TD: Application of biotechnology Flashcards
What cell produces ABs?
What are monoclonal ABs?
Plasma B-lymphocytes produce ABs - these only detect a small part of the molecule called the epitope.
A monoclonal antibody is the product of a single clone of plasma cells and will therefore react with a single specific epitope on an antigen.
In 1975 Kohler and Milsten developed a method for the production of …
In 1975 Kohler and Milsten developed a method for the production of unlimited amount of ABs of a pre-determined specificty from a single clone of cells (basically all cells produce that 1 monoclonal AB)
What are the properties of these cells
What is good about their production?
- The cell lines are immortal and can be grown in any lab to purify the AB.
- These AB are very much like a chemical reagent. They behave in a predictable way and are reproducibe from one lab and one time to the next
Although these AB are specific for only one epitope on one antigen, what problem can occur?
Cross-linking can occur i.e recognises a very similar sequence of AA therefore validation must be done
Describe the use of AB in diagnositic tests - what is used and why?
Monoclonal AB used in diagnostic tests often requires a cocktail of more than one AB, to avoid loss of detection due to mutation affecting one epitope
Describe the production on monoclonal AB
The animal of choice for monoclonal antibody production is the mouse, due mainly to the large number of mouse myeloma (plasma cell tumour) cell lines available for hybridoma formation. The initial stages of producing an immune response are identical to those required for polyclonal antibody production an antigen mixed with an adjuvant such as Freund’s, is introduced by subcutaneous or intraperitoneal injection into the mouse, followed by a secondary boost several weeks later. The mouse will produce an immune response but instead of bleeding the mouse to harvest the circulating antibody content, it is sacrificed and the antibody source, the B cells (from the lymph nodes and/or the spleen) are removed for processing.
The memory B lymphocytes contain the information allowing each individual cell to produce antibody of single specificity against a particular epitope (one epitope). Antibody producing B cells will produce the specific antibody required but cannot be maintained in vitro long enough to produce usable quantities of antibody.
In contrast, a mouse myeloma cell line will grow indefinitely in vitro. Myeloma cell lines from a syngeneic (genetically identical or closely related) mouse should ideally be selected as they do not secrete endogenous immunoglobulins. Hybrid cells can be formed between a memory B lymphocyte and a syngeneic mouse myeloma cell which will give rise to a hybridoma.
This hybridoma will possess the immortality of the myeloma cell and grow and divide in culture, with the ability to produce and secrete antibody, of a predetermined single specificity, from the B lymphocyte.
One of the problems in the production of monoclonal antibodies was the development of a system which would separate these hybrid cells from single myeloma cell and single B lymphocytes. In 1975 Kohler and Milstein developed a system which overcame these problems and forms the basis of most current fusion techniques.
Sendai virus was originally used as the fusion agent to bring the myeloma cell and the B lymphocyte into close proximity allowing the two membranes to fuse forming a hybridoma. Polyethylene glycol is now the fusion agent of choice as it is much easier to handle. The separation of hybridomas from myeloma cells is performed by selecting myeloma cell lines for the fusion process that are deficient in the enzyme hypoxanthine phosphoribosyl transferase (HPRT).
HPRT deficient cells will die in culture medium which contains hypoxanthine, aminopterin and thymidine, referred to collectively as HAT. In HAT medium, aminopterin acts to block the main pathway of DNA synthesis and the salvage pathway which utilises exogenous hypoxanthine and thymidine depends on the presence of the enzyme HPRT. Since unfused single B lymphocytes which do contain HPRT will die in culture anyway and single myeloma cells, which do not contain HPRT, die in HAT medium, the only cells which survive are hybrid cells in which the myeloma provides immortality in culture while the B lymphocyte provides the enzyme HPRT to allow the cell to utilise the DNA salvage pathway. This provides a system of cell fusion together with a method for positively selecting the hybridomas that are produced.
What needs to be done to these mice AB and what is this process called?
Genetic engineering of AB is required as otherwise the human body would evoke an immune response to the mice AB and produce AB against these
Describe the process of genetic engineering
- Genetic engineering can be used to modify monoclonal antibody genes to give them attributes more appropriate for particular applications.
- Human hybridomas are difficult to produce, so one can begin with a mouse antibody, produce probes for the CDR and insert the DNA for the CDR into genes for a human Ab.
- This allows human antibodies of a particular specificity to be produced in quantity.
- They can be used for therapy in humans without inducing an immune response as mouse antibodies would.
Fragments of mice AB can also be used as opposed to whole ABs - why might this be better?
•Other engineered versions include Fv (fragment variable). This includes no constant regions. Its pharmacological properties (more rapid and widespread distribution and longer half-life) are better than whole antibodies or even Fab fragments.
Describe the production of AB-phage display libaries
- By taking gene segment of antigens or antibodies and fusing them to the protein coat of phages, these phages will now express the anti-body in a fusion protein
- Phage Display Libraries of antigens can be created to create anti-body phage display libraries
Describe the steps of phage supply
What does this produce?
What is this used for?
- Different sets of genes are inserted into the genomes of multiple phages
- Proteins and peptides are fused to the Capsid (surface) of the phage
- The combination of the phage and peptide is known as a Fusion Protein
Used for cloning foreign genes anoung other applications
Once these Phages are isolated and recovered they can be used to infect bacteria such as Escherichia coli which will create a particle similar to a monoclonal antibody
Summary of phage disaply
Summary: Phage display
Use bacteriophages that propagate in E. coli to express various ligands/antibodies/peptides
Target gene is cloned into phage genome upstream of the genes encoding phage coat protein
Bacteriophage-expressed ligands are “displayed” on the surface of the phage particle produced in and released from E. coli.
Can be used for selection against a given target.
Cons:
Small- to mid-size proteins only
Lacks eukaryotic post translational modification may not be functional
What can mAB be used for?
•Diagnostic Applications
Biosensors & Microarrays
•Therapeutic Applications
•Future Applications
Fight against Bioterrorism
•Clinical Applications
Imaging the target
Possible problems with immunotherpay?
- Can you get enough antibody to the site required?
- Long term treatment causing alterations in the immune response
- Cost
What are the stages of vaccine development for tranditional vaccines
- Several stages in vaccine development prior to licencing
- Initial stage: identify and isolate natural or synthetic antigens.
- Antigen products are then in a cell culture or animal models
- Further assay development to determine the purity of raw materials, stability of the vaccine and potency of the vaccine product
- New vaccine must then seek appropriate approval from governing bodies such as Medicines and Healthcare products Regulatory Agency (MHRA) in the UK, Food and drug administration (FDA) in the US and European Medicines Agency (EMA).
- Once approval has been granted the process moves to clinical development where the focus is on the safety of the new medicine and whether the vaccine has any effects on the body or the body on the medicine
