I. Overview

Monoclonal antibodies have woven themselves into the fabric of basic research, diagnostic and biomarker assays, and therapeutics over the past 3-4 decades and truly transformed our understanding of the biological world.  There is likely no area of biology or medicine that has not benefited (or will benefit) from monoclonal antibodies.  The goal of this blog is to delve into, well—”The Biology of Antibodies”, to better understand the use and development of antibodies for research, diagnostics, and therapeutic purposes.  To begin, I wish to set the foundation of the development and use of the monoclonal antibody technology and then throughout the year I will focus on the specific biological processes that allow us to tune this technique to our advantage.  So, let’s begin.

II. A Very Brief History of Monoclonal Antibodies

There has been interest in using antibodies clinically since around 1890 when von Bering and Kitasato demonstrated passive immunity protected experimental animals against lethal doses of diphtheria1. It wasn’t until around the 1940’s however that antibodies began to be used to detect the presence of infections2, through the precipitin reaction discovered by Jacque Oudin, which led to the development of immunodiffusion assays. In the late 1950’s Roslyn Yalow and Solomon Berson developed the very sensitive radioimmunoassay3, which solidified the use of antibodies as a detection tool and revolutionized the immunoassay.  Developing specific antisera was limited to polyclonal antibody production in animals until 1975 when George Kohler and Cesar Milstein published the groundbreaking method for the production of cell lines that could be stably maintained in tissue culture to produce a monoclonal antibody4.  The key development was the establishment of an immortalized mouse myeloma cell line that could be fused with primary spleen cells to produce a hybridoma cell line that secretes a single (monoclonal) specific antibody. The immortalized myeloma cell line was critical here: a myeloma is a cancerous plasma cell, or antibody secreting cell, which was generated and selected such that it no longer secreted antibody and also was sensitive to HAT media selection (due to a mutation in the HGPRT gene).  Therefore successful fusion with a primary B cell to generate a hybridoma, resulted in antibody secretion and survival due to the immunoglobulin genes and wild type HGPRT gene transferred to the myeloma cell line, which conferred immortal growth.  While there have been advances in cell lines as well as selection and identification of antibody specificity and affinity, the overall process has generally remained the same since Kohler and Milstein originally described it over 40 years ago.

III. The Hybridoma Development Process for the Production of a Monoclonal Antibody

The identification and selection of a monoclonal antibody requires 4 steps (see Figure 1): 1) immunization, 2) somatic cell fusion, 3) selection & screening, and 4) subcloning.  Each of these steps will be discussed briefly below and in future articles in this blog we will go into detail regarding the biological process that drives this immunological process, manipulation and adaption of the general technique, and the utilization of the monoclonal antibody itself for research, diagnostics, and therapeutics.

Immunization picture1) Immunization. Mouse monoclonal antibody development relies on the generation of antibodies to a specific target (the antigen).  In general the naïve mouse immune system (or an immune system that has not been exposed to any or limited foreign antigens), does not produce antibodies with selective specificity or high affinity to a specific target.  In order to generate antibodies with specificity to a single or unique antigen, we must expose or immunize the mouse to develop a specific, hopefully high affinity, antibody response.  Immunization requires repeated exposure to the antigen, typically in an adjuvant mixture that helps to elicit a strong immunological response to the antigen.  This immune response will generate antibody production and through repeated exposures will increase the specificity to the antigen. Common routes of exposure are subcutaneous, intraperitoneal, and intravenous injections. To test the mouse to see if a response is being generated, blood is collected and the sera is tested, generally by ELISA (although other immunoassays can be used here as well), to determine if the mouse is responding to the antigen as well as determining the titer (concentration of the antibody in sera determined by serial dilution).  Once the appropriate antibody response or titer is achieved, the mouse is boosted one final time in preparation for the fusion.

