2.  Antibodies are normally obtained by injecting an animal with the antigen for which an immune response is sought.  A variety of antibodies appear each specific to a different part of the injected antigenic epitope molecule.  Blood serum taken from such animals contains the antibody mixture, called polyclonal antibodies.

3.  One of the most important contributions to immunology, that has a strong impact on biotechnology, was made by Kohler and Milstein in 1976.  This work has made possible to create pure and uniform antibodies against specific antigens.  This technology of producing pure (monoclonal) antibodies is called hybridoma technology

5. : Antibodies produced from a single clone of B cells. Produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture. 1. all have identical antigen-binding sites 2. they all bind to the same epitope with the same affinity 3.they are all of the same antibody class (isotype)

6. Normal Cells Are Fused With A Cancerous Cell Line whose own antibody synthesis has been stopped  E.x myeloma, lymphoma  Fusion Is Accomplished With PEG (polyethylene glycol)  The New Hybrid Cell Exhibits Properties Of Both Cell Types  Unlimited growth  Secretes monoclonal antibody  Or Secretes cytokines

7.  Hybridomas are hybrid cells capable of continuous production of monoclonal ab  Cultured myeloma (cancer) cells are fused with spleen cells (lymphocytes) obtained from an immunized animal. The spleen cells are the source of antibodies  Hybrid of B-Lymphocytes and myelomas

8. - B lymphocytes can mutate into tumor cells that result in a type of cancer termed myeloma. - Myeloma cells become “immortal” and will grow indefinitely in culture. - Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinitely in culture. Hybridomas Technique

10. Polyclonal antibodies Monoclonal Antibodies Produced by: Many B cell clonesA single B cell clone Bind to: Multiple epitopes of allA single epitope of a single antigens used in the antigen immunization Antibody class: A mixture of differentAll of a single Ab class Ab classes (isotypes) Ag-binding sites: A mixture of Abs withAll Abs have the same different antigen antigen binding site -binding sites Potential for cross-reactivity: HighLow

11.  Hybridoma: the B cell X myeloma cell  To be produce by using polyethylene glycol (PEG) to fuse cells  The myeloma cells : immortal growth properties  The B cells: to contribute the genetic information for synthesis of specific antibody  Selected by using HAT medium (hypoxanthine, aminoprotein, and thymidine)  Myeloma cells are unable to grow  B cells are able to survive

12. PRODUCTION OF MONOCLONAL ANTIBODY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum Spleen removed (source of cells)

13. PRODUCTION OF MONOCLONAL ANTIBODY Step 2: - Screening Of Mice For Antibody Production HYBRIDOMA TECHNOLOGY After several weeks of immunization Serum Antibody Titer Determined Technique: - ELISA Titer too low BOOST (Pure antigen) Titre High BOOST (Pure antigen) 2 weeks

14. Myeloma cells have been genetically engineered ( HGPRT - ) such that they can not use H ypoxanthine, A minopterin, and T hymidine (HAT medium) as a source for nucleic acid biosynthesis and will die in culture Only B cells that have fused with the engineered myeloma cells will survive in culture when grown in HAT medium Hybridoma Selection The “HAT Trick”

15. PRODUCTION OF MONOCLONAL ANTIBODY Step 3: - Preparation of Myeloma Cells HYBRIDOMA TECHNOLOGY Immortal Tumor Of Lymphocytes Myeloma Cells High Viability & Rapid Growth HGPRT - Myeloma Cells

16. PRODUCTION OF MONOCLONAL ANTIBODY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells HYBRIDOMA TECHNOLOGY FUSION PEG MYELOMA CELLS SPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium 1.Plating of Cells in HAT selective Medium 2.Scanning of Viable Hybridomas

17.  HGPRT : (hypoxanthine-guanine phosphoribosyl transferase).  HAT : (hypoxanthine-aminopterin-thymine) medium.  ELISA : (enzyme-linked immunosorbent assay) is a highly sensitive method forthe detection of antibodies or soluble antigens in sera and other body fluids.

18.  Step 1: Immunization of mice  Mice are immunized with an antigen (attached to adjuvant). The antigen can be whole cells, membrane fragment, or complex molecules.  Mice serum’s are screened using various techniques such as ELISA.  When sufficient titer is reached the mice are euthanize and spleen is removed as a source of cells for cell fusion.  Step 2: Preparation of Myeloma Cells  Myeloma cells are immortalized cells that are capable of dividing indefinitely.

19.  Step 3: Fusion of myeloma cells with Spleen cells  Spleen cells harvested from mice are fused with myeloma cells.  Fusion is done through co-centrifusing in polyethylene glycol.  Cells are plated in selection medium  Only fused cells with grow on HAT!

20.  Step 4: Cloning of Hybridoma cells.  A mouse is inoculated with the cell and thereby becomes a factory for producing the mAB.  Ascites are collected from the mouse.  Step 5: Ab are screened and Purified  Ab are screened using specific Ag binding.

22.  A pregnant woman has the hormone human chorionic gonadotrophin (HCG) in her urine.  Monoclonal antibodies to HCG (anti-HCG) have been produced. These have been attached to enzymes which can later interact with a dye molecule and produce a colour change.

