Monoclonal and Polyclonal Antibodies Production Methods and Differences

  • The ability of animal immune systems to antibodies production capable of binding specifically to antigens can be of use to develop probes for the detection of molecules of interest in a variety of research and diagnostic applications.
  • Nearly all medical or cell biology researchers use immunochemical techniques for molecular analysis.
  • Usually, all immunochemical methods depend upon the utilization of antibodies, and their effectiveness relies on the quality of the antibodies used.
  • The antibody production process involves the preparation of antigen samples and immunization of laboratory or farm animals to trigger the expression of antigen-specific antibodies within the serum at a high level, which then can be recovered from the animal.
  • Successful antibody production depends upon cautious planning and implementation concerning several steps and considerations.

Steps and Considerations of Antibody production

  • Synthesis and purification of the target antigen
  • Selection of an appropriate immunogenic carrier protein
  • Conjugating the antigen and carrier protein to make an immunogen
  • Immunization of animals using appropriate schedule and adjuvant formula
  • Screening of serum or hybridoma for antibody titer and isotype

There are two major classes of antibodies utilized in immunochemistry:

  1. Monoclonal
  2. Polyclonal

Differences between Monoclonal antibodies and Polyclonal antibodies

Monoclonal antibodiesPolyclonal antibodies
Monoclonal antibodies are epitope specific for an antigen.  Polyclonal antibodies are antigen specific.  
Derived from one specific clone of B cell, that recognizes one particular epitope on an antigen.  Derived from many B cell clones, that produce antibodies recognizing different epitopes of an antigen.
  Produced by animal cells artificially in tissue culture using hybridoma technique.  Produced by host animal immunized with the substance of interest usually three or four times.
    Expensive to produce and generally produced in mice.  Cheap to produce and generally produced in rabbits and guinea pigs for small amounts, and sheep or goats for large amounts.
Differences between Monoclonal antibodies and Polyclonal antibodies

Monoclonal Antibody

  • Monoclonal antibodies are antibodies that are made by identical immune cells, clones belonging to a single parent cell.
  • Monoclonal antibodies have monovalent affinity and bind to one particular epitope of an antigen.
  • Monoclonal antibodies can be produced in specialized cells through a method now referred to as hybridoma technology.
  • Kohler and Milstein in 1975, were the first to fuse lymphocytes to generate a cell line, both immortal and a producer of specific antibodies. They won the Nobel Prize for Medicine in 1984 for the development of ‘hybridoma’.
  • The value of hybridoma was not well acknowledged before 1987, around which the production of monoclonal antibodies in rodents for usage in diagnostics started.
  • B cells can mutate into tumor cells that result in a type of cancer called myeloma. Myeloma cells are immortal and grow indefinitely in culture. The fusion of a single activated B cell and a myeloma cell creates hybridoma that can grow continuously in culture and produce antigen-specific antibodies.

Production steps of Monoclonal Antibody

  • Mice are immunized with an antigen, and their blood is screened for antibody production.
  • The antibody-producing splenocytes are extracted for in vitro hybridoma production.
  • Myeloma cells are made ready for fusion.
  • Myeloma cells and isolated splenocytes are fused, creating hybridomas in the presence of polyethylene glycol (PEG). PEG causes the fusion of cell membranes1.
  • The hybridoma clones can be selected using HAT medium. Clones are screened and selected based on antigen specificity and immunoglobulin class.
  • Each positive clone is confirmed, validated, and characterized (Isotyping).
  • Positive clones are expanded, scaling up the production of the desired antibodies.
Production of monoclonal antibodies
Production of monoclonal antibodies

Figure: Production of monoclonal antibodies

HAT (hypoxanthine, aminopterin, thymidine) selection:

  • HAT medium contains drug aminopterin that blocks the de-novo synthesis of DNA nucleotides.
  • This makes the cells depend upon another pathway that needs HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) enzyme for DNA synthesis.
  • Myeloma cells which do not fuse with the B cells cannot grow in HAT medium, since they are HGPRT negative2.
  • B cells that are not fused with the myeloma cells also die as they have short life-span.
  • Therefore, the HAT medium allows the selection of hybridoma cells with the HGPRT gene and immortal property.

Polyclonal Antibody

  • Polyclonal antibodies are antibodies that are secreted by different B cell lineages within the body.
  • They are a collection of antibodies that react against a particular antigen, each binding to different epitopes.
  • Polyclonal antibodies are produced in the appropriate donor animals.
  • Usually, antigens are conjugated with an adjuvant before immunizing the animals.
  • Adjuvants are substances that increase the immunogenicity of the antigen, reducing the amount of antigen required as well as stimulating specific immunity to it.
  • Freund’s complete adjuvant (FCA), alum, bentonite, and Bacillus pertussis are some of the adjuvants that can be of use.

Production steps of Polyclonal Antibody

  • Preimmunize blood samples are collected to produce baseline IgG levels.
  • The first two immunizations are done within 14 days.
  • Later immunizations are spaced at intervals of 4-6 weeks to maximize the antibody production.
  • Blood samples are collected 10 days after the completion of immunization program.
  • The serum screened for presence of antibodies with specific activity to antigen. Method such enzyme-linked immunosorbent assay (ELISA) can be used for the activity testing.
Production of polyclonal antibodies
Production of polyclonal antibodies


1.        Wojcieszyn J, Schlegel R, Lumley-Sapanski K, Jacobson K. Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes. J Cell Biol. 1983;96:151-159. doi:10.1083/jcb.96.1.151

2.        Martínez P, Iborra A. Antibody Synthesis in Vitro. In: ; 2006. doi:10.1038/npg.els.0001115

Binod G C

I'm Binod G C (MSc), a PhD candidate in cell and molecular biology who works as a biology educator and enjoys scientific blogging. My proclivity for blogging is intended to make notes and study materials more accessible to students.

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