Chapter 6 Ag-Ab Interactions: Principles & Applications

Chapter 6 Ag-Ab Interactions: Principles & Applications

Nov 2, 2006

Fluorescent Ab staining reveals intracellular Ig

Chapter 5 Ab Engineering (p. 136 – 142)

Human Ab from mice bearing a human artificial chromosome that includes entire human H- and L-chain loci

Derivation of Single-Chain Fragment Variable (scFv) library

> 1011

Phage Display

Nature of the Ag-Ab interaction

Immunological assays

4 types of noncovalent forces

- The interactions of Ab with Ag are through non-covalent bonds.

a. Ionic (or electrostatic) bond

Ionic bonds form between surfaces of opposite charge.

b. Hydrogen bond Hydrogen bonds form between hydrogen atoms and two other electronegative atoms such as oxygen and nitrogen.

c. Van der Waals’ force

Van der Waals’ forces occur at very close ranges between two atoms. Fluctuations in the electrical charge within electron clouds can lead to attractive or repulsive forces between atoms, dependent on the distance between them.

d. Hydrophobic bond

Hydrophobic bond is created by the behavior of hydrophobic subunits in aqueous environments. These tend to be pushed together to minimize the instability they cause in the network of hydrogen- bonded water molecules.

AffinityThe combined strength of the noncovalent interactions between a single Ag-binding site on an Ab and a single epitope is the affinity of the Ab for that epitope.

AvidityThe strength of multiple interactions between amultivalent Ab and Ag is called the avidity.

Example: secreted pentameric IgM often has a lower affinity than IgG, but the high avidity of IgM, resulting from its higher valence, enables it to bind Ag effectively.

Cross-reactivity

Cross-reactivity occurs if two different Ags share an identical or very similar epitope.

Immunological Assays

1. Precipitation 2. Agglutination 3. Radioimmunoassay 4. Enzyme-linked Immunosorbent Assay 5. Western Blotting 6. Immunofluorescence 7. Flow Cytometry and Fluorescence 8. Immunoelectron Microscopy 9. Alternatives to Ag-Ab reactions

Precipitation – lattice formation (格子 )

Precipitation is inhibited at Ab excess or Ag excess

Precipitation :

Ab and soluble Ag interacting in aqueous solution form a visible precipitate.

Agglutination :

The interaction between Ab and a particular Ag results in visible clumping called aglutination.

Solid-phase radioimmunoassay (RIA)

Enzyme-linked Immunosorbent Assay (ELISA)

- Indirect ELISA

- Sandwich ELISA

- Competitive ELISA

- Chemiluminescence

- Elispot assay

Enzymes: alkaline phosphatase, horseradish peroxidase, -galactosidase, etc.

Substrates: chromogenic (呈色的 ) chemiluminescence – luxogenic (可產生光的 )

Elispot Assay

Western Blotting

Western Blotting

Immunofluorescence

Flow Cytometry

FACS:

Fluoresence-activatedcell sorter

Immunoelectron microscopy

500 nm

Alternatives to Ag-Ab reactions

Protein A: a cell-wall protein from Staphylococcus aureus (葡萄球菌 )

Protein G: a cell-wall protein in group C and G Streptococcus (鏈球菌 )

Avidin: an egg white protein

Streptavidin : a protein made by Streptomyces avidinii

- Protein A & protein G can bind to the Fc region of IgG molecules with high affinity (Kd ~ 10-8 M).

- Avidin (or strptavidin) binds biotin (a vitamin that is essential for fat synthesis) with Kd ~ 10-15 M.

Applications:

Ag + Ab + Protein A (labeled with flourochromes, radioactivity, biotin or conjugated on beads)

Ag + Ab + Protein G (labeled with flourochromes, radioactivity, or biotin or conjugated on beads)

Ag + Ab (labeled with biotin) + avidin or streptavidin (conjugated with enzymes,

flourochromes, radioactivity)