Enzyme-linked immunosorbent assay (ELISA) Technique

ELISA Service

Enzyme-linked immunosorbent assay (ELISA) is an immunochemical approach that has been widely applied to quantitatively detect the presence of a specific protein. In addition, by utilizing the anti-PEG immune response, evaluation of PEG and PEGylated proteins/particles can also be achieved.


The basis of ELISA is the immobilization of antigen or antibody and the enzyme labeling of antigen or antibody. The antigen or antibody bonded to the exterior of the solid-phase carrier can maintain its immunological activity, and the enzyme-labeled antigen or antibody can not only retain its immunological activity but also the enzyme activity.

When ELISA is carried out, the analyte in the mixture attach to the fixed antibody or antigen, so that the non-binding substance can be easily washed out by solvent. After that, the enzyme-labeled antigen or antibody is added. At this time, the amount of enzyme that can be immobilized is related to the amount of the analyte in the sample. By adding the substrate that reacts with the enzyme, the color will be developed, and the content of the analyte can be judged according to the color depth, thus qualitative or quantitative analysis can be performed.

Different Types of ELISA

  • 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.

Direct ELISA

  • 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.

Competitive ELISA

  • 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.

Sandwich ELISA

  • 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.



  1. In order to ensure the accuracy, experiments should be carried out as soon as possible after extracting the sample. Samples stored at 4°C should be tested within 1 week.
  2. If the experiment cannot be conducted immediately, the sample should be stored at -20°C or -80°C, but repeated freezing and thawing should be avoided.
  3. Sample containing NaN3, for NaN3 can inhibit the activity of horseradish peroxidase (HRP) so that it is not suitable for ELISA characterization.
  4. Hemolyzed specimen is not suitable for this test.

Strengths & Weaknesses of ELISA

Strengths and Weaknesses of ELISA


  1. Cheng T C, Chuang K H, et al. Sensitivity of PEGylated interferon detection by anti-polyethylene glycol (PEG) antibodies depends on PEG length. Bioconjugate chemistry, 2013, 24(8): 1408-1413.

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