Production of Monoclonal Antibodies Using Hybridoma Technology
Cells termed hybridomas manufacture monoclonal antibodies in a huge amount which is desirable. The mouse spleen cells are fused with mouse myeloma cells. Then, the secreted antibodies which show specificity are determined by parent spleen cell and the quantity properties are dictated by myeloma. Two researcher, Kohler and Milstein uncovered this technology in 1975. This technology creates monoclonal antibodies in distinct cells. Monoclonal antibodies are used in inhibiting, analysis and managing disease. Cancer antigens, alone or coupled with anticancer agents are marked by monoclonal antibodies. They are also used in saving organ transplantation where rejection of organ can occur.(Tokunaga, Chiba and Ohnishi, 2010)
Technique involved in hybridoma technology:
1. Necessary to initiating a hybridoma project:
– Sterility is necessary in cabinet where hybridoma production is introduced.
– An incubator is needed in which temperature is controlled, humidity is maintained and gas concentration is maintained. Liquid nitrogen storage should be facilitated as it is essential for hybridomas maintaining in a low temperature store for cryopreservation.
-Ease of animal holding, aseptic surgical equipment for mouse dissection, water baths of temperature at 37 and 56 degree Celsius, centrifugation machine, tissue culture ware are also needed.(Hurrell, 2018)
2. Materials and media:
– Cell growth medium is used. RPMI 1640 is used. In addition to it, FCS 10%, Glutamine 2mM, Penicillin 100 International Units/ml and streptomycin 100 microgram/ml are used.
– HAT selective medium is used.
– Polyethylene glycol is used.(Hurrell, 2018)
3. Selection of antigens and immunization schedules:
-Highly distinct antibodies against impure antigens are involved in purifying the antigens.
-Spleen cells are utilized in the antibody-producing hybrids.
-BALB/c mice strain are used.(Hurrell, 2018)
4. Choice of parent myeloma lines:
-It`s growth should be better and high concentrations of immunoglobulin must be secreted from it.
– The main biosynthesis pathway which does nucleic acid synthesis is inhibited by an aminopterin. Cell`s multiplications are continued by using salvage pathway in the presence of hypoxanthine and thymidine. If an enzyme needed in the salvage pathway that is absent in a mutant cell, growth of cell is impossible. Myeloma cells which are shortage of hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT) are applied. (Hurrell, 2018)
5. Hybridoma colony and antibody production:
A specific antigen is injected into mouse. Mouse spleen produces the antigen-specific plasma cells and myeloma, a cancerous cell is intermixed with these cells. This hybrid cell is thus duplicated and many identical daughter clones are produced and then secretion results in immune cells. Thus monoclonal antibodies are produced from only one type of cell. Then, monoclonal antibody is produced in HAT medium (hypoxanthine aminopterin thymidine). To obtain antibody against a specific antigen, that antigen is exposed to mice. Segregation of splenocytes from the mammal and fusion of the B cells with immortal myeloma cells that lack HGPRT gene is occurred. Polyethylene glycol or Sendai virus aids in fusion. Incubation of intermixed (fused) cells in the HAT medium occur. The mechanism depends on biosynthesis of nucleotides assures that only fused hybrids will survive. Tetrahydrofolate is essential for nucleotide synthesis and this can be acquired by dihydrofolate reductase. Folic acid analogue aminopterin blocks this enzyme. Incorporating 6-thioguanine or 8-azaguanine into nucleotides in the medium by HGPRT cause the death of cells. The cells where active HGPRT is absent can survive. So, only survival portions are B cell-myeloma hybrids. Thus, antibodies production by these cells (characteristics of B cells) and cell`s immortality (characteristics of myeloma cells) happen. Then, dilution of incubated medium into multi well plates are done so that only one cell can be present in each well and checking for desired antibody is taken place. (Tokunaga, Chiba and Ohnishi, 2010)(Kulkarni, 2002)
Adapted from: Tabll, A. et al. (2015) Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus, World Journal of Hepatology. Baishideng Publishing Group Inc. doi: 10.4254/wjh.v7.i22.2369. (Accessed: 4 November 2018).
