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Chapter 4- Antigens & Antibodies. Where we’re going- Immunogenicity vs antigenicity What makes for a good immunogen (aka antigen) Haptens. Some definitions:. Immunogenicity Immunogen, for practical purposes= antigen
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Chapter 4- Antigens & Antibodies Where we’re going- Immunogenicity vs antigenicity What makes for a good immunogen (aka antigen) Haptens
Some definitions: • Immunogenicity • Immunogen, for practical purposes= antigen • Antigenicity- ability to react with antibodies or Tcells; all immunogens are antigens, but haptens have antigenicity but not immunogenicity. (if this sounds confusing…)
Haptens • Loved by some immunologists-esp. old ones! Haptens aren’t immunogenic alone. BUT- when bound to a carrier, Ab’s are produced that DO bind to the unbound hapten
So how do we tell a good antigen from a poor one? Inject with antigen- isolate serum, and react with antigen to produce, say, precipitation; the more dilute the serum can be, and still ppt the Ag, the better the response. Discuss more in Chapter 6
Immunogen’s contribution- usually think “protein” • Foreignness- cow vs chimp serum albumin; “shielded” parts from us- mitochondrial proteins, sperm. • Size- > 100K better, < 10K worse, especially for proteins • Complexity- homopolymers poor, heteropolymers better • Easily processed better than poorly processed; adding L-amino acids to a D-polymer increases its immunogenicity.
“Our” Contribution- both the animal and how immunization is done • Genetics- mouse studies- responders and non-responder mice. • Dosage and route of administration- the pneumococcal polysaccharide story. • Route determines which lymph tissues meet the antigen- e.g., spleen vs local lymph nodes. (know the terms, p.80)
Our contribution- adjuvants • Something that stimulates the immune response to the antigen. • Alum- ppt’s, prolongs persistence • Inflammatory- dead Mycobacterium • Non-specific proliferation of cells- LPS,
Epitopes, or Ag determinants • Our cells don’t react to the whole molecule, but to parts- epitopes • B cells react to differently to epitopes than do T cells. • Remember- interaction is specific!
How B cells do it An epitope can be non-sequential- 56-62 and 15-21 can be brought together spatially, producing a single epitope. B cell eptitopes tend to be on the surface,.
This is a strange graph- by measureing inhibition, it essentially measures binding; antibody was to the native structure.
How T cells do it The binding is ternary- three components- MHCII, TCR, antigen. Only parts are displayed. No MHC binding= no antigenicity B cell eptitopes tend to be on the surface, but T cells can be from the interior of the molecule.
Antibodies • Where we’re going: • Learn about Ab structure: constant, variable, Fab, Fc, hypervariable, CDR regions. • Five different classes: G,M,A,E,D • Functional differences • Isotypes, allotypes, idiotypes • Monoclonals, and variants
In SERUM- liquid left from clotted blood Antibodies here- black is what happens when induced Ab’s are removed (anti-OVA)
Binds Ab still Binds complement, ppt’s in the cold, only constant region
We know these things from myelomas, and plasmacytomas induced in mice w/ mineral oil (MOPC)
Hypervariable= CDR= Ag binding regions- towards the outside of V domain. Complementarity determining region
Variability= # different amino acids at a given position- Frequency of most common AA e.g.- 10 different AA’s in a position, most frequent is at 0.2, variability= 50
What do Antibodies do? • Bind antigen- neutralize toxins, virus particles • Opsonization • Complement activation- IgG,M • ADCC • Transcytosis- movement across epithelial cells
Most abundant 4 subclasses Activates complement Crosses placenta See notes! Secreted Multiple forms (1-4) NOT inflammatory!
Antibodies can also be antigens- develop Ab’s agains mouse Ab’s in a bunny. Isotypic = class determinants Allotypic- allelic differences between the same Ig Idiotypic: differences due to antigen-binding differences
New topics The B Cell receptor (tails) and superfamilies, and monoclonals
New topics: Tails and Superfamilies Ig-α,Ig-β allow for signal transduction Tails of mIgM and mIgD are only 3 amino acids-too short for signal transduction
Some Ig superfamilies- similarity in structure, but not necessarily function. Thought to arise by gene duplication and evolution
Monoclonal Antibodies Ab’s from a single clonal line of B cells. The problem: differentiated B cells/plasma cells won’t divide. Solution: fuse with immortal cancer cell, to make LOTS of Mab
HAT medium: HGPRT- myeloma cells die HGPRT+ B cells don’t multiply.
Hypoxanthine and thymine from salvage HGPRT- X Myeloma cell-can divide, but poisoned! Aminopterin X De Novo synthesis
Hypoxanthine and thymine from salvage HGPRT+ B cell- resists poison, but can’t divide! Aminopterin X De Novo synthesis done in
Hypoxanthine and thymine from salvage HGPRT+ Fusion: can divide! Resists poison! Aminopterin X De Novo synthesis done in
Immunotoxins! A long-time promise. Actual monoclonals against B cells are out there, in use, now.
Key Points- See Learning Objectives! • General structure of Ab’s: L & H chains, V & C regions, Ab binding region, Fc, CDRs, etc. • Unique characteristics of each Ab • Superfamilies, Monoclonals