immuno L3-4 notes
Transcript of immuno L3-4 notes
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Immunology L3-4 Antibody Structure and Function
Jargons:
Blood fluid fraction = plasma (soluble small molecules and macromolecules, e.g.fribin)Take away clotting >>> serum
cellular fraction = red blood cells, leukocytes, platelets
Find out antibody in a particular serum protein (Tiselius and Kabat, 1939)
rabbit serum immunised with ovalbumin, 2 aliquots, electrophoresis
(1)Control: 4 peaks albumin, E-, F-, K-globulin(2)Added ovalbumin before electrophoresis, ppt removed: significant drop in K-globulin
@K-globulin fraction serum antibodies = immunoglobulins (Ig)
IgG, main class of Ab, mostly found in K-globulin fraction
Some IgG + other important classes of Ab found in E-, F- fractions
What is antibody (Ab)?
- Membrane bound (when act s as receptor) / Soluble secreted protein by plasma cells- Bifunctional
(1) binds antigen (Ag)
(2) binds receptors on cells and activate complements (C)
Structure of Ab
- H
eterdimer: 2 identical heavy(H
) chains [55kDa, 450aa], 2 identical light (L)chains[22kDa, 250aa])
- Inter-domain (L to H, then H to H):(1) disulphide bonds (CL-CH/ hinge-hinge)
(2) non-covalent bonds: electrostatic, salt linkages, hydrogen bonds, hydrophobic
interactions,
= dimer + dimer form a 4-polypeptide-chain unit
- Intra-domain: S-S (loop of ~60aa)- Hinge region (black): flexible region, proline-rich, arms move
Light (L) chain Heavy (H) chain
- 2 types- encoded by O and P - 5 subclasses (M, G, A, D, E)- encoded by 5 sets of genes (Q, K,E,H,and I)- VL: antigen binding - CH: control biological activities
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Variable Constant
Light y 1st 100-110 aa longy Single domainy Difference in complementarity-determining regions (CDRs)
H
eavy y
Single domainy y 3/4 domainsy CHO to CH2 = glycosylationo solubility (?!)affects rate Ab cleared from serum andqefficiency of interaction btw Ab and proteins
y amino-terminal variable (V) region - differsfrom one antibody to the next, bind to antigen
y constant (C) regions - limited variation,defines 2 light-chain subtypes, 5 heavy-chain
subclasses
y HK,H,E has a proline-rich hinge regiony HQ,I no hinge region, has additional C Hdomain
y effector functions are mediated by the otherdomains
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CH2 domains protrude because of the interior c arbohydrate
1o
= sequence of aa ofV/CH/L
2o = folding of extended polypeptide chain
series of antiparallel F-pleated sheets
alternating R = hydrophilic outside, hydrophobic inside
3o
= compact globular domains connected to neighbouring
domains by stretches of polypep chains btw regions ofF-
pleated sheets
4o
= globular domains of adjacentH and L polypep chains
functional antigen binding + effectors
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A. IgG proteotic experiment
show properties of Ab in relation to structure
(1) Papain digestion- cleave heavy chain on N-terminus of S-S3 fragments:
- 2 identical [45kDa]= Fragment antigen binding (Fab)
Monovalent 1 binding site to antigen each
- 1 fragment [50kDa] = Fc fragement,crystallised during cold storage
Binds & activates C
CH2 Binds to FcR on cells
(2) Pepsin digestion- Cut below S-S ofH2 fragments:
- Single [100kDa] Fab-like = Fab2- Fragmented Fc
B. Mercaptoethanol reduction + alkylation
deduce multi-chain structure by separating of
individual H and L
- Irreversibly cleave S-S2 fragments:
- H [55kDa]- L [22kDa]
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Subclass
IgG
IgM
IgA
IgE
IgD
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