Top 5 Steps Involved in Elisa Testing

Top 5 Steps Involved in Elisa Testing

The enzyme-linked immunosorbent assay, also known as EIA or popularly called ELISA, is a test to determine and quantify antibodies in biological systems, including the human body. With this test, scientists and researchers are capable of detecting whether your blood contains antibodies associated with specific infectious diseases and medical conditions. 

Antibodies are protein molecules produced by the body as part of a defense mechanism against harmful substances known as antigens. 

Some of the diagnoses in which Elisa tests are helpful include the following:

  • Lyme disease
  • HIV that leads to AIDs
  • Syphilis
  • Zika virus
  • Pernicious anemia
  • Squamous cell carcinoma
  • Toxoplasmosis
  • Varicella-zoster virus
  • Rocky Mountain spotted fever
  • Rotavirus

In all these cases, diagnoses with the Elisa test are carried out using several procedures. This post breaks down these procedures into five distinct steps.

Continue reading to learn how to do Elisa in five steps. 

Step #1 Plate Coating

Step #1 Plate Coating
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Plate coating is the first step in most Elisa tests, regardless of whether direct, indirect, or sandwich Elisa. In the procedure, experts immobilize specific capture antibodies on high protein-binding plates by incubating them overnight. The antigens are rendered stationary to the surface of polystyrene microplate wells. The entire procedure can be broken down as follows:

  • Dilution of the antigen using the coating buffer. 
  • The 96 well plates are coated with 100 µl/well of the antigen by serially diluting the plates. 
  • Plates covered and incubated overnight at 4°C. 
  • Get rid of the discard coating solution and use a solution like wash buffer to rinse plates no less than three times. 

Step #2 Plate Blocking

Irrelevant proteins such as albumin are used to block the plates. These proteins or other similar molecules help cover every unsaturated surface-binding site of the microplate wells. Despite being a relatively shorter procedure, it can further be separated into three processes that are covered below. 

  • Block every leftover protein-binding site found on the coated wells with 200 µl/well blocking buffer. 
  • The whole set is covered and incubated for one to two hours at room temperature.
  • Using a wash buffer, wash your plate thoroughly at least three times.

Step #3 Antibody Incubation

Step #3 Antibody Incubation
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This incubation involves antigen-specific antibodies that bind through affinity to the antigens. This step consists of the addition of standard dilutions and samples to the wells, which the bound antibodies will later catch. Like the previous steps, this entire procedure also occurs systematically and can be further broken down into three processes, as stated below. 

  • Dilute 100 µl/well of antibody in blocking buffer and add it. But remember that the end-user has to determine the suitable antibody, preferably purified antibodies will require 0.1 µg/ml final concentration. 
  • The whole setup is covered and incubated for between one to two hours at room temperature.
  • With a wash buffer, wash your plate at least three times thoroughly. 

Step #4 Detection

At this point, the wells will receive a specific biotinylated detection antibody to help detect the Custom Protein Synthesis that was captured. As the Elisa test draws to the final stages, extreme care is necessary to ensure you obtain the correct result. As such, experts carry out detection generally through the procedures stated below. 

  • The substrate solution is added, specifically 100 µl/well of it. 
  • Depending on what’s required, incubate and allow it to develop.
  • Add stop solution, specifically 100 µl/well.
  • With a suitable plate reader, read the optical density or absorbance.

In this process, the commonly used enzyme labels include horseradish peroxide (HRP) and alkaline phosphatase (AP). Other enzymes instrumental in Elisa tests are catalase, acetylcholinesterase, and β-galactosidase. 

Step #5 Analysis

Step #5 Analysis
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After measuring the absorbance with an Elisa reader and determining the quantity of the protein the samples contain, in-depth analyses are carried out. This analysis helps define several aspects of the signal produced through the direct or secondary tag on the antibody under consideration. This involves plotting a standard curve graph with the data recorded from your plate reader. In this graph, you’ll indicate the absorbance on the linear or Y-axis. On the other hand, you’ll label the log scale or x-axis with the concentration. With the help of the standard curve graph you plotted, you can interpolate the sample’s concentration. 


Even though Elisa tests are relatively straightforward, the waiting period or screening procedures for conditions like HIV can lead to severe anxiety. Always try remembering that each step above is crucial to the success and credibility of the final results. Therefore, the utmost responsibility and professionalism are required at each stage from the test subjects and those carried out the tests. Also, some of these diagnoses are voluntary, and it’s necessary to understand the laws relevant to your state regarding reporting positive results. So there you have it, the different steps used in carrying out a reliable Elisa test.  

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