The ELISA immunoassays are among the most versatile to detect, identify and / or quantitate a particular antigen present in our sample. It is a highly sensitive, robust and relatively inexpensive test, quick and easy to carry out.

But despite the apparent simplicity of its protocol, as with most biological assays, there are times when the results are not as expected.

In this post we will focus on  3 common problems when doing an ELISA , and we will try to give some guidelines to be able to  solve them successfully .

Some of the common problems when doing an ELISA are obtaining a weak , low intensity or even non-existent signal , generating a high background noise that distorts the results, or the high inconsistency of the results between wells .

Next we leave you a list of Possible Causes (PC) and Solutions (S) for these 3 common problems when doing an ELISA.


PC: Problems with the protocol

S: The essay may not have been prepared correctly, and to fix it it is necessary to review several points:

  • Check that each step of the protocol has been applied correctly
  • Check that the wavelength and filters used in the plate reader are appropriate. For this, the test can be repeated using a positive control.

PC: Antibody problems

S: Antibodies are the most critical reagents in ELISA tests, and it is important to pay attention to the following points:

  • Use the dilution recommended by the manufacturer, or failing that, optimize the dilution of the antibody to be used.
  • Make sure you use enough antibodies, testing different concentrations of the antibodies to identify the optimal concentration for your assay.
  • Make sure that the antibodies have been stored correctly and have not undergone excessive freeze / thaw cycles .

PC: Problems with antigen

S: When the plates are covered with the antigen and when adding the primary antibody the signal is weak or null, there are two main aspects to check:

  • Sometimes it is possible that a sufficient amount of antigen has not adhered to the plate, try adding more.
  • In the case of peptide antigens, they must be previously conjugated to a large carrier protein in order to facilitate the recognition of the epitope by the antibody.

PC: Problems with reagents

S: Always make sure that:

  • The reagents are at room temperature before starting the assay
  • Check expiration dates
  • Check that they have been prepared correctly and have been added in the corresponding order.
  • Make sure that they do not interfere with other buffers or compounds present in the sample such as sodium acid, EDTA, etc.
  • Avoid mixing reagents from different kits.

PC: Low incubation times and temperature

S: Be sure to incubate the samples for the time specified by the manufacturer and optimize the incubation temperature for your assay. Remember that the reagents must be at room temperature before starting the assay.

PC: Storage conditions

S: Stores reagents to your specifications, keeping in mind that all reagents may not need the same storage requirements.


PC: Antibody problems

S: Regarding antibodies, there can be several cases:

  • That the concentration of antibody used is too high. This will be resolved by reducing its concentration, or by carrying out an optimization test to determine the ideal concentration.
  • There are cross reactivity problems, for which a negative control should be used.
  • Non-specific binding where the antibody is detecting other molecules besides the target antigen. The solution is through the use of affinity purified antibodies and the use of the appropriate blocking buffers .

PC: Problems with reagents

S: Regarding the different reagents, we must make sure that:

  • The stop solution is added to avoid an over development of the reaction.
  • The detection reagent has been properly diluted, and if so, retest with an even higher dilution.
  • The appropriate blocking buffers have been used and the blocking time has been sufficient.

PC: Incubation problems

S: In case of obtaining high background noise, it may be necessary to reduce the incubation temperature. On the other hand, it is necessary to ensure that the incubation of the substrate is carried out in dark conditions.

PC: Insufficient washes

S: Carefully wash the bottom of the plate and repeat the reading.


PC: Inadequate incubation

S: Regarding the incubation step, it is important:

  • Do not stack the plates during the process to ensure that the temperature is evenly distributed.
  • Make sure there are no bubbles inside the wells before incubation.

PC: improper washing

S: Make sure to thoroughly wash all wells, as inhomogeneous washing could result in a high coefficient of variation.