex is then labelled with enzyme-linked antibody which produce colored
product upon addition of substrate
https://www.bosterbio.com/protocol-and-troubleshooting/elisa-
principle
https://www.sciencesource.com/archive/AIDS-Testing--ELISA-
plate-assay-SS2133121.html
Sakamoto, Seiichi et al. “Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of
plant secondary metabolites.” Journal of natural medicines (2018)72,1: 32-42.
Some history of ELISA ….RIA
❖ Radioimmunoassay (RIA) was developed in 1960 by Berson and Yalow.
❖ Radio-labelled insulin is used to measure the concentration of insulin in human plasma
❖ Based on competition between labelled insulin and unlabeled (natural) insulin
❖ The more natural insulin, the less radiolabeled insulin bind to the antibody
❖ The radiation level is inversely proportional to the
level of natural insulin in the human plasma
R. D. Grange, J. P. Thompson, D. G. Lambert, Radioimmunoassay, enzyme and non-enzyme-based
immunoassays, BJA: British Journal of Anaesthesia(2014)112,2 : 213–216
Advantages and Drawbacks of RIA
Advantages
❖ Precise
❖ Very sensitive
Drawbacks
❖ Short-life reagents
❖ Expensive equipment
❖ Health concerns
R. D. Grange, J. P. Thompson, D. G. Lambert, Radioimmunoassay, enzyme and non-enzyme-based
immunoassays, BJA: British Journal of Anaesthesia(2014)112,2 : 213–216
General principle of ELISA ❖ Solid phase is needed => 96 well plate
❖ Antigen or Primary Antibody (liquid phase) is immobilized on the solid phase
❖ Add sample containing analyte of interest
❖ Enzyme-linked secondary antibody is added (depends on type of ELISA)
❖ Types of enzymes used: 1. Alkaline Phosphates (substrate: p-nitrophenyl
phosphate)
2. Horseradish Peroxidase
(substrate: chromogenic substrate/chemiluminescent
substrates)
❖ The reaction usually takes 30-60min
❖ Add HCl/NaOH (stop solution) to stop the enzyme activity and stop the reaction
❖ The products are detected using a microtiter plate reader
https://www.laboratory-
equipment.com/microplate-reader-
agilereader-absorbance-actgene-8000-
54.html
https://www.bio-rad.com/en-
ca/product/96-well-eia-
plates?ID=b863247f-3c8e-4b65-
8c64-d300cd9f7460
Sakamoto, Seiichi et al. “Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of
plant secondary metabolites.” Journal of natural medicines (2018)72,1: 32-42.
https://www.moleculardevices.com/applications/enzyme-linked-immunosorbent-assay-elisa#gref
Blocking step Stop solution
Direct ELISA
❖ Target antigen is immobilized on solid phase
❖ Primary antibody/antigen is enzyme-linked
❖ Secondary antibody is not involved
❖ For qualitative analysis of macromolecules
❖ Analyze the immune response to antigen
❖ Pros : less prone to errors (less reagent/steps)
❖ Cons : - no signal amplification (less sensitive)
- antigen/antibody immobilization not specific
(higher background noise)
Sakamoto, Seiichi et al. “Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of
plant secondary metabolites.” Journal of natural medicines (2018)72,1: 32-42.
Indirect ELISA
❖ Target antigen is immobilized on solid phase
❖ Sample containing primary antibody against target
antigen added
❖ Secondary enzyme-linked antibody against primary
antibody added
❖ To find presence of antibody in antisera following
exposure to disease (vaccination)
❖ Pros : -highly sensitive
-more flexibility
❖ Cons : - longer procedure
- secondary antibody may cross-react
Sakamoto, Seiichi et al. “Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of
plant secondary metabolites.” Journal of natural medicines (2018)72,1: 32-42.
Competitive ELISA ❖ Competitive interaction between target molecule in
sample and enzyme-linked molecule
❖ Target antibody, antigen that is immobilized on the solid
phase
❖ Add sample mixture to the wells
❖ Add enzyme-linked antibody to the wells
❖ With increasing amount of natural antibody, the signal
decrease
❖ Used to measure the concentration of target molecule
❖ Pros: - easy , no secondary antibody required
- no sample preparation required
❖ Cons: - labelling the antibodies may render its inactivation
Sakamoto, Seiichi et al. “Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of
plant secondary metabolites.” Journal of natural medicines (2018)72,1: 32-42.
