immuno l3-4 notes

8/7/2019 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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immuno l3-4 notes

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