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Inexpensive to produce
Expensive to produce
Skills required for production are low
Training is required for the technology used
Relatively quick to produce
Hybridomas take a relatively long time to produce
Generate large amounts of non-specific antibodies
Generate large amounts of specific antibodies
Recognize multiple epitopes on any one antigen
Recognize only one epitope on an antigen
Can have batch-to-batch variability
Once a hybridoma is made, it is a constant and renewable source
No or low batch-to-batch variability
Recognize multiple epitopes on any one antigen. Serum obtained will contain a heterogenous complex mixture of antibodies of different affinity.
Polyclonals are made up mainly of IgG subclass.
Peptide immunogens are often used to generate polyclonal antibodies that target unique epitopes, especially for protein families of high homology.
Inexpensive and relatively quick to produce.
Production is less complex compared with monoclonal antibodies.
Polyclonal antibodies recognize multiple epitopes on any one antigen. This has the following advantages:
High affinity: polyclonals amplify signal from a target protein with low expression level, as the target protein will bind more than one antibody molecule on the multiple epitopes. However, this is disadvantageous for quantification experiments (eg in flow cytometry) as it generates inaccurate results.
Recognize multiple epitopes to give better results in immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP).
More tolerant of minor antigen changes (eg polymorphism, heterogeneity of glycosylation or slight denaturation) than monoclonals.
Can identify proteins of high homology to the immunogen protein, and can be used to screen for the target protein in species other than that of the immunogen.
Often the preferred choice for detecting denatured proteins.
Multiple epitopes generally provide more robust detection.
Prone to batch-to-batch variability.
Produce large amounts of non-specific antibodies, which can create background signal in some applications.
Multiple epitopes make it important to check immunogen sequence for cross-reactivity.
Not useful for probing specific domains of antigen because antiserum will usually recognize many domains.
High specificity. detect only one epitope on the antigen.
Consist of only one antibody subtype (eg IgG1, IgG2, IgG3). When a secondary antibody is required for detection an antibody against the correct subclass should be chosen.
Hybridomas are a constant and renewable source once created, and all batches will be identical, increasing consistency and standardization of experimental procedures and results.
Monoclonals detect one epitope per antigen. This has the following advantages:
Less background from staining of sections and cells. Specifically detecting one target epitope means they are less likely to cross-react with other proteins.
Due to their high specificity, monoclonal antibodies are excellent as the primary antibody in an assay and will often give significantly less background staining than polyclonal antibodies.
Homogeneity of monoclonal antibodies is very high relative to polyclonals. If experimental conditions are kept constant, results from monoclonal antibodies will be highly reproducible between experiments.
High specificity makes them extremely efficient for the binding of an antigen within a mixture of related molecules, such as during affinity purification.
May be too specific to detect across a range of species.
More vulnerable to the loss of epitope through chemical treatment of the antigen than polyclonal antibodies. This can be offset by pooling two or more monoclonal antibodies to the same antigen (eg cocktail antibodies).