Monoclonal antibodies.pptxsddjjhghgghthlh

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1. Monoclonal antibodies

2. Group: 5 (B) Saira Munir DPH-FA17-079. Sana Iftikhar DPH-FA17-080
Ayesha shabir Dph-fa17-075. Sufia Noor Dph-fa17-113
Rafia gulnaz Dph-fa17-069. Samra Bano Dph-fa17-135
Aiman Farooq DPH-FA17-146 Saman Ahsaan DPH-FA17-088
Zain-ul-hassan DPH-FA17-081 Aimen Fatima DPH-FA17-105

3. Monoclonal antibodies Antibody: An antibody, also known as an immunoglobulin, is used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Monoclonal antibodies: An antibody produced by a single clone of cells or cell line and consisting of identical antibody molecules.

4. Polyclonal antibodies: Polyclonal antibodies Polyclonal antibodies are antibodies that are secreted by different B cell lineages within the body. They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope.

5. Difference b/w monoclonal antibodies and polyclonal antibodies:

6. Characters of monoclonal antibodies: Monoclonal antibodies are single type of antibody that are identical and are directed against a specific apitopes and are produced by a B-cell clone of a single parent or a single hybridoma cell line.
Hybridoma cell line is formed by fusion of one B-cell lymphocytes with myeloma cells.
Some myeloma cells synthesize single mAB antibodies naturally.

7. Mechanism of monoclonal antibodies

8. Production of monoclonal antibodies: Monoclonal antibodies are produced by cell lines are clones obtained from the immunized animals with the substances to be studied. Cell lines are produced by fusing B cell from the immunized animals with myeloma cells. There are following steps involved in preparation of monoclonal antibodies: Step 1 : Immunization of mice and isolation of splenocytes: Mice are immunized with an antigen and later their blood is screened for antibody production. The antibody producing splenocytes are then isolated from in vitro hybridoma production.

9. Production of monoclonal antibodies: Step 2: Preparation of myeloma cells Myeloma cells immortalized cells that once fused with spleen cells , can result in hybridoma capable of unlimited growth. Myeloma cells are prepared for fusion. Step 3: Fusion Myeloma cells and isolated splenocytes are fused together to form hybridomas in the presence of polyethylene glycol which cause cell membrane to fuse. Step 4:Clone screening and picking Clones are screened and selected on the basis of antigen specificity and immunoglobulin class.

10. Production of monoclonal antibodies: Step 5: Functional characterization: Confirm , validate and characterize ( e.g ELISA ) each potentially high producing clonony. Step 6: Scale up and wean: Scale up clones producing desire antibodies and wean off selection agent Step 7: Expansion Expand clones producing desire antibodies ( e.g bioreactors)

11. Production of monoclonal antibodies:

12. Flowchart of production of monoclonal antibodies:

13. Preparation of monoclonal antibody by hybridomas technology: Immunization of specific animal which Which generate hybridoma cell with spleen cell. Isolation of myeloma cell Fusion between myeloma cell and spleen cell Selection of HAT medium Isolation of hybridoma cell Screening of hybridoma cell

14. Cont…

15. Evaluation of monoclonal antibodies Characterisation of monoclonal antibodies physio chemical characterization: Determination of class ,subclass and primary structure of monoclonal antibodies Immunological properties : To determine affinity Biological activity Invivo and Invitro assays performed Mechanism of action of drug determined Purity impurity and contaminants Process related impurities host cell DNA ,cell culture residuesContaminants microbial species, endotoxins

16. Evaluation Specifications : Identity Identify monoclonal antibodies by Biological assay and immnuochemical assay Potency Potency assay that measure binding assay of monoclonal antibodies. Quantity Quantity of the drug substance usually based on protein content is determined chromatographically by using reference standards. General test Appearance ,PH,solubility, osmolality

17. Purification Of monoclonal antibodies Contaminants: Media components: Hormones, growth factor, transferrins Viral, bacteria, endotoxins etc Methods of purification: Filteration for large particles Ultrafilteration especially for Low concentration sample Chromatrography Ion exchange chromatography Size exclusion chromatography

18. Purification of monoclonal antibodies

19. Applications of monoclonal antibodies Monoclonal antibodies are used for:
Diagnostic testing
Pregnancy testing
Radio-immunodetection (RID) of cancer
Radio-immunotherapy (RIT) of cancer
Treatment of cancer through drugs
Viral disease treatment
Identifying pathogens

20. Cont.. Tracing specific cells and their functions
Organ rejection
Rhesus disease immunization
Detection of cell surface markers
Sensitive detection assays

21. Side effects of monclonal antibodies Monoclonal antibodies are given intravenously (injected into a vein). The antibodies themselves are proteins, so giving them can sometimes cause something like an allergic reaction.
This is more common while the drug is first being given.
Possible side effects can include:
Fever
Chills
Weakness
Headache

22. Side effects of monclonal antibodies Nausea
Vomiting
Diarrhea
Low blood pressure
Rashes
Compared with chemotherapy drugs, naked mAbs tend to have fewer serious side effects. But they can still cause problems in some people. Some mAbs can have side effects that are related to the antigens they target.

23. Example related to side effects Bevacizumab (Avastin) is an mAb that targets a protein called VEGF that affects tumor blood vessel growth. It can cause side effects such as high blood pressure, bleeding, poor wound healing, blood clots, and kidney damage. Cetuximab (Erbitux) is an antibody that targets a cell protein called EGFR, which is found on normal skin cells (as well as some types of cancer cells). This drug can cause serious rashes in some people.

24. Advantages And disadvantages of monoclonal antibodies