23.  Pregnancy test contains three regions – reaction region, test region and control region  Reaction region : contains monoclonal anti-HCG antibodies linked to enzyme  Test region : contains polyclonal anti-HCG antibodies, which will bind to HCG molecules bound to monoclonal anti-HCG antibodies. Also contains dye molecules which will be activated if monoclonal antibodies bind to polyclonal anti-HCG antibodies  Control region : contains anti-mouse antibodies and dye molecules which will be activated if monoclonal antibodies bind here

24.  When pregnant woman’s urine travels up the pregnancy test, HCG will bind to monoclonal antibodies in reaction region  Movement of the urine will move the monoclonal antibodies up to the test region  Monoclonal antibodies with bound HCG will bind to antibodies in test region and activate dye molecules, producing a color change  Any unbound monoclonal antibodies will continue to travel to control region and will bind to anti-mouse antibodies, activating dye molecules and producing a color change

26.  Cancer cells carry specific tumour-associated antigens (TAA) on their plasma membrane.  Monoclonal anti-TAA antibodies have been produced.  Drugs which kill tumour cells or inhibit key proteins in tumour cells are attached to monoclonal anti-TAA antibodies.  Cancer cells are specifically targeted, avoiding damage to healthy host cells.

28.  Deep subcutaneous or intramuscular route (most vaccines)  Oral route (sabine vaccine, oral BCG vaccine)  Intradermal route (BCG vaccine)  Scarification (small pox vaccine)  Intranasal route (live attenuated influenza vaccine)

29.  Primary vaccination  One dose vaccines (BCG, variola, measles, mumps, rubella, yellow fever)  Multiple dose vaccines (polio, DPT, hepatitis B)  Booster vaccination To maintain immunity level after it declines after some time has elapsed (DT, MMR).

30. Hepatitis B is a germ (virus) that gets into your body and attacks your liver. Hepatitis B Virus What is hepatitis B? Your liver helps your body digest the food you eat and store energy. It also helps your body get rid of poisons. Liver

31. There are cases where hepatitis B can cause liver damage (cirrhosis [ sir-O-sis ]) that does not go away. Hepatitis B can also cause liver cancer, which may lead to death. Good medical care can make your risk less for these. It is passed by contact with the blood or other body fluids of someone who has the virus. Will I die from hepatitis B?

32.  Production of the HBV virion surface proteins is controlled by one virus gene, env, which has three start codons.  Thus, the HBV virion envelope contains three proteins – small (S; hepatitis B surface antigen [HBsAg], 226 residues), medium (M; preS2–HBsAg, 281 residues) and large (L; preS1–preS2–HBsAg, 389 residues)  The HBV S antigen is the major component of the virion surface

34. PLASMA DERIVED VACCINE RECOMBINANT DNA YEAST DERIVED

36.  Chinese hamster ovary ( CHO ) cells are a cell line derived from the ovary of the Chinese hamster.cell lineovaryChinese hamster  They are often used in biological and medical research and commercially in the production of therapeutic proteinsbiologicalmedical research

37. Pharmaceutical Production in Plants Genetically modified plants have been used as “bioreactors” to produce therapeutic proteins for more than a decade. A recent contribution by transgenic plants is the generation of edible vaccines. Edible vaccines are vaccines produced in plants that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then confer immunity against diseases. The first human clinical trial took place in 1997. Vaccine against the toxin from the bacteria E.coli was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no adverse effects.

38.  Prepared by introducing selected desired genes into plants  Allow the genetically modified plants o manufacture the encoded protein  This process is called transformation and the plant is called transgenic plant  As the proteins can produce immunogenic response when deliver orally –called edible vaccine.

39.  1984-transformation of tobacco, first plant to be transformed  This antigen has been entered in transgenic plant either through 1. Agrobacterium mediated transformation or 2. Particle gun bombardment (Biolistics) 3. encoding the hepatitis B surface antigen (HBsAg)

42.  Particle bombardment, or biolistics and microprojectile bombardment is a direct method of gene transfer  Involve the use of heavy metal and inert particles such as gold or tungsten coated with DNA to be transferred into plant.  The heavy metal and inert particles that coated with genetic materials are known as microcarriers. These microcarriers will be transferred with high speed into plants

46.  The "cold chain" is a system of storage and transport of vaccines at low temperature from the manufacturer to the actual vaccination site.  The cold chain system is necessary because vaccine failure may occur due to failure to store and transport under strict temperature controls.

47. Cold chain equipment consists of the following: (a) Walk in cold rooms: They are located at regional level, meant to store vaccines up to 3 months and serve districts. (b) Deep freezers (300 ltr) and Ice lined Refrigerators: supplied to all districts locations to store vaccines. Deep freezers are used for making ice packs and to store OPV and measles vaccines. (c) Small deep freezers and ILR (140 ltr)

48.  (d) Cold boxes: Cold boxes are supplied to all peripheral centers. These are used mainly for transportation of the vaccines.  (e) Vaccine carriers: Vaccine carriers are used to carry small quantities of vaccines (16-20 vials) for the out of reach sessions  (f) Ice packs: The ice packs contain water and no salt should be added to it.

49.  Among the vaccines, polio is the most sensitive to heat, requiring storage at minus 20 degree C.  Vaccines which must be stored in the freezer compartment are : polio and measles.  Vaccines which must be stored in the COLD PART but never allowed to freeze are : typhoid, DPT, tetanus toxoid, DT, BCG and diluents