Antibody Production

1. Antibody Production Monoclonal antibody Phage displayed antibody

2. 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

3. 1975, by Georges Köhler and Cesar Milstein - Be awarded a Nobel Prize in1984

4. 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

5. 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

6. (Most common screening techniques are ELISA and RIA) High concentration (1-10 mg/ml) Low concentration (1-20 ug/ml)

7. 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)

8. 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

10. 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.

11. 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.

12. 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.

13. 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.

14. 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

15. Phage Display Library • Antibody library • The sources of genetic material -from Immunology Today (2000), 21, 371-378

16. Phage Display Library • Ways of construction -from Annu. Rev. Immunol. (1994), 12, 433-455

17. 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.

18. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 -from Analytical Biochemistry (1996), 238, 1-13

19. 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 Flowchart of Phage Display Application

20. 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

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

22. 12 5 10 -from Biogerontology (2000), 1, 67-78

23. The Plantibody Approach - form Plant Molecular Biology (2000), 43, 419–428

24. 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

25. 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) Structures of Binding Peptides Isolated from Phage-Displayed Peptide Libraries - from Current Opinion in Biotechnology (2000), 11, 610–616

26. Biosynthetic Phage Display a novel protein engineering tool combining chemical and genetic diversity - from Chemistry & Biology (2000), 7, 263–274

27. 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