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
(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.
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.
Phage Display - Advantages
Filamentous 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.
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.

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
-from Annu. Rev. Immunol. (1994), 12, 433-455
Ways of construction
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.
Phage Display Library
-from Analytical Biochemistry (1996), 238, 1-13
Flowchart of Phage Display Application
Phage Display in Making Antibody
-from Annu. Rev. Immunol. (1994), 12, 433-455
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 Biogerontology (2000), 1, 67-78
12
5
10
The Plantibody Approach
- form Plant Molecular Biology (2000), 43, 419–428
Screening Panels of Monoclonal Antibodies Using Phage-Displayed Antigen
-from Analytical Biochemistry (1997), 248, 211-215
Miniplasminogen was displayed on M13-type phage by fusion to the NH2-terminus of the minor coat proteinIII.
Structures of Binding Peptides Isolated from Phage-Displayed Peptide Libraries
- 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