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					MONOCLONAL ANTIBODIES


  Five types : IgA, IgE, IgD, IgM, IgG
  Differ in aas composition, size, charge
  and CHO content
  Each Ab two heavy and 2 light chains
  linked by disulfide bonds
MONOCLONAL ANTIBODIES :


    Each Ab has constant (C-terminal) and
    variable region (N-terminal)

    One antigen can have more than one
    epitope (small antigenic part of a molecule
    which elicits an immune response)

    When antigens are injected, polyclonal
    Abs in the serum
HYBRIDOMA TECHNOLOGY

 Myeloma and B lymphocytes fused together

 Multiple myeloma: cancer of the B lymphocytes

  a) Overproduction of one type of antibody:
     limited MAB types

  b) Overgrowth of B cells w/ no Ab production
HYBRIDOMA TECHNOLOGY :


Two properties of myeloma cells to be used for
hybridoma technology:

  1. HPRT (hypoxanthine phosphoribosyl
     transferase) negative, an enzyme needed for
     DNA synthesis

  2. Do not produce antibodies
HYBRIDOMA TECHNOLOGY :

The technology:

  1. Obtain HPRT negative myleomas by selection on
     a medium of 8-azaguanine

  2. Obtain lymphocytes by immunizing an animal,
     obtain its spleen, homogenize and purify by
     density gradient centrifugation

  3. Fuse B cells w/ myeloma cells using PEG
HYBRIDOMA TECHNOLOGY :

The technology:

  4. Select the hybrids on a HAT medium. Only
     hybrids will survive because they obtain the
     HPRT enzyme from the B lymphocytes

  5. Remove PEG after fusion (cytotoxic)

  6. Clone and select single Ab-producing hybridoma
     (screening)
LARGE SCALE PRODUCTION OF MAB


  1. Grow the hybridomas in microspheres
     (protect from contamination)

  2. To prepare the microsphere, place the cells
     in gelling liquid (sodium alginate), then
     add coating solution (poly-L-lysine)

  3. Culture the cells in large vessels, v. high
     yields of MAB in vessel
PROBLEMS w/ MABs


 Retroviral infection (mouse) is a common

 Problem (Quality control)- How detect?

 Safer to prepare MAB by ------------------
ADVANTAGES of MAB over PAB:


 1. No batch to batch variation (mouse
    reacts differently to each injection)

 2. Faster immune reaction (Ab may be
    dilute in the polyclonal preparation),
    less reaction time

 3. More specific and sensitive
APPLICATIONS OF MAB:

  1. Protein purification by Immunoaffinity
     Chromatography

  CAUTION:
     Use appropriate Buffer for optimal binding &
      Elution

     Two proteins with similar antigenic
      determinants may bind

     Inactive protein, if degraded but Ag are intact
APPLICATIONS OF MAB:



2. Organ transplants: used to suppress the immune

     system of the recipient after organ transplant

     e.g. use anti-T cell MAB that destroys T-cells

     usually shortly before the transplant (risks)
APPLICATIONS OF MAB:

  3. Cancer treatment: MAB attached to toxin (As),
     or radionuclide (I131)-Half life=8 days

    Criteria: MAB is against Ag specific for tumor
              cells, or abundant on tumor cells

             Ag should not be secreted into
             Circulation

              Radionuclide should have short half
              life, low level of penetration and high
              energy
APPLICATIONS OF MAB:

   4. Tumor Necrosis treatment: necrotic areas
      of normal cells (direct MAB to Ag specific to
      these areas): Advantages


   5. Diagnosis of certain diseases or in kits
CHIMERIC MAB:

  Can produce hybrid MAB (bind constant region

  of one organism with the variable region of

  another organism) / More stable
    PLANT BIOTECHNOLOGY

 I. BASIC BIOLOGY OF PLANTS
 II. PLANT CELL/TISSUE / ORGAN CULTURE
 III. REGENERATION OF WHOLE PLANTS
 IV. SOMATIC EMBRYOGENESIS
 V. CLONAL PROPAGATION
 VI. SOMACLONAL VARIATION
 VII.APPLICATIONS OF PLANT CELL CULTURE
VIII.GENETIC ENGINEERING OF PLANTS
   •   PROTOPLAST FUSION/ VIABILITY
   •   MICROPROJECTILE
   •   PLANT VECTORS
PLANT BIOTECHNOLOGY

 PLANT BIOLOGY:

   Germination: seed to seedling, imbibition of
    Water

   Indeterminate growth: roots and stems, grow
                          Continuously, apical
                          Meristems

   Determinate growth: flowers and leaves
   Xylem v.s Phloem
   Primary v.s secondary growth
PLANT BIOTECHNOLOGY

