Chapter 4 Antibodies

• Ab’s are Ag-binding proteins secreted by plasma cells; found on surfaces of B cells and free in the blood/IF/lymph

• Ab’s triggered by an Ag are heterogeneous -produced for dif’t epitopes on the Ag (polyclonal Ab’s)-from several clones of B cells – each producing monoclonal Ab’s

Antibody isolation• Centrifuged blood contains:

• Cells

• Liquid (plasma)

• If left to clot – serum

• Serum is Ab rich

• Electrophoretic peaks shown by Tiselius and Kabat (1939)

Injection of ovalbumin

Antibody structure

• 4 peptide chains:– 2 identical Light chains– 2 identical Heavy chains

• Ea. light chain is bound to a heavy chain by di-S bond + noncovalent attractions

• Similar di-S bonds link 2 H-L chain combo’s

(H-L)2

Antibody structure

• 1st 110 aa’s @ NH3 end exhibit variation in both H + L chains -(V)ariable regions

• w/i V regions are CDR’s – specific binding sites for Ag

• w/i the same Ab class, there is relatively constant aa seq thru rest of molecule – (C)onstant regions

• Glycolsylation of Fc fragments – Ab’s are glycoproteins

Discovery of aa sequences in H and L chains• Each Ag carries multiple epitopes (even haptens!)

so that initial efforts hindered

• Discovery of multiple myeloma (and Bence Jones proteins) allowed isolation of large quant. of homogenous Ab

• Can stimulate MOPC’s in mice from intra-peritoneal mineral oil injections stim production of malignant plasma cells

Light Chain sequencing:

• V regions (amino terminal half) vary in aa sequence

• C regions (carboxyl terminal half) show 2 basic aa sequence patterns– Led to typing of L chains – kappa (κ) and

lambda (λ)– minor differences in λ chains results in further

sub-typing ( λ1, λ2, and λ3)

Heavy Chain sequencing:

• Like L chains, amino terminal end reveals variation in 1st 100-110 aa’s

• Remaining portion revealed 5 seq. patterns corresponding to 5 dif’t C regions: labelled γ, α, μ, δ, and ε

• Each of these 5 C chains creates an Isotype• Heavy chains of Ab’s determine the class of the

Ab – IgG, IgA, IgM, IgD, IgE• Minor differences in α and γ chains (α1,2 and γ1,2,3,4)

called “subisotypes”

Critical interactions of Ab domains dictates 4° structure

Diversity of Variable Regions is concentrated in CDR’s

• Max variation is seen in those aa sequences in loops joining β sheets of the proteins of the VL and VH regions Ag binding sites

• As Ag-binding occurs between complementary aa’s = “complementarity-determining regions” (CDR’s)

• there are 3 such loops on both chains• Variation in length and sequence of the 6 CDR’s creates

wide range of Ab specificities!!

Constant region domains

They perform a variety of functions:• CH and CL domains support and orient (V)ariable regions

contrib to > diversity• “Hinge” region between CH1 and CH2 rich in proline;

di-S bonds between cysteines• IgG, D, A have 3 C domains (CH1-3)• IgM, E have 4 C domains (CH1-4)• CH2’s of G,D,A and CH3’s of M, E are separated by

polysaccharides (solubility) and Complement binding sites• CH3’s and CH4’s exhibit carboxyl-terminal ends• Secretory Ig has hydrophilic aa’s @ COOH end• Membrane Ig contains 3 parts:

Roles of Antibody:

Both ends of Ab are functional (NH3-end: Fab; COOH-end: membrane + protein

binding)

• Opsonization

• Complement activation

• Transcytosis to mucosal surfaces