Friday, 7 March 2014

Key differences between polyclonal and monoclonal antibodies

Polyclonal and monoclonal: A comparison




Summary

Polyclonal antibodiesMonoclonal antibodies
Inexpensive to produceExpensive to produce
Technology required is lowHigh technology required
Skills required are low Training is required for the technology use
Time scale is shortTime scale is long for hybridomas
Produces large amounts of non specific antibodiesCan produce large amounts of specific antibodies but may be too specific
Recognizes multiple epitopes on any one antigenRecognizes only one epitope on an antigen
Can be batch to batch variabilityOnce a hybridoma is made it is a constant and renewable source and all batches will be identical

Polyclonal antibodies

Facts:
  • Recognise multiple epitopes on any one antigen. Serum obtained will contain a heterogeneous 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
Antibody production:
  • Inexpensive to produce
  • Technology and skills required for production low
  • Production time scale is short
  • Polyclonal antibodies are not useful for probing specific domains of antigen because polyclonal antiserum will usually recognize many domains
General advantages: 

Polyclonals will recognize multiple epitopes on any one antigen which has the following advantages:
  • Polyclonals can help amplify signal from target protein with low expression level, as the target protein will bind more than one antibody molecule on the multiple eptitopes. This would not be an advantage for quantification experiments e.g. in flow cytometry, as the results would become inaccurate.
  • Due to recognition of multiple epitopes, polyclonals can give better results in IP / ChIP
  • More tolerant of minor changes in the antigen, e.g., polymorphism, heterogeneity of glycosylation, or slight denaturation, than monoclonal (homogenous) antibodies.
  • They will identify proteins of high homology to the immunogen protein or to screen for the target protein in tissue samples from species other than that of the immunogen e.g. Polyclonal antibodies are sometimes used when the nature of the antigen in an untested species is not known. This also makes it important to check immunogen sequence for any cross-reactivity.
  • Polyclonal antibodies are often the preferred choice for detection of denatured proteins.
  • Multiple epitopes generally provide more robust detection.
  • Polyclonal antibodies not useful for probing specific domains of antigen, because antiserum will usually recognize many domains.
Disadvantages:
  • Prone to batch to batch variability.
  • They produce large amounts of non-specific antibodies which can sometimes give background signal in some applications.
  • Multiple epitopes make it important to check immunogen sequence for any cross-reactivity. 

Monoclonal antibodies

Facts: 
  • Detect only one epitope on the antigen.
  • They will consist of only one antibody subtype. Where a secondary antibody is required for detection, an antibody against the correct subclass should be chosen.
Antibody production 
  • High technology required.
  • Training is required for the technology used.
  • Time scale is long for hybridomas.
Advantages:
  • Once hybridomas are made it is a constant and renewable source and all batches will be identical – useful for consistency and standardization of experimental procedures and results
Monoclonals detect one epitope only on any one antigen which has the following advantages:
  • Monoclonals usually have less background from staining of sections and cells. As they are more specifically detecting one target epitope, they are less likely to cross-react with other proteins.
  • Because of their specificity, monoclonal antibodies are excellent as the primary antibody in an assay, or for detecting antigens in tissue, and will often give significantly less background staining than polyclonal antibodies.
  • Compared to polyclonal antibodies, homogeneity of monoclonal antibodies is very high. If experimental conditions are kept constant, results from monoclonal antibodies will be highly reproducible, between experiments.
  • Specificity of monoclonal antibodies makes them extremely efficient for binding of antigen within a mixture of related molecules, such as in the case of affinity purification
Disadvantages:
  • They can produce large amounts of specific antibodies but may be too specific (e.g. less likely to detect in across a range of species) 
  • More vulnerable to the loss of epitope through chemical treatment of the antigen than are polyclonal antibodies. This can be offset by pooling two or more monoclonal antibodies to the same antigen.