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

Question