A summary of the advantages and disadvantages of the different ELISA techniques

ELISA

Strengths

Weaknesses

Indirect

1) High sensitivity due to the fact that multiple enzyme-conjugated secondary antibodies can bind to the primary antibody

1) High background signal may occur because the coating of the antigen of interest to the plate is not specific (i.e., all proteins in the sample will coat the plate)

2) Many different primary antibodies can be recognized by a single enzyme-conjugated secondary antibody giving the user the flexibility of using the same enzyme-conjugated secondary antibody in many different ELISA (regardless of the antigen being detected)

3) Best choice when only a single antibody for the antigen of interest is available

Sandwich

1) The use of antigen-specific capture and detection monoclonal antibody increases the sensitivity and specificity of the assay (compared to the indirect ELISA)

1) Optimizing the concentrations of the capture and detection monoclonal antibodies can be difficult (especially for non-commercial kits)

2) Best choice for detecting a large protein with multiple epitopes (such as a cytokine)

Competitive

1) Impure samples can be used

1) Requires a large amount of highly pure antigen to be used to coat plate

2) Less sensitivity to reagent dilution effects

3) Ideal for detecting small molecules (such as a hapten)