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ANTIBODIES

Chapter 4

http://bcs.whfreeman.com/immunology5e/default.asp?s=&n=&i=&v=&o=&ns=0&uid=0&rau=0

Ig M

What do you know about Abs so far?

• B-lymphocytes• Plasma cells• 2 major functions?

– Binding foreign Ags– Effect neutralization or elimination

• Plasma vs. serum?• Ab response to specific Ag?

– Heterogeneous– polyclonal

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Serum electrophoresis

Tiselius and Kabat: 1939…describe experiment with rabbits

Ab amino acid sequence

• Heterogeneous population• Multiple myeloma• Myeloma cell• Myeloma protein• Bence-Jones proteins• Plasmocytomas: PC• MOPCs: mineral oil PCs

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Antibody (immunoglobulin) structure: 4 protein chains

Carboxyends

Aminoends

lSSl

lSSl

lSSl

lSSl

light

light

heavy

heavy

Heterodimer

Immunoglobulin structure

IS

S l

IS

Sl

IS

Sl

ISSl

I S S l

IS

Sl

IS

Sl

IS

Sl

ISSl

ISSl

ISSl

IS

Sl

IS

Sl

IS

Sl

I S S lI SS

l

Antibody details

IS

S l

IS

Sl

IS

Sl

ISSl

I S S l

IS

Sl

IS

Sl

IS

Sl

ISSl

ISSl

ISSl

IS

Sl

IS

Sl

IS

Sl

I S S lI SS

l

214 residuesLIGHT CHAIN

446 residuesHEAVY CHAIN

variable

constant

NH3

COO

Antigen binding regions

4

Papain activity

IS

S l

IS

Sl

IS

Sl

ISSl

I S S l

IS

Sl

IS

Sl

IS

Sl

ISSl

ISSl

ISSl

IS

Sl

IS

Sl

IS

Sl

I S S lI SS

lvariable

constant

NH3

COO

papain FC: biological effector functions

FAB

Pepsin

IS

S l

IS

Sl

IS

Sl

ISSl

I S S l

IS

Sl

IS

Sl

IS

Sl

ISSl

ISSl

ISSl

IS

Sl

IS

Sl

IS

Sl

I S S lI SS

l

214 residues

446 residues

variable

constant

NH3

COO

Pepsin

F(ab’)2

FC fragments

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Domains

IS

S l

IS

Sl

IS

Sl

ISSl

I S S l

IS

Sl

IS

Sl

IS

Sl

ISSl

ISSl

ISSl

IS

Sl

IS

Sl

IS

Sl

I S S lI SS

l

214 residues

446 residues

Variable:Ag binding

Constant:

NH3

COO

VL

VH

VL

VH

CL CL

CH1CH1

CH2

CH3 CH3

CH2

HINGE

Intra-chain disulfide bonds

Inter-chain disulfide bonds

Ab structure

Carbohydrate role: 4-9

6

Light Chain SequencesNH3

COO

1

110

214

Variable

2 types of constant regionsKappa (κ) orLambda (λ)

Heavy chain sequences

V

5 different constant regionsM(μ), A(α), D(δ), G(γ), E(ε)(ISOTYPES OR CLASSES)

NH3

COO

Constant regionsIgA, IgD, IgG IgE, IgM

CH1 CH1 CH1 CH1

Hinge region Hinge region

CH2

CH3

CH2

CH3

CH2 CH2

CH3

CH4

CH3

CH4

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Immunoglobulin domains; p. 89

Class M and E domains

Ig G model

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Immunoglobulin classes: p. 87

B-pleated sheet structurein domains

Ab quaternary structure

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Light chains: Ig fold structure

CDR’S IN BLUEComplimentary Determining

Regions

Variability equalsnumber of different amino acids at a given

positiondivided by

frequency of most common amino acid at given position

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Hypervariable regions: CDR’s

Interaction between Ab and flu Ag

Conformation: HIV protease and anti-protease Ab

allosterism

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Ab-mediated effector functions

• Opsonization– FcR: Fc receptors on macrophage surfaces

• Complement activation• Ab-dependent cell-mediated cytotoxicity

(ADCC)– Fc receptors on natural killer cells

Immunoglobulin activities

Classes and subclasses: p. 96

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Classes structures compared

IgG subclasses

Secretory IgA

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Secretory IgA formation

Breast milk

IgE and allergens

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Isotypes/allotypes/idiotypes

Isotypes

Constant-region determinants that identify each heavy chain class and subclass and light chain type and sub-type

Allotypes

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Allotypes• Alleles of the isotypes within species

γ: Gm {G1m(1), G2m(23), G3m(11), G4m(4a)} :25 different onesα: A2m (1), A2m(2) :2 different onesκ: κm(1), κm(2), κm(3) :3 different ones

Idiotypes

Unique amino acid sequences of VL and VH chains.Determine specificity of the immunoglobulin.

B cell receptor

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Human Fc receptorsPart of secretory component

Ig superfamily: common primordial domain gene structures

More superfamily

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Monoclonal Antibodies

• Ab derived from a single B-cell clone and has a single binding site

• Pure antibody of one specificity• All have same molecular structure

Nucleotide biosynthesis

• De novo pathway• Phosphoribosyl PPi + Uridylate

• Nucleotides• Salvage pathway HGPRT

• Hypoxanthine + thymidine

aminopterin

HAT Media

• Aminopterin (to block de novo pathway)• Hypoxanthine and thymidine ( to allow

salvage pathway)

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Monoclonal Antibody ProductionAg

HGPRT+, Ig+

Myeloma cells

HGPRT-, Ig-spleen

Can’t use salvage pathway

Myeloma cells+ spleen cellsin polyethylene glycol

fusion

HAT media

Unfused myeloma cells dieUnfused spleen cells dieHybridomas live HGPRT+, Ig+, immortal growth

+ +

Assay for desired Ab,select positive clones

HypoxanthineAminopterinThymidine

Monoclonal Ab

Abzymes

• Antibody acting like an enzyme and catalyzing a reaction

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Clinical uses

More clinical uses

Passive Ab therapy:p.84

• 1890:Behring and Kitasato• Tetanus-immunized rabbit serum• Tetanus-infected mice protection