Elisa Protocol | Step by step procedure of Elisa test technique

Elisa Protocol is sequential and has an orderly procedure to be followed out in carrying the test.

Elisa test is an immunoassay that relies on the interaction between a specific antibody and antigen.

For this, factors like time of reaction, interfering substances, temperature, pH, buffer composition, treatment, and washing play an important role in the assay’s successful outcome.

Hence the Elisa assay must be carried out in a particular sequence and operating procedure for best results.

Any deviation from this Elisa protocol can affect the results greatly and sometimes; one may even fail to get proper results.

Most ELISA tests are done using commercially available ELISA kits, which have a definite protocol mentioned on a leaflet.

In general, these kits contain an antibody tagged with an enzyme, a substrate reagent, Elisa plates with wells, i.e., 96 places or few, washing buffer, etc and also a step-by-step Elisa protocol for the given sample.

Once received, these kits will be stored at recommended temperatures for the kit’s contents’ long-life.

Once opened, the Elisa kit contents must be used within a specified time or short life mentioned by the supplier for effective analysis.

Read ELISA vs. Westernblot analysis.

Elisa Protocol steps:

• Take out the ELISA kit’s contents on to suitable work table inside a temperature-controlled room or lab. Then wash the ELISA plate and let it dry (do only if recommended in the kit’s protocol brochure from the supplier).

• The given test sample under analysis is taken preferably in a homogenate or solution form.

• A fixed volume of the test sample is drawn into a micropipette and loaded into the Elisa plate’s wells.

• The samples are placed in such a way to accommodate a blank, a standard (if available) and test sample in the wells of the Elisa plates.

A brief video  demo for competitive ELISA procedure

• Then wait for a specified time, preferably half-hour, for the antigens in the sample to fix to the walls in the well.

• Next, rinse (wash) the samples inside the wells with buffer. This step removes unbound part of the sample from the wells, i.e., only antigens remain fixed on the well’s wall in Elisa plates.

• Then, the fixed volume of enzyme-linked anti-body is placed into the washed wells and allowed to stay for a half-hour. This lets firm binding of enzyme-linked antibodies with antigens, if any, fixed to the walls of the wells.

• Again, rinse the wells with washing buffer to remove any unbound enzyme-linked antibodies.

• Now load the washed wells with a specified amount of Elisa substrate and incubate for a specified time for the reaction to proceed and generate a color.

• In the meantime, switch on the Elisa instrument or Elisa plate reader and set the defined wavelength filter. Mark the sample locations on the plate wells on the computer screen, i.e., blanks, standards, and test sample’s positions, to avoid later confusion.

• After the incubation time, immediately place the plate into the socket of the Elisa instrument, close the socket door and take the reading after detection.

The reading can be had in an excel sheet or print out based on the Elisa instrument you use.

Then discard the used plate. That’s the end of Elisa’s protocol.

It is so simple and can be done in a couple of hours without much strain.

Leave a comment

  1. Do you have to manipulate your controls to make the plate (samples) “pass” the reader. In my lab, we sometimes manipulate the controls to make our test pass the reader. Are these results valid?

    • @Gina! Ya every biological material we eat is made of carbohydrates, proteins and fats. So every food we eat has carbohydrates. But, the proportion of it varies. In pulses, protein is more and carbohydrates are less.

  2. I want to find out how to figure whether any diagnostic kit is Elisa method or not.How many types of Elisa kits are there.

    whether absence of conjugate in the kit will mean NOT Elisa.Pl advice

    • @karunakar! In general elisa kits have everything supplied. Conjugates are meant to produce colour for spectroscopical measurement. Some times suppliers use different conjugates to minimise the price. But when the kit is supplied as ELISA kit it performs only ELISA.. Try to purchase only from reliable suppliers.


Leave a Comment