Cells called hybridomas manufacture antibody in huge amount which is desirable and create monoclonal antibodies.The mouse spleen cells that are antibody-producing are fused with mouse myeloma cells.The obtained secreted antibodies which show specificity are determined by parent spleen cell and the quantity properties are dictated by a myeloma.Here, some chromosomes are lost and some characters of each of the parental cells are preserved in the hybrids.Two scientists, Kohler and Milstein uncovered the hybridoma technology in 1975. this technology introduces monoclonal antibodies in specific cells. Monoclonal antibodies are beneficial for study of parasites antigens. They are used in inhibiting,analysis and managing disease. Cancer antigens,alone or coupled with anti cancer agents are indicated by monoclonal antibodies. They are also used in saving organ transplantation where rejection of organ can occur.
Technique involved in hybridoma technology:
1. Prerequisites to starting a hybridoma project-
– Sterility must be maintained in cabinet where hybridoma production will be introduced .
– An incubator is required 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 maintainment 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 celcius, centrifugation machine , tissue culture ware are also needed.
2. Materials and media:
-Cell culture medium is used. RPMI 1640 is used,. In addition to it, FCS 10%, Glutamine 2mM, Penicillin 100 IU/ml and streptomycin 100 microgram/ml are used.
– HAT selective medium components ( hypoxanthine, aminopterin, thymidine) are used.
-polyethylene glycol are used.
3. Choice of antigens and immunization schedules:
-Highly specific antibodies against impure antigens are obtained and antibodies can be used to clarify the antigens.
-Spleen cells are used in the antibody-producing hybrids.
-Increasing number of cells in blast transformation ,and depleting insignificant cells are beneficial.
-The ratio of hybrid specific for aimed antigen is enhanced by using purify antigens.
-BALB/c mice strain are used cause myeloma cells set for fusion are of origin of BALB/c.
4. Choice of parent myeloma lines:
-It`s growth should be better and high concentrations of immunoglobulin must be secreted from it. Hybrids must be delayed from parent cells.
-HAT selective medium is required. The main biosynthesis pathway which cause nucleic acid synthesis is blocked by 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 lack in a mutant cell,growth of cell is impossible. Generally, myeloma cells which lack hypoxanthine guanine phosphoribosyl transferase ( HGPRT) are applied. The HGPRT from spleen cell parent is present in fusion product and multiplication occurs in HAT medium.
5. Hybridoma colony and antibody production:
Basic outline of hybridoma technology is following:
A specific antigen is injected into mouse , the antigen-specific plasma cells are produced from the mouse `s spleen and myeloma, a cancerous cell is fused with this cells. This is called hybridoma . The hybrid cell is thus duplicated and innumerable identical daughter clones are obtained and then secretion results in immune cells. These antibodies are called monoclonal antibodies as they produce from only one type of cell. Then, monoclonal antibody is prepared by HAT medium ( hypoxanthine administering thymidine) . To obtain desire antibody against specific antigen, mice are exposed to that antigen.Isolation of splenocytes from the mammal, fusion the B cells with deathless myeloma cells that lack HGPRT gene occur. Polyethylene glycol or Sendai virus helps in fusion. Incubation of fused cells in the HAT medium cause aminopterin in the myeloma cells death as nucleotide inhibition occur. So, death of myeloma cell occur in culture that lack HGPRT enzyme. Death of unfused B cells happen for shorter life span. Only survival portions are B cell-myeloma hybrids because of functional HGPRT gene originated from the B cells. Thus, antibodies are produced by these cells ( characteristics of B cells) and cells are immortal ( characteristics of myeloma cells). 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 are taken place.The antibodies in a all came from the same B cell ,will be in the direction of same epitope and so, this is called monoclonal antibodies. Now, a hybrid oma colony is created ,continuous growth takes place in culture medium and antibody is obtained.
6.Rapid primary screening process:
Hybridomas that create antibodies of appropriate specificity is identified and selected only. Secondary enzyme that is labelled with conjugate , hybridoma that culture supernatant and chromatogenic substrate can be breed and a colored product is formed. it indicates a positive hybridoma. Alternatively, ELISA can be done. Here, adsorbtion of antigen to the the bottom of 96-well plates,incubation for growth of hybridomas occur. The desired antibody in the sample remain bound to antigen and is detected by an immunoconjugate. Immunoconjugate is consists of two components, one antibody is specific for an epitope that remains in the constant domain in the first antibody. It acts as anti-antibody.Second one is alkaline phosphatase enzyme. Immunoconjugate 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.
After screening , cloning can be done by three different tenchniques( cloning by technique of limiting dilutions,cloning using semi-solid agars and cloning and selection using the fluorescence-activated cell sorter).
It is essential against the loss of beneficial lines.Hybridoma should be freezed down as soon as possible to reduce chromosome loss.
9. Characterization of monoclonal antibodies:
Monoclonal antibodies should be characterized with respect to following parameter:
-specificity is determined for specific antigen.
-titer: By chosing for screening and concluding the highest dilution at which a positive outcome can be achieved.
-affinity of binding can be measured.
-storage and stabilty 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 labelling of monoclonal antibodies:
Monoclonal antibody can be manufactured by two processes ( tissue culture or by maturing the hybridoma as a tumor in mice). Then, ammonium sulfate can precipitate antibody and ion-exchanging chromatography and antigen affinity chromatography can be used for higher extend of antibody purity. At last, Labelling of monoclonal antibodies can be achieved by radioistopic labelling or by fluorescence labelling.