Elisa Protocol: Step by step procedure of Elisa test technique


Elisa Protocol is the sequential and orderly steps to be followed in carrying out an ELISA Test or assay.

Elisa test is an immunoassay and depends on reaction between specific antibody and antigen.

For detailed principle check “ELISA TEST PRINCPLE“.

For this factors like time of reaction, interfering substances, temperature, pH, buffer composition, treatment, washings play an important role in successful outcome of the assay.  Hence the Elisa assay requires to be carried out in 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 definite protocol mentioned on a leaflet. These kits in general contain an anti-body tagged with 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. These kits once received are to be stored at recommended temperatures for long-life of the kit’s contents. Once opened, the contents of the Elisa kit are to be used within specified time or short life mentioned by the supplier for effective analysis.

Elisa Protocol steps:

• Take out the contents in elisa kit 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 protocol brochure of the kit from supplier).

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

• A fixed volume of test sample is drawn into a micro pipette and loaded into the wells of Elisa plate.

• 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 specified time, preferably half hour for the antigens in the sample to fix to the walls of 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 wall of the well in Elisa plates.

• Then fixed volume of enzyme linked anti-body is placed into the washed wells and allowed to stay for half hour. This lets firm binding of enzyme linked anti-bodies 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 specified time for the reaction to proceed and generate colour.

• In the mean time switch on the Elisa instrument or Elisa plate reader and set the defined wave length 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 time of incubation, immediately place the plate into the socket of 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 protocol.

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

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