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Monoclonal vs Polyclonal Antibodies
Article

Monoclonal vs Polyclonal Antibodies

Monoclonal vs Polyclonal Antibodies
Article

Monoclonal vs Polyclonal Antibodies

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Antibodies are immunological proteins that play fundamental roles in host defense against infectious agents such as viruses, bacteria and fungi. The immunological functions of these Y-shaped proteins are determined by their ability to bind antigens, the molecules released by or found upon cells that stimulate an immune response.

B
eyond their roles in host immunity, antibodies prove to be valuable for research and therapeutic purposes too. Two types of antibodies – polyclonal and monoclonal – provide research scientists with distinct ways to detect or quantify target antigens largely due to differences in specificity and affinity. In this article, we describe polyclonal and monoclonal antibodies in more detail and consider how each type may be used for various applications.

What are polyclonal antibodies?


Once surveilling immune cells detect antigens in a host, the immune system undergoes a cascade of molecular events that induce the production of polyclonal antibodies by stimulated B cells. Polyclonal antibodies
1 consist of biochemically dissimilar paratopes, the antigen-binding sites on the extended “arms” of an antibody, that enable specific binding to different binding sites on the same target antigen.

Polyclonal antibodies are attractive for research applications due to a combination of their high affinity towards the antigen of interest and their relative tolerance to minor changes exhibited by the target antigen in solution. For example, polyclonal antibodies generally display higher sensitivity than their monoclonal counterparts in diagnostic assays due to their biophysical diversity towards a target antigen. These features can also contribute to the clearance of pathogens more effectively
2 than monoclonal antibodies. However, the feasibility of using polyclonal antibodies at scale is limited by factors such as our inability to industrially reproduce these proteins with consistency.

How do you generate polyclonal antibodies for research?


To generate polyclonal antibodies suitable for research purposes, a laboratory animal must first be injected with a peptide, protein, molecule or a whole cell that stimulates an immune response. Once the host immune system generates antibodies against the injected antigen, they must be harvested and purified from the animal’s antiserum. One way that polyclonal antibodies are purified from animal serum is through the application of antigen-coated magnetic beads, precipitation or chromatography methods after immunization
3. After this affinity-based purification method reduces nonspecific antibodies in the final polyclonal population, these antibodies are ready to be used for detection-based methods like western blotting, immunohistochemistry and enzyme-linked immunosorbent assays.

Differences between monoclonal and polyclonal antibodies


What are monoclonal antibodies?


Unlike polyclonal antibodies, monoclonal antibodies
4 consist of highly specific proteins that recognize a single epitope on an antigen. Monoclonal antibodies are produced ex vivo by hybridomas, a type of immortalized cell line with the ability to produce antibodies like a B cell.

How do you generate monoclonal antibodies for research?


The production of hybridomas involves the fusion of isolated spleen cells from immunized mice with immortalized myeloma cells.
Clones of the resulting hybridomas are then screened and selected for based on target antigen specificity and reactivity. Since monoclonal antibodies share high specificity to a single epitope, they can prove useful to research scientists and clinicians alike.

Monoclonal vs polyclonal antibodies - key differences

Properties

Monoclonal Antibodies

Polyclonal Antibodies

Population of antibodies

Homogeneous

Heterogeneous

Binding specificity

Recognize a single epitope on a target antigen

Recognize multiple epitopes on a target antigen

Sensitivity

Better for protein quantitation

High for low-quantity proteins

Cross-reactivity

Lower due to high specificity

Higher due to biophysical diversity

Cost to produce

More expensive

Inexpensive

Origin

Single lineage of stimulated B cells; hyridomas

Multiple lineages of stimulated B cells

 

Uses of polyclonal and monoclonal antibodies


Two of the key features that define the usefulness of polyclonal and monoclonal antibodies involves their respective specificity and sensitivity to an antigen. The specificity of an antibody is determined by the relative affinity between its binding domain and the target antigen while other molecules are present. The exploitation of this specificity is essential for immunological researchers and clinicians since many applications utilize polyclonal and/or monoclonal antibodies to specifically detect target molecules. In conjunction with antigen specificity, the sensitivity of an antibody is an important parameter that determines its usefulness in the lab.

