What is an Indirect ELISA?

Posted Jun 13, 2019

How is it different from the Direct ELISA?


There are 4 common types of ELISA testing: Direct, Indirect, Sandwich, and Competition/Inhibition. The Direct test was the first 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 ELISA adds another step to the baseline ELISA process. The antigen of choice is bound to the experimental surface, and a primary antibody specific for it is added and binds to the antigen. Subsequently, an enzyme-labeled secondary antibody (e.g. HRP-labeled goat anti-mouse IgG (Cat# 16728)) 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. Once the enzyme-labeled secondary antibody binds to the primary antibody, the conjugated secondary antibody catalyzes a reaction with its respective substrate (e.g. ReadiUse™ TMB Substrate Solution (Cat# 11012)) resulting in a visible colorimetric output that is measured by a spectrophotometer or absorbance microplate reader. Although more complex to perform, the Indirect ELISA shows an improvement in sensitivity, since the signal can be amplified.

A researcher must consider multiple criteria when deciding which assay to include in an experimental protocol. A selection of factors to weigh for the Indirect ELISA are included in the table below. 


Figure 1. Illustrates the setup of an indirect ELISA.

  1. Antigen is immobilized onto the wells of a 96-well polystyrene plate via passive adsorption.
  2. An unlabeled primary antibody specific for the target antigen is added to the wells and directly binds to the antigen.
  3. An enzyme-labeled secondary antibody ( e.g. HRP-labeled goat-anti mouse) against the host species of the primary antibody is added to the wells and binds to the primary antibody.
  4. A respective enzyme substrate (e.g. ReadiUse™ TMB Substrate Solution) is added, which upon reaction with HRP, produces a visible colorimetric output that can be measured using a spectrophotometer or absorbance microplate reader.


 Table 1. Advantages and Disadvantages of indirect ELISA.

Advantages Disadvantages
  • Accessible - A wide selection of pre-labeled secondary antibodies are commercially available
  • Economical - Fewer labeled antibodies are needed
  • Highly sensitive - Primary antibodies contain various epitopes for binding several labeled secondary antibodies resulting in signal amplification
  • Very flexible - A single secondary antibody can be used to detect different primary antibodies
  • Maximum immunoreactivity of the primary antibody is retained
  • Different visualization markers can be used with the same primary antibody
  • Lengthier protocol compared to direct ELISA format as an extra incubation step is required in the procedure
  • Potential cross-reactivity for secondary antibody, resulting in nonspecific staining

For more information about assays and related details, please see 'Additional Resources' below. 

Additional resources

Biochemical Assays
HRP Conjugation
HRP-labeled goat anti-mouse IgG (H+L)
ReadiUseā„¢ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*

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