ELISAs

In an ELISA, the antibody is usually bound to a surface, and linked to some type of enzymatic reporter system (for instance, horseradish peroxidase). Typically, the enzymatic reporter systems are linked to the surface of 96-well

FIGURE 2 RIAs and ELISAs. These assays are ligand-based assays. The triangle represents the analyte of interest. In the RIA, the analyte displaces the binding of a known quantity of radiolabeled analyte (triangle with 125I). The oddly shaped molecule with a triangular edge represents a potential interference, namely a molecule with a similar hapten as the analyte of interest. In the ELISA, once the analyte binds the antibody (which is bound to a surface), the enzyme linked to the antibody is activated to signal the interaction.

FIGURE 2 RIAs and ELISAs. These assays are ligand-based assays. The triangle represents the analyte of interest. In the RIA, the analyte displaces the binding of a known quantity of radiolabeled analyte (triangle with 125I). The oddly shaped molecule with a triangular edge represents a potential interference, namely a molecule with a similar hapten as the analyte of interest. In the ELISA, once the analyte binds the antibody (which is bound to a surface), the enzyme linked to the antibody is activated to signal the interaction.

plates. Samples are added along with the necessary reactants, and gently mixed. After a defined period of incubation, the reaction in each well is "stopped" and the amount of analyte is quantified (often using a spectrophotmetric plate reader). One of the major drawbacks with ligandbased assays is antibody binding to nonanalyte entities. This type of binding will produce overestimates of the analyte quantity. It can be difficult to determine whether this process has occurred because unlike chromatography, there is no visual output to assess. Therefore, greater care has to be taken to ensure that no interference occurs in these types of assays.

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