High sensitivity antibodies are well suited for diagnostic applications
5 like immunoprecipitation, western blot and enzyme-linked immunosorbent assay (ELISA) due to their ability to recognize low levels of target antigen. Immunoprecipitation is an analytical technique that isolates an antigen from a mixture by using an antibody that specifically binds the antigen. The resulting antigen-antibody complexes can then be further processed by an immobilized antibody or magnetic beads, which allows the antigen-antibody complexes to be separated from the mixture prior to analysis.

Like immunoprecipitation, ELISAs
6 can use polyclonal or monoclonal antibodies to detect a target antigen in solution. Indirect and sandwich ELISAs are two immunoassay forms that use both types of antibodies throughout their procedures: monoclonal antibodies are typically applied first (primary) due to their high specificity to the target antigen; polyclonal antibodies are more useful as the secondary reagent due to their ability to amplify a low signal with high sensitivity7. Beyond ELISAs, antibodies are useful for detection techniques like immunohistochemistry and flow cytometry since they can detect antibody-antigen constructs within a complex tissue or cell with high resolution.

Confirmation of antibody specificity and sensitivity by manufacturers allow immunoassays like ELISAs to take on different forms depending on how the antigens and antibodies are used, which make these tests highly versatile. For example, an immunoassay that requires a highly sensitive test to detect an unknown pathogen would benefit from polyclonal antibodies due to their ability to recognize multiple epitopes on an antigen. If a pathogen or antigen has previously been characterized, then it may be more appropriate to use monoclonal antibodies in downstream applications for further characterization.

Outside of the laboratory, antibodies can be used to enhance, mimic or restore the immune system’s ability to attack disease targets. With some exceptions, monoclonal antibodies are better suited for therapeutic purposes than polyclonal antibodies due to their homogeneity, high specificity to a single epitope and their low degree of cross-reactivity
8. These properties are especially important for effective cancer therapies since tumor cells are capable of evading or blocking the immune system. Some of the ways monoclonal antibody therapies function against cancer cells include mechanisms that involve direct binding to induce cell death and indirect mechanisms that block tumors from growth factors and blood supply.9

References

  1. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR Journal. 2005;46(3):258-268. doi:10.1093/ilar.46.3.258
  2. Bregenholt S, Jensen A, Lantto L, Hyldig S, Haurum JS. Recombinant human polyclonal antibodies: a new class of therapeutic antibodies against viral infections. Curr. Pharm. Des. 12(16), 2007–2015 (2006). doi: 10.2174/138161206777442173
  3. Grodzki AC, Berenstein E. Antibody purification: ammonium sulfate fractionation or gel filtration. Methods MolBiol. 2010;588:15-26. doi:10.1007/978-1-59745-324-0_3
  4. Nelson PN, Reynolds GM, Waldron EE, Ward E, Giannopoulos K, Murray PG. Monoclonal antibodies. Mol Pathol. 2000;53(3):111-117. doi:10.1136/mp.53.3.111
  5.  Ascoli CA, Aggeler B. Overlooked benefits of using polyclonal antibodies. Biotechniques. 2018;65(3):127-136.doi:10.2144/btn-2018-0065
  6. Hornbeck, P. Enzyme-Linked Immunosorbent Assays. Curr Protoc Immunol. 1992;1: 2.1.1-2.1.22. doi: 10.1002/0471142735.im0201s01
  7.  Alhajj M, Farhana A. Enzyme Linked Immunosorbent Assay. [Updated 2021 Feb 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK555922/
  8. Shepard HM, Phillips GL, D Thanos C, Feldmann M. Developments in therapy with monoclonal antibodies and related proteins. Clin Med (Lond). 2017;17(3):220-232.doi: 10.7861/clinmedicine.17-3-220
  9.  Zahavi D, Weiner L. Monoclonal Antibodies in Cancer Therapy. Antibodies (Basel). 2020;9(3):34. doi:10.3390/antib9030034

Meet The Author
Jonathan Dornell, PhD
Jonathan Dornell, PhD
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