Antibody Production Antibody Production •Monoclonal antibody •Phage displayed antibody Poly and Mono Clonal Antibodies • Polyclonal antibody – Antigens possess multiple epitopes – Serum antibodies are[.]
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 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 10 11 12 13 14 15 16 17 18 19 20 21 22 23 -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