Chapter 4 Antibodies

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

Chapter 4 Antibodies