What Characteristics To Look For In An Elisa Kit

The kit ELISA designed to determine the presence and / or concentration of a particular antigen in a test sample, are immunoassays which can be designed in the laboratory. For this, it is necessary to acquire the different components (plates, antibodies, buffers …), and to fine-tune a functional protocol through adjustments and tests under different variables until it is optimized.

Sometimes this is a complex, expensive and laborious process, so resorting to commercial kits that have already been developed to carry out the immunoassay can be a good option. If this were the case, what would be the most important characteristics that we should take into account to choose a more suitable ELISA kit ?

In this post we explain four of the most important aspects that we should look at when buying a commercial ELISA kit .

Factors To Consider When Choosing An Elisa Kit

1.- Essay Format

Based on aspects such as flexibility, sensitivity, complexity of the sample or the results we want to achieve, we can choose different types of ELISA kit that best suit our needs.

ELISA DIRECTO

  • Background : The antigen is immobilized in a microwell plate, to be later detected directly by means of an antibody conjugated, for example, to an enzyme like HRP.
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  • Strong points :
    • Speed
    • Less probability of making mistakes during the test, due to the fewer number of steps and reagents used.
  • Weak points :
    • It can generate greater background noise because all the proteins in the sample can be immobilized on the plate (both our antigen of interest and the rest), resulting in non-specificity.
    • Less flexibility, since it requires the use of a specific conjugated primary antibody for each antigen of interest.
    • Lower sensitivity, since there is no signal amplification.
  • Most frequent use : To analyze the immune response against a certain antigen.

INDIRECT ELISA

  • Background : In this case, the antigen that is immobilized on the plate is subsequently detected in two steps. First, an unlabelled primary antibody specifically binds to the antigen, and then an enzyme-conjugated secondary antibody is added that will bind to the primary, amplifying the signal. (Remember this guide to select secondary antibodies ).
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  • Strong points :
    • High sensitivity, since several conjugated secondary antibodies can bind to the same primary antibody.
    • Greater flexibility, being possible to use different primary antibodies with a single conjugated secondary.
  • Weak points :
    • Possible cross-reactivity due to the use of secondary antibodies that could increase the background noise.
    • The procedure is longer than in a direct ELISA, with additional incubation steps.
  • Most frequent use : To determine the total concentration of antibody in a sample.

ELISA SANDWICH

  • Background : This assay requires the use of pairs of primary antibodies that recognize different epitopes of the same antigen. The plate is covered with one of them, which will act as a capture antibody. Subsequently, the sample with the analyte of interest will be added, and finally the second antibody that will act as the detection antibody. This detection antibody may be labeled with an enzyme (direct sandwich ELISA), or unlabelled by subsequent use of a conjugated secondary antibody (indirect sandwich ELISA).
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  • Strong points :
    • High sensitivity
    • High specificity, by the use of two primary antibodies
    • Flexible, the signal can be detected by direct or indirect method.
  • Weak points :
    • It is not always easy to get an adequate pair of antibodies, and it may lead to cross-reactivity between the two. (Remember the keys to select pairs of antibodies ).
  • Most frequent use : For the analysis of complex samples, since it is not necessary to previously purify the antigen.

2.- Antibodies

  • Assess the use of monoclonal or polyclonal antibodies . Both are valid for carrying out ELISA tests, prioritizing polyclonal when sensitivity is sought, and monoclonal in case of importing more specificity. In the sandwich ELISA, it is common to use a polyclonal antibody for capture and a monoclonal antibody for detection. (Remember the differences between monoclonal and polyclonal ).
  • In the case of the sandwich ELISA, it is necessary to ensure that there is a pair of antibodies validated for this purpose. That is, they recognize different non-overlapping epitopes within the same antigen.

3.- Cross Reactivity And Interferences

  • To avoid cross-reactivity problems in sandwich ELISAs, each of the primary antibodies should be generated on a different host . In this way, the secondary antibody will react only with the primary detection antibody.
  • Attention should also be paid to the composition of the ELISA kit buffers , as they may contain compounds that may interfere with antigen-antibody binding.

4.- Detection Method

  • Another aspect to evaluate before choosing an ELISA kit is the detection method you want to use. Currently ELISA kits can use radioactive, fluorescent, chemiluminescent, or chromogenic forms of detection.

Before buying an ELISA kit, it is important to pay attention to the four aspects that we have summarized in this entry, and which we hope have been helpful to you. If in doubt, do not hesitate to contact us and we will advise you in this regard.