What is the difference between direct and indirect ELISA?

The major difference between direct and indirect ELISA is that only one antibody is used in direct ELISA, while indirect ELISA requires two antibodies.

In direct ELISA, the antibody is conjugated directly to the detection enzyme, which saves time to complete the assay and is also more cost effective because only one antibody is needed. However, the reactivity of antibody may be compromised due to the attachment of an enzyme. Besides, an enzyme labeled antibody that is specific for the antigen of interest should be prepared for each ELISA experiment.

In indirect ELISA, two antibodies are required: a primary antibody for the antigen of interest and an enzyme-linked secondary antibody that is complementary to the primary antibody. Although the secondary antibody costs more time and money, it offers more flexibility in choice of labeled secondary antibodies and enzyme-substrate detection systems. The labeled secondary antibody could be used for multiple ELISA experiments, as long as there is an appropriate primary-secondary antibody combination. Meanwhile, different enzyme-substrate detection systems can be used for the same primary antibody, allowing for fine-tuning and improving the ELISA protocol. In addition, the primary antibody has more binding sites for the secondary antibodies than the antigen has for an antibody, making it possible to exhibit a higher signal than that with direct ELISA.