Monoclonal versus Polyclonal antibody use in Sandwich ELISA

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.

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.

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.

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.

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.