Monoclonal Antibody Techniques

What is Monoclonal Antibody Technology

Monoclonal antibodies (mAbs) are monovalent antibodies which bind to the same epitope and are produced from a single B-lymphocyte clone. Monoclonal antibodies are important tools used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer.

What is the value of monoclonal antibody technology? Monoclonal antibody technology allows us to produce identical antibody molecules in large scale or industrial yields. It should be noted that the emergence of monoclonal antibody technology makes it possible for a variety of applications of monoclonal antibodies.

Our four platforms for monoclonal antibody development

  • Hybridoma technology
    Hybridoma technology
  • Antibody phage display
    Antibody phage display
  • Human naïve antibody library
    Human naïve antibody library
  • Single B cell antibody technologies
    Single B cell antibody technologies

Which Technology is for Your Monoclonal Antibody Development

Methods Advantages Disadvantages

Hybridoma

・Well-established processes
・A well-recognized method with Low R&D costs
・High specificity
・Long turnaround time
・Genetic rearrangement resulting in non-functional light chains
・Require humanization of the Ab sequences

Phage display

・Provides a direct linkage between phenotype and genotype of the antibody
・Antibodies derived from a wide variety of B cell types
・Flexible and specific screening schemes available according to the experimental design and antibody applications.
・Antibody sequence information to avoid loss of clones
・ Wide range of applications (epitope analysis, vaccine development, protein interaction analysis, peptide drugs, CAR-T…)
・Unnatural pairings of VH/VL with preference by the phage system
・Post-translational modification is restricted due to prokaryotic expression
・Only qualitative assays available during panning. Full-length antibody expression required for complicated activity assays.

Single B cell

・Natural VH/VL pairing
・Antigen-specific B cells sorting
・High specificity, high affinity, rich genetic diversity…
・Fresh samples, such as PBMCs, required for best sorting efficiency
・Antigen-specific cells exist at a low level that requires screening with high-quality antigens
・Strict operating environment
Antibody Development

Interested in mAb Generation Guide?

Get an in-depth overview of featured technology platforms, including hybridoma technology, antibody phage display and single B cell technologies.

Technologies for Monoclonal Antibody Production

Hybridoma technique

Monoclonal antibodies were first generated in mice in 1975 using a hybridoma technique. The generation of hybridomas involves immunising a certain species against a specific epitope on an antigen and obtaining the B-lymphocytes from the spleen of the animal. The B-lymphocytes are then fused (by chemical- or virus-induced methods) with an immortal myeloma cell line lacking the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) gene and not containing any other immunoglobulin-producing cells. These hybridoma cells are then cultured in vitro in selective medium (i.e. medium containing hypoxanthine-aminopterin-thymidine) where only the hybridomas (i.e. the fusion between the primary B-lymphocytes and myeloma cells) survive as they have inherited immortality from the myeloma cells and selective-resistance from the primary B-lymphocytes (as the myeloma cells lack HGPRT, they cannot synthesise nucleotides de novo as this is inhibited by aminopterin in the selective medium).

The initial culture of hybridomas contains a mixture of antibodies derived from many different primary B-lymphocyte clones, each secreting its own individual specific antibody into the culture medium (i.e. the antibodies are still polyclonal). Each individual clone can be separated by dilution into different culture wells. The cell culture medium can then be screened from many hundreds of different wells for the specific antibody activity required and the desired B-lymphocytes grown from the positive wells and then recloned and retested for activity. The positive hybridomas and monoclonal antibodies generated can then be stored away in liquid nitrogen. View more about "Hybridoma technique".

Antibody phage display

Another method of generating monoclonal antibodies is by using phage display. This involves isolating B-lymphocytes from the blood of humans and then isolating the mRNA and converting it into cDNA using PCR to amplify all the VH and VL segments. These segments can then be cloned into a vector (usually as scFv) next to the PIII protein of a bacteriophage before being used to infect E. coli in order to generate a library containing approximately 1010 cells by inoculating the library with an additional helper phage.

E. coli can then secrete the bacteriophage containing the VH and VL segments as part of the bacteriophage coat. Specific VH and VL segments against the antigen can then be selected and used to reinoculate E. coli with the bacteriophage. Cells containing the plasmid can then be isolated and sequenced. Its advantages include: once the library is made, the same library can be used to generate new antibodies and does not have to be remade, no immunisations are required as the entire process is done in vitro, antibodies can be obtained much more quickly than the traditional hybridoma technique and the library can be used to generate antibodies to toxic antigens that could not be used to immunise an animal.

Single B cell antibody technologies

This approach to produce monoclonal antibodies from single human B cells is based on the analysis of the immunoglobulin gene repertoire and reactivity at the single-cell level by the application of reverse transcription-polymerase chain reaction (RT-PCR) and expression vector cloning.

By recognition of selected cell surface markers, individual mouse or human B cells are isolated (e.g., by fluorescence-activated cell sorting), and genes coding for VL and VH fragments are separately amplified by RT-PCR and combined by PCR. For the final production of human mAbs in vitro, H and L chain gene transcripts from each cell are amplified by RT-PCR before cloning and expression in a mammalian system. This method has the virtue of being able to produce many specific human mAbs in a short period. View more about "Single B cell antibody technologies".

References

1. Baldo, B. A., & Baldo. (2016). Safety of Biologics Therapy. Springer.
2. Liu, J. K. (2014). The history of monoclonal antibody development–progress, remaining challenges and future innovations. Annals of Medicine and Surgery, 3(4), 113-116.
3. Siegel, D. L. (2002). Recombinant monoclonal antibody technology. Transfusion clinique et biologique, 9(1), 15-22.

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