AAT Bioquest

What is a Direct ELISA?

Posted January 6, 2020


A direct ELISA (enzyme-linked immunosorbent assay) is a plate-based immunosorbent assay intended for the detection and quantification of a specific analyte (e.g. antigens, antibodies, proteins, hormones, peptides, etc.) from within a complex biological sample. Of the four different ELISA formats, direct ELISA is the simplest and quickest to perform, but there are some disadvantages associated with this method (see Table 1).

In a direct ELISA (Figure 1), the antigen is immobilized directly onto the surface of a multi-well microtiter plate such as a 96-well polystyrene plate, and then complexed with an enzyme-labeled primary antibody specific for the antigen. Once the enzyme-labeled primary antibody binds to the antigen, the conjugated primary antibody catalyzes a reaction with its respective substrate resulting in a visible colorimetric output that is measured by a spectrophotometer or absorbance microplate reader. Direct ELISAs are suitable for qualitative and quantitative antigen detection in samples of interest, antibody screening, and epitope mapping.


Direct ELISA

Figure 1. Illustrates the setup of a direct ELISA:

  1. Antigen is immobilized onto the wells of a 96-well polystyrene plate via passive adsorption.
  2. An enzyme-labeled primary antibody (e.g. HRP-labeled primary antibody) specific for the target antigen is added to the wells and directly binds to the antigen.
  3. A respective enzyme substrate (e.g. TMB (11012) a suitable substrate for HRP) is added, which upon reaction with the enzyme, produces a visible colorimetric output that can be measured by a spectrophotometer or absorbance microplate reader.


Table 1. Advantages and disadvantages of direct ELISA.

  • Less reagents and fewer steps are required making this ELISA format simple and quick while minimizing potential user error
  • Cross-reactivity of secondary antibody is eliminated
  • Antigen immobilization is not specific resulting in potentially high background interference
  • Primary antibody must be labeled individually, which is time-consuming and expensive
  • No signal amplification
  • Low flexibility – a specific conjugated primary antibody is needed for each target protein
  • Immunoreactivity of the primary antibody may be adversely affected by labeling with enzymes
Additional resources

ReadiUse™ Preactivated HRP NHS ester

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