Nanotechnology-Based Reagents for ELISA

Enzyme-linked immunosorbent assay (ELISA) is an analytical technique in which, usually, one of the reaction components is adsorbed to the surface of a microtiter well of a multi-well plate to facilitate the separation of bound and free-labeled reactants. In the most employed analises, the sample containing the antigen to be detected, is added to the microtiter wells and allowed to bind with the antibody-coated bottom. After washing, an enzyme-labeled secondary antibody is added to generate a “sandwich” of coated Ab-Ag-Ab enzyme. Unbound reagent is then flushed away, and enzyme substrate is added to each well. The amount of product generated is proportional to the quantity of antigen in the sample and it is revealed using appropriate instruments.

On the other hand, specific antibodies in a sample can be detected and quantified using an ELISA modified procedure in which the antigen is coated to the microtiter well bottom. Ini this case the reagent to be added is an enzyme-labeled antibody specific to the analyte antibody. The successive phases of the analyses are the same described above.

The competitive ELISA is a particular analysis to detect presence of an antibody specific for antigens in the biological fluid under exam. This subtype of ELISA utilizes two specific antibodies, an enzyme-conjugated antibody and another antibody eventually present in the biological fluid (in case of positivity of the unknown sample). Combining the two antibodies into the wells will establish a competition for the antigens. If the color of the solution in the wells changes, it means that the enzyme-conjugated antibody bound the antigens due to the absence, in the unknown sample, of the unconjugated competitor antibody. The sample is ratified as negative for the antibody of interest. This particular ELISA has a lower specificity and is advised against to be used in low concentration samples.