AAT Bioquest

What is an Indirect ELISA?

Posted March 13, 2020

How is it different from the Direct ELISA?


An enyzme-linked immunosorbent assay (ELISA) can be done with a varitey of modifications to its basic protocol. For example, the direct ELISA uses an enzyme-labeled primary antibody conjugate to detect immobilized antigens. 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 direct ELISA process. The antigen of choice is bound to the experimental surface, and an unlabeled 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. A coloreless substrate is introduced to the sample, which reacts with the enzyme conjugate, and produces a measurable byproduct. Depending on the choice of substrate, this byproduct can be either colorimetric, chemiluminescent or fluroescent.

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 colorimetric indirect ELISA.

  1. Coat ELISA plate with testing antigens, seal plate and incubate overnight at 4°C
  2. Remove coating solution and wash plate 2 times with desired buffer
  3. Block plate with desired blocking solution for 1 hour at 4°C
  4. Wash plate 2 times with buffer
  5. Incubate with unconjugated primary antibody at room temperature for 1 hour
  6. Wash plate 4 times with buffer
  7. Incubate with HRP-labeled secondary antibody (in blocking buffer) at room temperature for 1 hour
  8. Wash plate 4 times with buffer
  9. Incuabte plate with ReadiUse™ TMB Substrate Solution (Cat No. 11012) at room temperature for 15-30 minutes.
  10. Stop reaction using Signal Guard ™ HRP reaction stopping solution (Cat No. 11020)
  11. Measure the absorbance signal at 650 nm with an ELISA microplate reader


Indirect ELISA detection
  • 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 (e.g. biotin/streptavidin)
  • Potential cross-reactivity from secondary antibody, resulting in non-specific staining
  • Lengthier protocol compared to direct ELISA format as an extra incubation step is required in the procedure
Additional resources

Biochemical Assays

HRP Conjugation

HRP-labeled goat anti-mouse IgG (H+L)

ReadiUse™ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*