2) Somatic Cell Fusion. The final antigen boost, or fusion boost, is initiated to induce an expansion of memory B cells to become antibody forming cells or plasmablasts; in addition to the expansion of the antibody specific B cells the fusion boost isFusion Picture typically injected intraperitoneally to encourage the cells to migrate into the spleen.  Three to four days after the fusion boost, the spleen is harvested from the mouse and all of the spleen cells, or splenocytes, are collected. The splenocytes are then fused with a myeloma cell line grown to the proper density.  We typically use F0 cells, however there are several excellent myeloma cell lines derived specifically for hybridoma development. The fusion is mediated with either polyethylene glycol (PEG) or electrofusion(inactivated Sendai virus has also been used to fuse somatic cells); both of which induce the splenocytes and myeloma cells to combine and generate a hybrid cell, or hybridoma.  The chromosomes from the two fused cells will reassort into a single nuclei with chromosomes originating from both cells.  However it will not contain all of the chromosomes from each cell, some of the genetic material will be discarded (to generate a normal diploid chromosome number) and each cell will have a unique genetic composition.  The cells that contain the genetic material necessary to produce immunoglobulins (obtained from the B cells of the spleen), the wild type HGPRT gene (obtained from the primary splenocytes), and the ability to grow immortally and secrete antibody (obtained from the myeloma cell line) will allow for selection of stable B lymphocyte hybridoma cell lines (generally just referred to as hybridomas or B hybridomas) that produce the antibody with the specificity of interest.

3) Hybridoma Selection and Screening Cells have the ability to synthesize nucleotides de novo (from scratch) or through the salvage pathway which recycles degraded nucleic acids within the cell. The HGPRT gene is required for the cells to generate nucleosides (for the generation of nucleotides) from nucleic acids through the salvage pathway, which means that the myeloma cells used for hybridoma development are unable to use the salvage pathway due to a gene defect in HGPRT.  The HAT selection media (culture media containing Hypoxanthine, Aminopterin, & Thymine) requires that the salvage pathway is intact as the de novo pathway for nucleotide synthesis is blocked. Therefore the only hybridoma cells that will survive are the cells that contain a functioning HGPRT gene.  The HAT selection will result in the non-HGPRT containing cells (myeloma cells that didn’t properly fuse with B cells) to die off after several days.  Primary B cells will not survive in culture very long and will also die off if they are not properly fused with the myeloma cell.  The selection process will yield healthy hybrid cells that contain the HGPRT gene. These resulting hybridoma cells can now be screened for specific antibody production.  Using an immunoassay that allows for the detection of antibodies specific for the intended antigen, cell populations are identified that contain the appropriate antibody or antibodies.  Typically the hybridoma cells are plated in 96-well culture plates so that the cells can be separated and screened efficiently.  It is the culture supernatant from these wells that is used in an immunoassay to identify the wells with hybridoma cells producing the appropriate antibody.

4) Subcloning. The wells that are positive in the fusion screen generally contain more cells than just those that are producing the antibody of interest.  We refer to these as polyclonal wells, as there are multiple cell colonies or clones.  In order to remove the unwanted cells, a process of subcloning is undertaken, which typically involves one or more rounds of limiting dilution.  The general principle is to dilute the cells in cell culture dishes or typically 96-well culture plates so that there is an average of less than one cell per well.  This results in many wells only having one cell in the well (some with multiple cells and many with no cells), and therefore is monoclonal.  Once these individual cells have been allowed  to divide and expand they can be screened (typically using the same method as for the serum and fusion screens) for antibody production and specificity towards the original antigen used in immunization of the mice.  As these hybridoma cells are monoclonal (although sometimes multiple rounds of limiting dilution subcloning are required depending on how multiclonal the original parental well was at the start of the subcloning process), the antibody produced by these cells are referred to as monoclonal antibodies.  At this stage the hybridoma cells can be scaled up for production of large quantities of a specific monoclonal antibody for a wide variety of uses.  

Depending on how well the antigen elicits an immune response, the process, while seemingly straightforward, has several delicate steps and requires a capable cell culturist and a good immunoassay screening strategy to yield positive results.  We here at BBI, have 25-years of experience and would welcome the opportunity to assist you in any way possible for any of your antibody development requirements.


  1.  Von Bearing, E. A. & Kitasato, S. Uber das Zustandekommen der Diphtherie- Immunitat und der Tetanus-Immunitat bei Thieren. Dtsch Med Wochenschr 49, 1113–4 (1890).
  3. Yalow, R. S. & Berson, S. A. Immunological specificity of human insulin: application to immunoassay of insulin. J. Clin. Invest. 40, 2190 (1961).
  4. Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).