AAT Bioquest

Are there different kinds of ELISA?

Posted June 12, 2019

If so, what kind/type do I use/choose?


There are 4 common types of ELISA testing: Direct, Indirect, Sandwich, and Competition/Inhibition.


  • The Direct test is the fastest and simplest, which is a binding of the ELISA antigen on the test surface with its enzyme-labeled primary antibody conjugate. However, since it is just a 1:1 stoichiometric ratio, amplifying or improving the signal strength for ease or reading and more exact measurements is impossible.


  • The Indirect test adds another step to the first process. The ELISA antigen is bound to the experimental surface as usual, and a primary antibody specific for it is added and binds to the antigen. Subsequently, an enzyme-labeled secondary antibody is added and binds to the primary antibody. This not only gives a customizable signal but allows for more adaptability of the process for the specific experiment, since the secondary antibody can be directed against more than one primary antibody. Although more complex to perform, the Indirect ELISA shows an improvement in sensitivity, since the signal can be amplified.


  • The Sandwich ELISA, the most popular choice for many research applications, is unique. Although it also uses two antibodies in conjugation with the target antigen like the Indirect test, one of the antibodies will be used to coat the experimental surface first. After the microplate is thoroughly coated, the target antigen is added to the sample wells and binds to the first antibody. The second antibody is then added to the microplate and binds to the target antigen as well, effectively ‘sandwiching’ the target protein between the two antibodies. This test is highly specific, highly sensitive, and has the advantage of not requiring any form of sample purification, since only the target antigen will be effected by both the antibodies, allowing for excellent experimental readings of analyte concentration. The expense and difficulty of finding suitable paired antibodies is a major detractor, however.


  • The Competition/Inhibition ELISA approaches the problem from the opposite direction. First, a known antigen is used to coat the microplate sample wells. Secondly, the test sample material is added, and lastly the enzyme-labeled antibody is introduced that allows detection of results. Since the test analyte is ‘competing’ to bind with the enzyme-labeled antibody, the presence of the analyte will decrease the signal output commensurate with its concentration. This inverted approach also allows for testing of an unknown sample without need for purification, and is applicable to a wide range of experiments. The complexity and difficulty of this technique is similar to the Indirect or Sandwich ELISA protocols in regards to time and expense.


In summary, the researcher must take into account the restrictions inherent in the protein they are studying (some analytes may be too small to bind to more than one antibody, for example) the limitations of their laboratory resources (space, time, personnel) and the complexity of their experiment when making a decision of what type of ELISA test to use. Customization may be necessary in some cases.

Additional resources

What is a Direct ELISA?

Types of ELISA tests

US Patent for ELISA tests not employing microplates

Screen Quest™ Colorimetric ELISA cAMP Assay Kit

Screen Quest™ Fluorimetric ELISA cAMP Assay Kit

Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*

Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*