SI LEC - Antibodies Flashcards
composition of AB
82-96% polypeptide
2-14% CHO
Antibody molecule has 2 separable functions:
1) specific binding to antigen eliciting response
2) biological activity
- Discovered that hyperimmunizing rabbits resulted in increased gamma
- Purification revealed antibody activity resided in this serum portion
kabat and tiselius
Antibodies Are Made Up Of:
2 Light Chains (identical)
2 Heavy Chains (identical)
Each Light Chain bound to Heavy Chain (L-H) by
Disulfide Bonds
Heavy Chain bound to Heavy Chain (H-H) by
Disulfide Bonds
First a/a of amino terminal vary for both H and L Chains (Variable Region)
__
First 100 a/a of amino terminal vary for both H and L Chains (__ Region)
Variable
: Ag-binding site
CDR (Complementarity Determining Regions)
The folded domains of the H chain line up w/ the L chains forming an ___
immunoglobulin fold/barrel
Antigen is captured w/in barrel by binding to a small number of amino acids at strategic locations known as
hypervariable regions.
Immunoglobulins (with possible exception of IgM & IgE) contain hinge between
CH1 & CH2
Is there homology between AA sequence of hinge & heavy chains
None
residues in hinge region
proline and cysteine
hinge region residue involved in formation of interchain disulfide bonds
Cysteine
hinge region residue
prevents folding in a globular structure, allowing flexibility between
two Fab arms of the Y-shaped antibody; ;
Proline
hinge region residue
allows open & close to accommodate
binding to two epitopes
Proline
hinge region residue
because it is open, it can be cleaved by proteases
(e.g. papain) to generate the Fab & Fc fragments
Proline
: centered on using analytic ultracentrifuge on the basis of MW
Gerald Edelman
: worked on the use of papain to cleave IgG into 3 pcs.
Rodney Porter
Used 7M urea to unfold IgG : exposed the sulhydryl bonds
Gerald Edelman
Gerald Edelman Used ___ to unfold IgG : exposed the sulhydryl bonds
7M urea
unfolding IgG exposed __
sulhydryl bonds
Treatment with mercaptoethanol yielded
Treatment with mercaptoethanol = 4 chains:
breaks S-S bonds
mercaptoethanol
used PEPSIN :
Alfred Nansoff
Treatment with proteolytic enzyme, papain,
2 capable of ag rxn (fragment antigen binding)
1 could be crystallized (fragment- crystallizable)
specifically bind antigen,
can’t precipitate
univalent/one binding site each,
identical to each other
Fab
: crystallizable (thus homogenous) , can’t bind ag,
responsible for biological activity of molecule
after Ag bound to Fab portions
Fc
This proteolytic enzyme was found to cleave IgG at the carboxy-terminal side of the interchain disulfide bonds, yielding one single fragment (Fc) and antigen binding fragment called F(ab)2.
pepsin
found in urine of patients with MM
Bence Jones Protein :
BJP: peculiar behavior ppt at __
60C,
BJP: peculiar behavior
reappears upon ___
cooling
BJP: dissolves at __
80C
Two main types of LC
Lambda
Kappa
First 110 amino acids at terminus : constant or variable?
highly Variable (V)
First 110 amino acids at terminus of HEAVY CHAINS: highly Variable (V)
110 AA
Remaining aa can typically be divided up to 3 or more constant regions
WHAT CHAIN
HEAVY
Immunoglobulin variations class of Ig, unique aa sequence that is common to all Ig molecules of a given class in a given species
Isotype
Immunoglobulin variations
genetic difference, depends on existence of allelic forms of the Ig , result of different forms of the same gene, involve changes of 1-3 AA in constant region
Allotype
Immunoglobulin variations
one of several thousand Ig, each of which directed toward specific epitope
Idiotype