Sandwich ELISA ❖ Immobilization of capture antibody on the solid phase
❖ Antigen of interest in sample mixture will bind to capture
antibody
❖ Detection antibodies is added and will bind to antigen at a
different epitope
❖ Direct or indirect Sandwich ELISA
❖ Pros: - highly sensitive
- highly specific
❖ Cons: - expensive
- labor-intensive
https://www.aatbio.com/catalog/enzyme-
linked-immunosorbent-assay-elisa-direct-
indirect-and-sandwich-assays
Sakamoto, Seiichi et al. “Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of
plant secondary metabolites.” Journal of natural medicines (2018)72,1: 32-42.
Application of ELISA
Disease diagnostic (HIV, cancer, viral infection, food allergy)
Detection and quantitation of antibodies in response to infection (vaccine design)
Detection and quantitation of antigens of interest (Pregnancy kit)
Detect allergen in commercial products
Research laboratories
Test for crop allergens
Manenti, A.; Maciola, A.K.; Trombetta, C.M.; Kistner, O.; Casa, E.; Hyseni, I.; Razzano, I.;Torelli, A.; Montomoli, E. Influenza Anti-StalkAntibodies: Development of a New Method for the Evaluation of the Immune Responses to Universal Vaccine. Vaccines 2020, 8, 43.Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with special reference to ELISA techniques. J Clin Pathol. 1978;31(6):507–520
https://en.wikibooks.org/wiki/Method
s_and_Concepts_in_the_Life_Science
s/Immunoassays
Influenza A & B => annual epidemics in human race due to
antigenic change at hemagglutinin (HA) and neuraminidase (NA)
antibody binding site (antigen drift)
Problems:
-Composition of vaccines needs to be updated every year (takes
6-8 months)
-Current available influenza vaccine not able to protect against
emerging influenza virus
Universal Vaccine : vaccine against stalk domain(HA2) of HA
Pros: -avoid the need of annual re-formulation and re-
administration of vaccine
-allow time for more effective vaccine to be made in event
of pandemic
https://www.vanderbilt.edu/vicb/DiscoveriesArchiv
es/targeting_flu_through_host_protein.html
https://microbenotes.com/differences-
between-antigenic-shift-and-antigenic-drift/
Manenti, A.; Maciola, A.K.; Trombetta, C.M.; Kistner, O.; Casa, E.; Hyseni, I.; Razzano, I.;Torelli, A.; Montomoli, E. Influenza Anti-StalkAntibodies: Development of a New Method for the Evaluation of the Immune Responses to Universal Vaccine. Vaccines 2020, 8, 43.
Manenti, A.; Maciola, A.K.; Trombetta, C.M.; Kistner, O.; Casa, E.; Hyseni, I.; Razzano, I.;Torelli, A.; Montomoli, E. Influenza Anti-StalkAntibodies: Development of a New Method for the Evaluation of the Immune Responses to Universal Vaccine. Vaccines 2020, 8, 43.
• Competitive ELISA => able to detect stalk specific
antibodies
• Standard serological assay => insufficient to detect
stalk-specific antibodies accurately (stalk-induced
antibodies have various functions)
• These methods could only detect HA1 domain
• Limitation: - not able to tell if the antibodies bound
are functional (Need to couple with
other assay, not able to tell whether
antibodies detected are alive or dead)
Results
Manenti, A.; Maciola, A.K.; Trombetta, C.M.; Kistner, O.; Casa, E.; Hyseni, I.; Razzano, I.;Torelli, A.; Montomoli, E. Influenza Anti-StalkAntibodies: Development of a New Method for the Evaluation of the Immune Responses to Universal Vaccine. Vaccines 2020, 8, 43.
Advantages and Disadvantages of ELISA
Advantages
Simple procedure
High specificity and sensitivity
High efficiency
Simultaneous analysis can be
performed
Safe and eco-friendly
Cost-effective
Disadvantages
Labor-intensive
Antibody is expensive
Possibility of false positive/negative
Antibody instability
Antibody needs to be refrigerated
Sakamoto, Seiichi et al. “Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of
plant secondary metabolites.” Journal of natural medicines (2018)72,1: 32-42.
Current development of ELISA
Automation of ELISA assays (Agilent Bravo Platform)
- coating/delivery of reagent/plate washing
-to reduce time and steps
Instant ELISA
-add sample to pre-coated ELISA
-one hour, one wash
New technique? CyVek Analyzer
-just add sample into cartridge & go!
Comley, J. Enzyme-linked Immunosorbent Assays(ELISA): Recent Innovations Take Analyte Detection To New
Levels. Drug Discovery World(DDW). 2012. https://www.ddw-online.com/screening/p191009-enzyme-linked-immunosorbent-assays-(elisa):-recent-innovations-take-analyte-detection-to-new-levels.html
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