Sandwich ELISA Assays

Monoclonal versus Polyclonal antibody use

What is Sandwich ELISA?

Sandwich ELISA (Enzyme-Linked Immunoassay also known as EIA) is a diagnostic technique used to find antigens or antibodies in biological samples. This technique is one of four types of microplate tests: Direct, Indirect, Sandwich, and competitive ELISA. Sandwich ELISA allows for the use of monoclonal versus polyclonal antibodies to capture antigens.

Should we use monoclonal or polyclonal antibodies as the primary antigen in Sandwich ELISA testing?

A question most often asked in technical forums. In this article, we take a look at this question, the advantages, and disadvantages of monoclonal versus polyclonal antibodies as primary or capture antigens.

Monoclonal Sandwich ELISA Antibodies

Sandwich ELISA antibodies - Monoclonal Antibody

Monoclonal antibodies are single antibodies from producing B-cells, therefore it only binds with 1 unique epitome.

Technically speaking, individual antibodies in a polyclonal mix are monoclonal antibodies.

However, the terminology describes a process where the actual B-cell is isolated. Once isolated, the B-cell fuses into a hybrid cell line. This way, technicians produce large volumes of identical antibodies.

Monoclonal Antibody advantages

  • The use of monoclonal antibodies produces better results when it is used in assays that require protein quantification levels.

  • It is possible to produce identical antibodies in large volumes with batch-to-batch similarity.

  • Monoclonal antibodies reduce cross-reactivity probability. It is highly specific to a single epitope.

Monoclonal Antibody Disadvantages

  • The slightest change in monoclonal antibodies’ epitome structure might lead to target antigen detection problems.

  • Producing monoclonal antibodies requires laboratories with purification and cell culture capabilities.

  • Monoclonal antibodies are not as robust as polyclonal antibodies. Challenges to detect denatured proteins or proteins with altered conformation.

  • Monoclonal antibodies are more sensitive to buffer and acid levels.

  • These antibodies are susceptible to binding changes when labeled.

  • Significantly more expensive to produce.

  • Requires significantly more time to produce and develop the hybridized clone.

  • Monoclonal antibodies are less ideal to use when a quick capture of target proteins is required.

  • Economics: time to produce monoclonal antibodies, the volume required, and storage conditions make it more expensive.

Polyclonal Sandwich ELISA Antibodies

Sandwich ELISA Polyclonal antibody

Polyclonal antibodies are manufactured from different B-cells, recognizing several antigenic determinants (epitope) on the same antigen. The epitope is unique to the antigen. Each antibody recognizes this unique epitope located on the specific antigen.

Polyclonal Antibody advantages

  • Inexpensive to produce.

  • Quick to produce. Purified antibody ready to use in under four months.

  • Easy to store.

  • Highly stable and tolerant of pH or buffer changes.

  • Higher overall antibody affinity against the antigen due to recognition of multiple antigenic determinants.

  • In general, the ability to detect multiple epitopes gives more robust detection.

  • Offers greater sensitivity for detecting proteins that are present in low quantities in a sample, since multiple antibodies will bind to multiple epitopes on the protein.

  • Ideal to use as the capture antibody in sandwich ELISA. Greater ability to quickly capture the target protein.

  • Superior antibody affinity generally results in quicker binding to the target antigen. Ideal in assays requiring quick capture of the protein, such as IP or ChIP.

  • Significantly more robust when assaying proteins that show slight variations in individual epitopes such as denaturation, polymorphism, or conformational changes.

  • Superior for use in detecting a native protein in multiple assay types.

  • Much easier to couple with antibody labels. Less likely to affect binding capability.

Polyclonal Antibody disadvantages

  • Variability between different batches produced in different animals at different times

  • Higher potential for cross-reactivity due to recognizing multiple epitopes.

  • Affinity purification of the serum will typically be required to minimize cross-reactivity.


Should I use Monoclonal or Polyclonal antibodies as binding antibodies?

Firstly, decide on the type of test that you have to develop. Secondly, it will determine the type of antibodies you will need. Thirdly, ensure that the antibodies work for the specific test that you develop.

Practical tips when developing a test
  • Traditional sandwich ELISA tests use polyclonal antibodies is as capture antibody as it pulls down the antigen (preferred method). Use Monoclonal antibodies for specificity testing.

  • Ensure that the capture antibody does not change the antigen’s immunogenic properties. It will affect antibody binding to other epitopes.

  • The same antibody and antigen (capture and detection) are used for testing. Self-sandwich ELISA testing works only for large molecules where the epitopes are far apart.

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About Marinda Stuiver

Dip, NHDip Microbiology (Vet) TUT (1989)

Marinda Stuiver is a qualified Veterinary Microbiologist with more than 12 years of laboratory experience.

Her experience includes histology, Electron Microscopy, and ELISA testing. Following the sale of the laboratory where she worked, she ventured into the commercial side of the business.

Marinda sold IDEXX ELISA test kits to various laboratories in Southern Africa (Zimbabwe, Botswana, Malawi) and trained laboratory staff to set up and conduct testing.

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