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Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol

Protocols & Procedures

Tips and Tricks

Reconstitution & Storage Instructions  
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Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol ELISA Tips & Tricks
Blocking Peptide Competition Protocol (BPCP)  
Immunoprecipitation (IP) Protocol IP Tips & Tricks
Antibody Array (AA) Protocol


Enzyme-Linked ImmunoSorbent Assay (ELISA)



An enzyme-linked immunosorbent assay (ELISA) is used to detect the presence of an antigen in a sample. The antigen is immobilized to the well of a plate by adsorption, or captured with a bound, antigen-specific antibody. A detection antibody is then added forming a complex with the antigen, if present. The detection antibody can be covalently linked to an enzyme, or itself be detected by a secondary, enzyme linked antibody. Enzyme substrate is then added to the wells producing a visible signal that is correlated with the amount of antigen and measured by a spectrophotometer.


ELISA Sandwich Assay Summary

Sandwich ELISA Summary


For general ELISA reference only.

For ELISA/EIA kit-specific protocol questions, please refer to the kit instructions, or email

  1. 100 ul peptide (@4ug/ml) in coating buffer is added to individual wells of a microtiter plate. Incubate the plate for 2 hours at 37C or overnight at 4C.
  2. Remove the coating solution and wash the plate three times by filling the wells with 100 ul PBS-0.05%Tween20. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.
  3. Block the remaining protein-binding sites in the coated wells by adding 100ul blocking buffer, 3% skim milk in PBS per well. Incubate for 1 hour at RT with gentle shaking.
  4. Wash the plate three times with 100ul PBS-0.05% Tween 20.
  5. Add 50 ul of diluted antibody to each well. Incubate the plate at 37℃ for an hour with gentle shaking.
  6. Wash the plate six times with 100ul PBS-0.05%Tween 20.
  7. Add 50ul of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use. Incubate at 37C for an hour.
  8. Wash the plate six times with 100ul PBS-0.05%Tween20.
  9. Prepare the substrate solution by mixing acetic acid, TMB and 0.03% H2O2 with the volume ratio of 4:1:5.
  10. Dispense 50ul of the substrate solution per well with a multichannel pipe. Incubate the plate at 37℃ in dark for 15-30mins.
  11. After sufficient color development, add 100ul of stop solution to the wells (if necessary).
  12. Read the absorbance (optical density at 450nm) of each well with a plate reader.


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