Enzyme-linked immunosorbent assay (ELISA) Technique

• Direct ELISA

Direct ELISA is beneficial for calculating the concentration of PEGylated antibodies in serum samples. In this approach, a specific antigen is coated in the wells of microtiter plates for the capture of PEGylated antibodies in the sample. The quantity of antibody bound to the antigen is then determined by adding a detection antibody, typically a horseradish peroxidase (HRP) conjugate. Moreover, direct ELISA can also be employed to measure the concentration of other PEGylated compounds by using a competition assay format.

• Competitive ELISA

In the competition assay format, PEGylated compounds are coated on the surface of a solid carrier. The degree that serum samples block binding of a specific antibody to plate-coated PEGylated compound provides a measure of the serum concentration of the PEGylated compound. However, both direct ELISA and this kind of competitive assay cannot be able to distinguish between PEGylated and de-PEGylated metabolites.

• Sandwich ELISA

Sandwich ELISA is a powerful approach to measure the concentration of PEGylated conjugates in complicated biological samples. Hereby two antigen-specific antibodies are needed, one for capturing and the other for recognizing the target antigen. The analyte must possess at least two antigen-binding sites, usually distinct binding epitopes on the analyte. Sandwich ELISA is usually more sensitive (concentration can down to pg/mL) and more specific (when using two distinct antibodies) than direct and competitive ELISA, especially for the determination of analyte with very low concentrations.

• Anti-PEG ELISA

Anti-PEG ELISA provides a sensitive and accurate technology for the analysis of PEGylated molecules, which under some circumstances are difficult to be tested by other methods. Anti-PEG antibodies can be generated by attaching PEG to highly immunogenic carrier proteins and immunizing mice multiple times to overcome the very low immunogenicity of PEG. Anti-PEG sandwich ELISA is a more general tool which allows detection of a wide variety of PEGylated species by direct binding to PEG independent of the attached compound.