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Antibody Production (PowerPoint download)

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					Antibody Production

   •Monoclonal antibody
   •Phage displayed antibody
Poly- and Mono- Clonal
Antibodies
  • Polyclonal antibody
     – Antigens possess multiple epitopes
     – Serum antibodies are heterogeneous,
         • To increase immune protection in vivo
         • To reduces the efficacy of antiserum for various in vitro uses
     – To response facilitates the localization, phagocytosis, and
       complement-mediated lysis of antigen
     – To have clear advantages for the organism in vivo
  • Monoclonal antibody
     – Derived from a single clone, specific for a single epitope
     – For most research, diagnostic, and therapeutic purposes
1975, by Georges Köhler and Cesar Milstein
          - Be awarded a Nobel Prize in1984
Formation and Selection of
Hybrid Cells
   • 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, but can not live for extended
          periods
Two different pathways to synthesis nucleotide in mammalian cells




                 (Folic acid analog)
                                             Myeloma cells used in
                                             hybridoma technology are
                                             double mutants, they lack
                                             the HGPRTase and lose
                                             the ability to produce Ig
                    (Most common screening
                    techniques are ELISA and RIA)




Low concentration            High concentration
   (1-20 ug/ml)                 (1-10 mg/ml)
Human Monoclonal
Antibodies
  • Production of human monoclonal antibody
     – There are numbers of technical difficulties
         • The lack of human myeloma cells to exhibit immortal growth, be
           susceptible to HAT selection, to not secrete antibody, and support
           antibody production in the hybridoma made with them
         • Human B cell sometimes have immortality
         • That is the difficulty of readily obtaining antigen-activated B cells
  • To culture human B cells in vitro to produce human
    monoclonal antibody
     – Transplant human cells with immune response into SCID mice
       (lack a functional immune system)
Clinical Uses for
Monoclonal Antibodies
  • Very useful as diagnostic, imaging, and
    therapeutic reagents in clinical medicine
     – Monoclonal antibodies were used primarily as in vitro
       diagnostic reagents
     – Radiolabeled monoclonal antibodies can also be used in
       vivo detecting or locating
  • Immunotoxins
     – To compose of tumor-specific monoclonal antibodies
       coupled to lethal toxin
     – Valuable therapeutic reagent
Phage Display -
Introduction

  • The display of functional foreign peptides
    or small proteins on the surface of
    bacteriophage particles.
  • As an important tool in protein engineering
  • As a powerful way to screen and select for
    peptides on the basis of binding or
    molecular recognition.
Phage Display -
Advantages
 • More efficiently than through conventional hybridoma system.
 • Cheaper to produce recombinant antibodies using bacteria, rather than
   mammalian cell line.
 • Easier to maintain and grow bacterial cultures for recombinant antibody
   production.
 • Bypass immunization in antibody selection.
 • Bypass the use of animal cells for production of antibodies.
 • Producing the combinatorial library (ideally with 108 to 109 members) of
   functional antibodies to generate a larger repertoire of antibodies than
   those available through conventional hybridoma technology.
 • Easy isolation and expression of the cloned gene in a bacterial host.
 • Excellent potential to further improve binding properties of the selected
   antibody by protein engineering techniques.
Filamentous Phage
• long, thin, and flexible particles
  that contain a closed circular
  single-strained DNA molecule,
  such as fd, f1, and M13.
• The major coat protein is pVIII.
  The minor coat proteins pIII and
  pVI are located at one end of the
  phage; pVII and pIX are located
  at the other end of the phage.
• to infect Gram-negative bacteria
• to adsorb specifically to the tip of F pili on male
  cells.
• Be able to accommodate foreign DNA fragments.
• its nonlysogenic characteristic to permits the
  extrusion of recombinant phage into the culture
  supernatant.
Filamentous
Bacteriophage Vector
 • To construct filamentous bacteriophage vectors enabled
   the ‘‘biological’’ generation of the hundreds of millions of
   unique peptides.
 • Be the double-stranded replicative form from a culture of
   inserted host cell.
 • Be inserted an antibiotic resistance gene.
 • Be introduced a pair of specifically situated endonuclease
   restriction sites to allow cloning of DNA insert into a
   position to express a foreign fusion protein with capsid
   protein.
a For additional information of phagemid vectors, see (1).
b The restriction sites were created to allow cloning of the foreign DNA encoding the fusion protein
on the capsid subunit indicated.



                                                - from Analytical Biochemistry (1996), 238, 1-13
Phage Display Library
•   Antibody library
     The sources of genetic material




                         -from Immunology Today (2000), 21, 371-378
Phage Display Library
   Ways of construction




                           -from Annu. Rev. Immunol. (1994), 12, 433-455
Phage Display Library

 •   Random peptide library
        the phage vector with which the library is produced,
        the phage capsid component displaying the peptide,
        the length of the insertion sequence,
        the choice of invariant residues that flank the random
         sequence.
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     -from Analytical Biochemistry (1996), 238, 1-13
Flowchart of Phage Display
Application
                                                  Expression the Protein Fragment
 Displaying Targets on   Panning (Selection) of
                                                   or Isolation of Affinity-Purified
  Filamentous Phage       The Phage Library
                                                             Phage Clones




 Library clones
                                                   Expression



                                                    Isolation
Phage Display in Making
Antibody
    Generation of antibodies by immune system and phage
                      display technology




1: rearrangement od assembly of germline V
genes
2: surface display of antibody (fragment)
3: antigen-driven or affinity selection
4: affinity maturation
5: production of soluble antibody (fragment)


-from Annu. Rev. Immunol. (1994), 12, 433-455
- from Annu. Rev. Immunol. (1994), 12, 433-455
12




5



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     -from Biogerontology (2000), 1, 67-78
The Plantibody Approach




               - form Plant Molecular Biology (2000), 43, 419–428
Screening Panels of Monoclonal Antibodies
Using Phage-Displayed Antigen

                         Miniplasminogen was displayed
                         on M13-type phage by fusion to
                         the NH2-terminus of the minor
                         coat proteinIII.




                     -from Analytical Biochemistry (1997), 248, 211-215
Structures of Binding Peptides Isolated from
Phage-Displayed Peptide Libraries


                                     A turn-helix conformation is adopted
                                     by a peptide that binds to the insulin-
                                     like growth factor binding protein 1
                                     (1998 in Biochemisty)



           A peptide that binds to the
           IgG-Fc is a b-hairpin (2000 in
           Science)
- from Current Opinion in Biotechnology (2000), 11, 610–616
Biosynthetic Phage Display
  a novel protein engineering tool combining chemical and
                      genetic diversity




                       - from Chemistry & Biology (2000), 7, 263–274
Further…..

Modification of Technique
• Panning of a Phage VH Library Using Nitrocellulose
   From J. Biochemistry (2001), 129, 209–212
• Developments in the use of baculoviruses for surface
  display
   From Trends in Biotechnology (2001), 19, 231–236
Development of phage display application
• Applying Phage Antibodies to Proteomics
   From Analytical Biochemistry (2000), 286, 119–128
• Applying phage display technology in aging research
   From Biogerontology (2000), 1, 67–78

				
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