Course title: Pharmaceutical Biotechnology
Course code: PHRM 407
Department of pharmacy,EWU Submitted by:
Date of submission:05.11.18
Monoclonal Antibody and Hybridoma technology
Monoclonal antibody is different from polyclonal antibody because of its specificity to the antigen. Monoclonal antibody derives from identical single B-cell clones. They are very much specific because they can bind with a specific epitopes or binding sites of an enzyme.
Monoclonal antibodies production method was first discovered by Gorge’s jf kholer and Ceaser Milstein in 1975. The technique is known as Hybridoma Technology. Leonard Harzenberg first termed it as hybridoma. Hybridoma cells are the cells that can be found by fusion of B cell with a myeloma cell. Myeloma cells are tumor cell which can multiply indefinitely.
Production of monoclonal antibody by hyrbridoma technology:
There are several steps that are involving in these process. The process is described below:
In repetitive way microgram to milligram immunogen are mixed with suitable adjuvant and injected to mouse. The animal is bled at times to find the amount of desired antibody is produced or not. If the produced antibody crosses the desired level the animal is sacrificesd and its spleen is collected because the speeln is rich in B cell.
On the other hand an another mouce with tumor or cancer was sacrificed and collect the myeloma cell of that mouse. (1)
For the production of hybridoma cell there will need two types of cells.they are
B Cells that are collected from the spleen of the muse. The spleen is supposed to be chemically and enzymetically disrupted and the spleenocytes are collected as they are rich in B cells that can produce antibody. But the antibody has short life span.
Myeloma cell: they are cancerous B cells. Their two genes are mutated. they are the HGPRT gene and immunoglobulin gene. As a result they can’t synthesis nucleotide in Denovo pathways and can multiply in salvage pathways and can’t produce antibody.
The spleenocytes is then mixed with myeloma cells in 50% PEG medium. Thus hybridoma cells are produced.(2)(3)
After cell fusion there will be several types of cells. Example: fused B cells, fused myeloma cells, infused B cells, infused myeloma cells, hybridoma cells. To separate the hybridomas from the cells HAT (hypoxanthine aminopterin thymidine) medium is used. HAT medium consist of a drug aminopterin, this drug will block the De novo pathways for multiplying and allows only the multiplication vaia salvage pathway which is dependent to HGPRT enzyme, this enzyme is inherited in the hybridoma from the B cells. that means only the hybridoma cells can survive In this condition and other cells will die eventually.(2)
This step is done for the selection and iolation of desired antbody of desired specificity. The screening technique is known as ELISHA(Enzyme linked immonosorbent assay) technique. Another techniques are also used, limitng dilution method.
In a solid support (EX: Polystyrene micrititer plate) or non specific surface ,the samle with unknown amount of antigen are immobilized.
Addition of the detection antibody on the Elisha mediam.
The detection antibody will bind with the antigen and form complex. The desired antibody can be covalently linked with the enzyme.
By change in color or any other hint the presence of desired antibody can be detected and then it is isolated and cloned.(3)
The specific antibody will then cloned via limiting dilution method and soft agar method in order to produce more antibodies. In soft agar medium over the semi solid medium which contains a low amount of agar,all the malignant cells multiply rapidly. But if the sample can spread in such a way that each single cell will dispersed on the agar medium then it will be easier.
In this method the culture is distributed in well culture plate at a very low density so that each well plate can eventually contains only one hybridoma.
Characterization involves in three process. It is useful for establishing the monoclonality of the antibody and it requires a series of biochemical and biophysical characterization. The very first step in characterization is screening which has been already done by ELISA method. In this method the desired antibody is selected from others antibody.
The second step is tittering. In this process the concentration of the antibody is measured by immune globuline specific method or general protein assay. In this method the functional potency and suitability of an antibody also can be determined. The third step is iso typing. This is done for determining the classes ( igV/ igM) or subclass (igG1/ igG2) of the desired antibody. It requires further purification for storage.(4)
The antibody must be stored in liquid nitrogen and in frozen state to avoid the disruption of the antibody. (4)
Figure: monoclonal antibody production using hybridoma technique
( HYPERLINK “https://biocyclopedia.com/index/genetics/images/figure/f43.1.jpg” https://biocyclopedia.com/index/genetics/images/figure/f43.1.jpg)
Application of monoclonal antibody:
monoclonal antibody can be used in diagnostic perpous. Such as: it is used in the diagnosis of myocardial infraction, deep vein thrombosis, atherosclerosis.
It can diagnosis different kind of cancer by targeting different tumor marker.
Monoclonal antibody are also used to deliver the potent drugs toxins.
To treat the rejection of organ transplants by T cells, monoclonal antibody is used.
In autoimmune diseases it is also used (4)
1.En.wikipedia.org.(2018). Hybridoma technology. online Available at: https://en.wikipedia.org/wiki/Hybridoma_technology Accessed 5 Nov. 2018.
2. Tortora, G. (2010). Instructor resource DVD to accompany Microbiology, an introduction, 10th ed., by Tortora, Funke, Case. Harlow: Pearson Education.
3. En.wikipedia.org. (2018). ELISA. online Available at: https://en.wikipedia.org/wiki/ELISA Accessed 5 Nov. 2018.
4. Kulkarni, G. (2002). Biotechnology and its applications in pharmacy. New Delhi: Jaypee Bros Medical Publishers.