Transcript Antibody Production

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
(1-20 ug/ml)
High concentration
(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
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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
Displaying Targets on
Filamentous Phage
Panning (Selection) of
The Phage Library
Expression the Protein Fragment
or Isolation of Affinity-Purified
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
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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 insulinlike 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