the difference here is that before adding the sample in the wells (in this case covered with the antigen), the sample is incubated with unlabeled antibodies. Then the complex of antigen-antibody is added to the wells and incubated. The more antigen I present in the sample, the more antibody-antigen complexes will be form and the less unbound antibodies will remain. The unbound antibodies will attach to the antigen that is covered on the well. Then a wash follows. Afterwards a secondary antibody is added that recognizes the primary antibody. The secondary antibody is conjugated with HRP. After another wash an HRP substrate is added. The more free antibodies were available in the sample as a result of less antigen presence (less antigen-antibody complexes formed), the more antibodies will remain free to bind to the antigen on the well and remain there. In turn, this means that more secondary antibodies (conjugated with HRP) will bind to the primary antibodies and remain in the wells. This will lead to higher HRP activity and more intense color. In this case the higher the intensity of the color, the less antigen was present in the analyzed sample.
In conclusion, despite the various modifications, all ELISA tests, in their essence, are a clever utilization of antibodies’ ability to recognize antigens with high specificity. This property coupled with an appropriate color/fluorescence signaling molecules conjugated to the antibodies allow us not only to prove the presence or absence of a molecule in a sample but also to calculate with great accuracy its concentration.