Figure: Schematic diagram of production of monoclonal antibodies using hybridoma technology
6. Rapid primary screening process:
ELISA can be done. Here, adsorbtion of antigen to the bottom of 96-well plates, incubation for growth of hybridomas occur. The desired antibody in the sample remains bound to antigen and is detected by an immune-conjugate. Immune-conjugate is consists of two components, one antibody is distinct for an epitope that remains in the constant domain in the first antibody. It acts as anti-antibody. Second one is alkaline phosphatase enzyme. Immune-conjugate is retained in the well during the immobilization at first incubation of antibody. After washing colorless substrate of enzyme (ex. p-nitrophenyl phosphate) is converted to a colored product (ex. p-nitrophenol) by the enzyme (ex. alkaline phosphatase). After incubation and the termination of enzyme function, ELISA reader quantify the optical density of product.(Kulkarni, 2002)
Adapted from: ELISA Guide – Creative Diagnostics (no date). Available at: https://www.creative-diagnostics.com/ELISA-guide.htm (Accessed: 4 November 2018).
Figure: Screening process by ELISA (Enzyme-linked immunosorbent assay)
After screening, cloning can be done by three different techniques (cloning by technique of limiting dilutions, cloning using semi-solid agars and cloning and selection using the fluorescence-activated cell sorter). The limiting dilution method allows the enumeration of cells in the culture, dilution and fractional adsorption into new wells in which each well is consist of only one cell. Then, cell regeneration process is repeated to assure presence of monoclonal property. Another method is soft agar method that allows the proliferation of enormous malignant cells in a low agar content semi-solid medium. The dispersion of culture into single cells and the presence of spaced colonies due to such cell concentration ensure the presence of monoclonal antibody. Both techniques are used by combining these methods.(Hurrell, 2018)
Adapted from: Cloning Method. EuroMAbNet (no date). Available at: https://www.euromabnet.com/protocols/cloning.php (Accessed: 4 November 2018).
Figure: Cloning process by technique of limiting dilutions
It is essential against the loss of beneficial lines. Hybridoma should be froze down as soon as possible to reduce the loss of chromosome.(Hurrell, 2018)
9. Characterization of monoclonal antibodies:
Monoclonal antibodies should be characterized with respect to following parameter:
-Specificity is determined for specific antigen.
-Titer: By choosing for screening and determining the highest dilution at which a positive outcome can be achieved.
-Affinity of binding can be measured.
-Storage and stability are determined to know the effect of manipulations, such as lyophilization .
-Immunoglobulin class/subclass: For recognition of IgG1, igG2a, IgG2b, IgG3 and IgM , sets of antisera are used.
-Monoclonality : Only one subclass of antibody/ only one cell type should be present in hybridoma.
10. Production, purification and labeling of monoclonal antibodies:
Monoclonal antibody can be manufactured by two processes (tissue culture or by maturing hybridoma as a tumor in mice). Then, ammonium sulfate can precipitate antibody and ion-exchanging chromatography technique and antigen affinity chromatography technique can be used for higher extend of antibody purity. At last, Labeling of monoclonal antibodies can be achieved by radio-isotopic labeling or by fluorescence labeling.(Hurrell, 2018)
Adapted from: Affinity Purification – Biologicscorp Blog (no date). Available at: https://www.biologicscorp.com/blog/protein-purification-methods-based-on-bioproperties-affinity/#.W98qUJMzbIU (Accessed: 4 November 2018).
Figure: Selective adsorption of the desired antibody is enabled by Affinity chromatography
Use of monoclonal antibody:
-Recognition of ABO blood groups
-Diagnosis (ELISA test is used for determination of viruses and imaging)
-Immunopurification that separates one substance from a mixture of very analogous molecule(Tokunaga, Chiba and Ohnishi, 2010)
A monoclonal antibody can be prepared if the series of procedure can be done successfully. Immunization of mice, a myeloma cell line for fusion and maintaining in culture are the core procedures. The fusion products are allowed to grow in culture where other dividing cells are absent. The growing hybridoma colonies are appeared and then screened to obtain the desired antibody. The interesting hybrids are undergone cloning process for ensuring monoclonality. Cloned hybrids that produce antibodies of interest are manufactured in large quantities so that useful quantities of antibody can be obtained and then the cryopreservation of the cells are done. Finally, it is essential to characterize, purify and label the monoclonal antibody. (Hurrell, 2018)
Hurrell, J. G. R. (2018) Monoclonal Hybridoma Antibodies: Techniques and Applications – John G.R. Hurrell – Google Libri. doi: 10.1109/34.713358.
Kulkarni, G. T. (2002) ‘Production of Monoclonal Antibodies’, in Biotechnology and Its Applications in Pharmacy. 1st ed. New Delhi: Jaypee Brothers, Medical Publishers, pp. 4–51.
Tokunaga, T., Chiba, J. and Ohnishi, K. (2010) ‘Attempts to improve hybridoma technology for the production of human monoclonal antibodies’, Gan to kagaku ryoho. Cancer & chemotherapy, 14(2), pp. 2198–2204.