Antibodies or immunoglobulin’s are protein molecules made through a specialized cluster of cells referred to as B-lymphocytes (plasma cells) in mammals. Antibodies are part of the defense system to guard the body against the invasive foreign substances specifically antigens. Every substance has unique substance determinants (epitopes) located thereon. The antibodies have complementary determinative regions (CDRs) that are in the main accountable for the protein specificity. In response to an antigen (with many totally different epitopes), B-lymphocytes change and manufacture various antibodies. These forms of antibodies which might react with the identical antigen are known as polyclonal antibodies.
The polyclonal antibody production is variable and relies on factor like epitopes, response to immunity etc. Due to loss of specificity and heterogenic nature, there are many obstacles on the application of polyclonal antibodies for therapeutic and diagnostic functions.
Monoclonal antibody (MAb) is a single kind of antibody that is directed against a particular antigenic determinant (epitope). It had been a dream of scientists to provide MAbs for various antigens. Within the early years, animals had been immunized against a particular antigen, B-lymphocytes have been isolated and cultured in vitro for generating MAbs. This approach turned no longer successful since culturing normal B-lymphocytes is troublesome, and also the synthesis of MAb was short-lived and restrained.
It had been in 1975, George Kohler and Cesar Milstein (Nobel Prize, 1984) achieved massive scale manufacturing of MAbs. They could efficaciously fused antibody—producing B-lymphocytes with myeloma cells in vitro and produce a hybridoma.
The result is that, by artificial means immortalized B-lymphocytes will multiply indefinitely in vitro and manufacture MAbs. The hybridoma cells possess the expansion and multiplying properties of myeloma cells however secrete antibody of B-lymphocytes. The production of monoclonal antibodies by the hybrid cells is remarked as hybridoma technology. Hybridomas are cells that are designed to provide a desired antibody in massive amount, to provide monoclonal antibodies.1
The production of MAbs involves the following steps :
2. Cell fusion
3. Selection of hybridomas
4. Screening the products
5. Cloning and propagation
6. Characterization and storage.1
The first step in hybridoma technology is to immunize an animal (usually a mouse), with applicable antigen. The injections at multiple sites are continual many times.
This enables exaggerated stimulation of B-lymphocytes that are responding to the antigen. Three days before killing of the animal, a final dose of antigen is intravenously administered. The immune-stimulated cells for synthesis of antibodies have fully grown maximally by this approach. The concentration of the specified antibodies is assayed within the bodily fluid of the animal at frequent intervals throughout the course of immunization.
When the bodily fluid concentration of the antibodies is perfect, the animal is sacrificed. The spleen is aseptically removed and disrupted by mechanical or enzymatic method to release the cells. The lymphocytes of the spleen are separated from the remainder of the cells by density gradient centrifugation.1
The totally washed lymphocytes are mixed with HGPRT(Hypoxanthine-guanine phosphoribosyltransferase) enzyme defective myeloma cells.The mixture of cells is exposed to polyethylene glycol (PEG) for a brief amount (a few minutes), since it’s poisonous. PEG is removed by washing and therefore the cells are kept in a fresh medium. These cells are composed of a mix of hybridomas (fused cells), free myeloma cells and free lymphocytes.1
Selection of Hybridomas
When the cells are cultured in a HAT (hypoxanthine-aminopterin-thymidine medium) medium, solely the hybridoma cells grow, whereas the remainder will slowly disappear. This happens in 7-10 days of culture. Choice of one antibody manufacturing hybrid cells is extremely necessary. This can be possible if the hybridomas are isolated and grown separately. The suspension of hybridoma cells is thus diluted that the individual aliquots contain on an average one cell each. These cells, once grown in a very regular culture medium, produce the required antibody.1
Screening the Products
The hybridomas should be screened for the secretion of the antibody of desired specificity. The culture medium from every hybridoma culture is periodically tested for the required antibody specificity. The foremost common screening assay is that the ELISA (enzyme-linked-immunosorbent serologic assay).The antigen is absorbed to the underside of 96-well plates. In the assay, the antibody binds to the specific antigen (usually coated to plastic plates) and therefore the unbound antibody and different parts of the medium may be washed off. Thus, the hybridoma cells manufacturing the required antibody may be known by screening. The antibody secreted by the hybrid cells is termed as monoclonal antibody.1
Cloning and Propagation
The single hybrid cells manufacturing the required antibody are isolated and cloned. Two techniques are normally utilized for cloning hybrid cells-limiting dilution method and soft agar method.
Limiting dilution method:
In this procedure, the suspension of hybridoma cells is serially diluted and therefore the aliquots of every dilution are placed into small culture wells. The dilutions are thus created that every aliquot in a well contains only one hybrid cell. This ensures that the antibody made is monoclonal.
Soft agar method:
In this technique, the hybridoma cells are cultured in soft agar. It is possible to grow several cells in semi solid medium at the same time to create colonies. These colonies will be monoclonal type. 1
Characterization and storage
The antibody needs to be subjected to biochemical and biophysical characterization for the required specificity. It is additionally necessary to elucidate the MAb for the immune globulin class or sub-class, the epitope for that it is specific and therefore the range of binding sites it possesses.
The stability of the cell lines and therefore the MAbs are necessary. The cells (and MAbs) should be characterized for their ability to withstand freeezing and thawing. The required cell lines are frozen in liquid nitrogen at many stages of cloning and culture.(Biology Discussion, 2018)
Conclusion: Hybridoma technology is a suitable method for preparing monoclonal antibody in vitro condition. Monoclonal antibody have opened remarkable new approaches for preventing, diagnosing, and treating disease.2
1.Biology Discussion. (2018). Monoclonal Antibodies: Production, Advantages and Limitations. online Available at: http://www.biologydiscussion.com/antibodies/monoclonal-antibodies-production-advantages-and-limitations/10068 Accessed 4 Nov. 2018.
2.Global-research-online.net. (2018). online Available at: http://www.global-research-online.net/volume1issue2/Article%20017.pdf Accessed 4 Nov. 2018.