PLANT TISSUE CULTURE:

   Start w/ explant (plant piece, undifferentiated)
    e.g. buds, seeds, nodes

   Trim the explant, cut a small piece of it

   Surface sterilize in sodium hypochlorite

   Wash several times in sterile distilled water
   (autoclaved) (remove hypochlorite: toxic)
PLANT BIOTECHNOLOGY

PLANT TISSUE CULTURE:


   Final trim (remove dead tissue on surface)
    and culture

   Incubate in light and subculture (37 C)

   Obtain a callus: mass of undifferentiated
    cells from which a whole plant can be
    regenerated (totipotency)
PLANT BIOTECHNOLOGY

PLANT TISSUE CULTURE:

     Culturing media is MS (Murashige and Skoog)
      media with defined concentrations of:

        a) growth hormones: auxins, gibberellins,
           cytokinins

        a)   inorganic salts: Na, Ca, Mg, K
        b)   trace metals: Zn, Cu
        c)   vitamins & organic nitrogen
        d)   sucrose
PLANT BIOTECHNOLOGY

PLANT CELL CULTURE:

  - Start w/ a callus, get individual cells by
    Agitating callus

  - Culture cells in conditioned media

  - Prepare protoplasts from plant cells: treat w/
    cellulase to break cell wall, then pectinase to
    break aggregates. Can also get protoplasts
    from callus or homogenized leaf (O.N. at 20 C)
PLANT BIOTECHNOLOGY

PLANT CELL CULTURE:

  - Wash protoplasts 3x in DDW to wash off enzymes

  - Sucrose density gradient to separate: protoplasts

  - whole cells and cell debris (protoplasts: top)

  - Suspend in isotonic sucrose to get viable
    protoplasts, Re-centrifuge and obtain top layer,
    damaged protoplasts settle to bottom
PLANT BIOTECHNOLOGY

PLANT ORGAN CULTURE:

   Technique known as micropropagation

   Culturing an organ (root or stem): start w/ auxiliary bud
    found in axil of leaf

   Place bud on growth medium for 3-5 weeks

   Dissect it for the 1st time to accelerate growth

   Allow growth for 2-3 weeks, then dissect further to get
    daughter generations
PLANT BIOTECHNOLOGY
REGENERATING WHOLE PLANTS:

 - Can use protoplasts, plant cells or calluses
   as starting material

 - On a solid medium w/ proper culture solution,
   protoplasts develop into whole cells w/in 10 days


 - Grow plant cells on solid medium of low auxins
   and cytokinins to get calluses
PLANT BIOTECHNOLOGY

REGENERATING WHOLE PLANTS:


  - Place calluses in media containing high levels of
    auxins, low cytokinins (roots develop)

  - Then place in media containing high levels of
    cytokinins, low auxins (shoots form)

  - Grow the plantlets to whole plants
  **** This process of developing roots on shoots and
       vice versa is known as ORGANOGENESIS
PLANT BIOTECHNOLOGY

SOMATIC EMBRYOGENESIS:


    The technique of obtaining embryos from

    somatic rather than from germ cells. The

    obtained embryoids can generate whole plant
PLANT BIOTECHNOLOGY

SOMATIC EMBRYOGENESIS:

 CONDITIONS:

    Culture plant cells in 2,4-D as the only auxin

    source and then transfer them to a 2,4-D free

    medium  Embryoids are formed.
PLANT BIOTECHNOLOGY

CLONAL PROPAGATION:

   The propagation of plants on a large scale whereby
    a clone of plants w/ favorable properties could be
    generated:

       1.Generate a callus from an explant

       2.Obtain cells from the callus /suspension

       3.Generate embryoids from the cells
PLANT BIOTECHNOLOGY


SOMACLONAL VARIATION:


    Genetic variation that occurs in plant somatic cells
    in culture. It is specific for plants.


    Results in new genotypes: some good/some bad
PLANT BIOTECHNOLOGY

APPLICATION OF PLANT BIOTECH:

   Herbicide tolerant plants

   Improve plant quality/ N2 fixation

   Insect/viral/fungal resistant plants (inoculate
                                          w/ pest)
   Ornamental plants

   Basic research: Restoration

   Stress resistant/heavy metal tolerance
PLANT BIOTECHNOLOGY

APPLICATION OF PLANT BIOTECH:


   Cell culture application: shikonin from the roots of
                            Lithospermum erythrorhizon

   Plants as Bioreactors: biodegradable plastic
                           (polyhydroxybutyrate), MAB

   Germplasm banks: seed banks, field plantation

				
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