Enzyme-Linked Immunosorbent Assay 

An Enzyme-linked Immunosorbent Assay (ELISA) is a biochemical analysis method used 

to detect concentrations of antibodies (a protein produced by an individual’s immune 

system found in the blood to counteract a specific antigen). This technique is widely 

considered the “gold standard” of assays due to its consistency and easily interpreted 

results. This technique can identify antibodies, antigens (proteins from outside intruders to 

the body), proteins, glycoproteins, and hormones. ELISA is commonly used to test the 

concentration of Human Immunodeficiency Virus antibodies in a person or more recently 

to test if an individual has contracted SARS-Covid-19 (CoronaVirus) in the past. This 

technique cannot and should not be used to test if an individual currently has a virus.  

History 
Two separate teams invented the Direct ELISA technique at the same time. ELISA came 

about from the modification of a similar process called radioimmunoassay (RIA). The 

critical difference is attaching an enzyme flag rather than using iodine 125[3][5]. This 

change reduces radioactive materials used in a laboratory while producing more reliable 

results. Older versions of an ELISA used color as flag, however more modern ELISA use 

flags that are easier to quantify at a higher fidelity such as fluorescent tags or generation of 

energy in heat. 

 
Background  
The basis of ELISA lies in the interaction antibodies and antigens have. Once an antigen (a 

virus or another intruder) enters the body, it reacts by making an antibody that 

compliments the intruder. The antibodies generated fits with the antigen like a lock and 

key. The antigen “key” fits with the developed antibody “lock.” 

The next question is: what do we need to test to see if there are antibodies? The answer: 

most fluids from the body. Blood and saliva are the most common things tested. 

Procedures and Mechanism 

There are four base ways scientists perform an ELISA immunoassay. All four use the same 

foundational techniques to reach similar outcomes. Each starts with a coating step where 

either an antibody or antigen coats the wells of a microplate, then a blocking step to fill the 

gaps left from the coating step. Next, a detection step is followed by a final reading of the 

results[1].  

 
  

Figure 1 Empty 96 Well Microplate 

Lilly_M, CC BY-SA 3.0 <https://creativecommons.org/licenses/by-sa/3.0>, via Wikimedia Commons 

Figure 1 depicts an example of an empty microplate. A pipette ( an apparatus with a slim tube typically with a bulb used to transfer 

liquid samples) fills each well with varying substances. 

Direct ELISA: 

The antigen is fixed on the microplate, and only the primary antibody is needed. The 

enzyme linked to the primary antibody reacts with an added substrate[1]. The substrate is 

another chemical added to the reaction. The reaction occurs between the enzyme and the 

substrate. If there is a reaction, antibodies are present. However, if no response occurs, 

there are no antibodies present.  

 
Figure 2 Direct ELISA [2] 

Indirect ELISA: 
The antigen is fixed to the microplate (blue circle in Figure 3) like a direct ELISA and a 

primary bonds to that antigen (blue Y-shape in Figure 3). However, in an indirect ELISA, a 

secondary antibody tagged with an enzyme (yellow Y-shape in Figure 3) bonds to the 

primary antibody.   

 
Figure 3 Indirect ELISA [2] 

Sandwich ELISA: 

An antibody is immobilized by a capture antibody that is fixed to the microplate with a 

primary and secondary antibody binding to the antigen. Referred to as a Sandwich ELISA 

because the antigen is in between two antibodies. 

 
Figure 4: Sandwich ELISA [2] 

Competitive ELISA: 

An inhibitor antigen is also added, and all previously mentioned ELISA can be adapted to 

fit in a competitive ELISA. Unlike the previous kinds of ELISA depicted above, the first step 
is to coat the wells with an inhibitor antigen. Another difference is that the antigen of 

interest is incubated with the primary antibody to form a bond like in a Direct ELISA. The 

only difference is this bonding does not occur in the well.  

 
Figure 5: Competitive ELISA [2] 

Pros and Cons of the Different ELISA 

Direct ELISA: 

The Direct ELISA, like the name says, has a direct procedure and is time and reagent 

saving. It is simple compared to the others. Since no secondary antibody is needed there is 

no change for cross-reactivity with the substrate near the end.  

However, a Direct ELISA has no signal amplification since only a primary antibody is used 

and is not as flexible as other forms of ELISA. 

Indirect ELISA: 
A secondary antibody amplifies the signal and is highly flexible since the same antibody 

can bind to various different primary antibodies. 

Being able to bond to different primary antibodies also leaves room for cross-reactivity. 

This ELISA is also more complex than the Direct ELISA 

Sandwich ELISA: 

A sandwich ELISA is very flexible and very sensitive. This is especially sensitive since only 

a limited number of antibodies can bind to the same antigen[5].  

The target antibody must be large in size to allow for two antibodies to bind to it. Also 

finding two different antibodies that can bond to an antigen and suitable for ELISA is 

difficult. 

Competitive ELISA: 

This type is well suited for smaller antigens or for samples with low concentrations. 

However, the inclusion of an inhibitor antigen makes this easily one of the more complex 

protocols[6]. 

Uses 
The ELISA immunoassay is a valuable tool that simply tests for the presence of a protein. 

This broad test group can identify many things and is not at all limited to the measurement 

of antibodies. Other than tracking past exposures from a variety of viruses or tracking 

disease outbreaks, it can also estimate hormone levels such as Testosterone or Prolactin, 
screen donated blood for viral contaminants, and detect drug abuse- namely 

Methamphetamine, Cocaine, and Benzoylecgonine. 

Limitations 
Utilizing ELISA also has its risks. At the forefront, human error is always a possibility and 

can never be fully eradicated, only minimized. The tools used in the assay affects the 

quality of the results as well. The shape of the wells in the plate, the material the wells are 

made. 

In terms of what can be tested, only past infections can be tested. Antibodies are 

generated post-infection. To test for an ongoing infection another quantitative test called 

RT-PCR is required.  

   
  

 
[1] Alhajj, M., & Farhana, A. (2020). Enzyme Linked Immunosorbent Assay (ELISA). 

Retrieved 15 September 2020, from 

https://www.ncbi.nlm.nih.gov/books/NBK555922/ 

[2] Four Types of ELISA-CUSABIO, (2020). Diagram of ELISA [Image]. Retrieved from Four 

Types of ELISA-CUSABIO 

[3] Hosseini S., Vázquez-Villegas P., Rito-Palomares M., Martinez-Chapa S.O. (2018) 

Fundamentals and History of ELISA: The Evolution of the Immunoassays Until 

Invention of ELISA. In: Enzyme-linked Immunosorbent Assay (ELISA). SpringerBriefs 

in Applied Sciences and Technology. Springer, Singapore. 

https://doi.org/10.1007/978-981-10-6766-2_1 

[4] Musiani, M., Gallinella, G., Venturoli, S. et al. Competitive PCR–ELISA protocols for the 

quantitative and the standardized detection of viral genomes. Nat 

Protoc 2, 2511–2519 (2007). https://doi.org/10.1038/nprot.2007.312 

[5] Rudolf M Lequin, Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay 

(ELISA), Clinical Chemistry, Volume 51, Issue 12, 1 December 2005, Pages 

2415–2418, https://doi.org/10.1373/clinchem.2005.051532 

[6] ("What is ELISA Mechanism? | Sino Biological", 2020)