Direct ELISA, Simple and Time-Saving

Initially in a direct ELISA test which is considered to be the simplest type of ELISA the antigen is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block all the other binding sites. While an enzyme is linked to an antibody in a separate reaction, the enzyme-antibody complex is applied to adsorb to the antigen. After excess enzyme-antibody complex is washed off, enzyme-antibody bound to antigen is left. By adding in the enzyme's substrate, the enzyme is detected illustrating the signal of the antigen.

However, in terms of direct ELISA versus indirect ELISA, in an indirect ELISA, the steps are similar, but with important differences and an additional step. After the antigen is adsorbed to the plate (and after the BSA step), the next antibody to be added is the antibody that recognizes the antigen (this antibody does not have the enzyme attached to it).

Then, an enzyme-antibody conjugate is prepared, which is added to the plate and detects the antibody that is adsorbed to the antigen (in a direct ELISA, the enzyme-antibody conjugate directly adsorbs to the antigen), then the substrate is added which detects the presence of the enzyme and thus the antigen. So, in the indirect ELISA, the enzyme-antibody conjugate uses an antibody against the type of antibody that is used to detect the antigen, kind of like a sandwich.

For instance, if the antigen is HIV-1 gp120, then an anti-HIV antibody (HIV-1 gp120 Antibody) is prepared (let's say from a mouse). Then, in a separate reaction, an enzyme is attached to an anti-mouse antibody. So, in order to detect the HIV in the assay, an anti-mouse antibody is used to detect the antibody attached to the antigen.


Direct ELISA, when compared to other forms of ELISA testing, is performed faster because only one antibody is being used and fewer steps are required. This can be used to test specific antibody-to-antigen reactions, and helps to eliminate cross-reactivity between other antibodies.

Disadvantages of direct ELISA

The primary antibody must be labeled individually, which can be time-consuming and inflexible when performing multiple experiments. Also, the signal is less amplified in direct ELISA, which means a lower sensitivity and could be viewed as a disadvantage to some.