抗体噬菌体展示技术ppt课件

▪ pIII protein is essential for infection of bacteria
▪ Helper phage: wild-type pIII helper phage and
special helper phage
▪ Antigen immobilized on magnetic beads,
▪ Affibodies ▪ Anticalins ▪ DARPins ▪ Avimers ▪ Affimers ▪ Monobodies
Antibody Formats
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▪ Multivalent fragments ✓ Miniantibodies are scFvs or Fabs connected via a flexible linker
▪ A number of starting material: proteins, peptides, haptens, cell
lines, tissue slides, or virus particles
Antibody Formats
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▪ The most commonly used format: single-chain variable
exposed to an antigen.
✓ Useful in analyzing natural humoral responses, for
example, in patients with autoimmune disease, viral infection, neoplastic diseases, etc.
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
polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and later captured by immobilized streptavidin
Advantages of Phage Display for
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
used for phage display, primarily the M13 and Fd strains.
▪ Proteins to be selected are infused to all five
coat proteins, with pIII and pVIII most commonly used.
▪ Amber codon TAG: supE strains (glutamic acid
codon), non-suppressor strains (stop codon)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein translocation, crucial
separately, and secreted into the periplasm where they fold to form heterodimers.
✓ Fab exhibit higher stability than scFvs ✓ Possess better PK and PD qualities than scFvs ✓ Easier to convert into full-length antibodies ✓ Clinical applications: abciximab, lucentis, cimzia.
✓ Fab-A is created by genetic fusion of the Fab Fd gene with the
alkaline phosphatase (PhoA) gene and coexpressing the light chain gene.
✓ scFv-Fc are scFvs dimerized by the Fc domain.
Recombinant Antibody Selection
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▪ More efficiently than through conventional hybridoma system. ▪ Cheaper to produce recombinant antibodies using bacteria, rather
packing sequence and replication origin of minus and plus strands
▪ Molecular tag: to facilitate library screening and for
protein analysis
▪ Restriction enzyme recognition sites: useful for
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Antibody Phage Display
Meiling Xiong 20180629
Contents
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▪ Introduction of Ab phage Display Technology ▪ Ab Formats for Phage Display ▪ Ab Libraries Construction ▪ Phage Ab Selection Methods & Strategies ▪ Phage Ab Screening Applications ▪ In vitro Affinity Maturation ▪ Expression & Purification of Phage Ab Fragments
for display level
▪ Selective marker: for selection of infected host cells
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Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the most often
Antibody Libraries
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▪ Immune libraries: first, immunize an animal with an
antigen and isolate the mRNA from B lymphocytes ( for immunized animals) or peripheral blood B cells (for immunized donors). The mRNA is then reverse transcribed into cDNA, and the variable regions of expressed antibodies are amplified via PCR and cloned into a phage display vector.
DNA recombination and gene manipulation; multiple cloning sites (MCS)
▪ Coat protein: PIII (larger protein, less than 5 copies,)
PVIII (more than 5 copies, decreased length)
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Introduction of Phage Display Technology
The Ff bacteriophage structure
Introduction of Phage Display Technology
The scheme of phagemid vector
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▪ IG region: intergenic region, usually contains the
▪ Advantages: ✓ Matured in vivo ✓ Immune libraries can be generated from any animal and
even humans: mouse, human, chicken, rabbit, camel…
✓ Any species that have been immunized, infected, or
fragment (scFv)
✓ Simplicity of cloning process ✓ Fast and easy library generation ✓ A high display rate (small protein size ~25 kDa) ✓ Less stable than Fab fragments ✓ Tend to form dimers (can be reduced with linker more than