Antibody

Yanjun Lei

Ph.D & Associate Prof.

Antigen

Antibodies are major effector molecules and

representative for specificity in humoral immunity

Membrane antibody: BCR

Membrane-bound receptors on the surface of B cell

Bind foreign antigens encountered by the host

Secreted antibody/Soluble circulating antibody

Secreted by plasma cells.

Present in serum and tissue fluids

Mediate effector functions to neutralize or

eliminate antigen.

Content of Antibody Chapter

The concept of Antibody/immunoglobulin, monoclonal/polyclonal antibody;

The structure of immunoglobulin ; The domain of immunoglobulin and the function of

each domain; The biological function of immunoglobulin; The characteristic and function of each Ig class

Kabat and Tiselius - 1938

immunized rabbit with ovalbumin

Before

After

-globulin fraction was identified as containing serum antibodies.

Harvest serum

No treatment

Mix with Ag to remove

precipitation

Electrophoresis

Immunoglobulin (Ig) Globulins( a family of glycoproteins) that have antibody functionImmunoglobulin superfamily (IgSF) Protein molecules that shared similar structural features with Immunoglobulin. TCR, CK,CKR

1. Concept of antibody and Immunoglobulin

Antibodies (Ab) Antibodies are globulins that are produced by plasma cells in response to an antigen and react specifically with the antigen that stimulated their production.

5 class: IgG, IgM, IgA, IgD and IgE

2. Antibody Structure

2. “Y” shape: linked by disulfide bonds

1. Consist of 4 polypeptide chains Two heavy (H) chains Two light (L) chains

3. Two terminal amino-terminal end (N) carboxyl-terminal end (C)

Disulfide bond

Fab

Fc

4. Two regions

Constant (C) region: The C terminal sequence (~ 110 residues of L chain and remaining of H chain) was constant for all antibodies

L chain 1/2-VL

H chain 1/4-VHN-end

L chain 1/2-CL

H chain 3/4-CHC-end

Variable (V) region : the sequence of the ~110 N terminal residues of L chain and H chain was seen to be unique for each antibody with differernt binding specificity.

Hypervariable regions/ complementarity-determining regions(CDRs) form the antigen-binding site

The amino acids of V region varies greatly among antibodies with different antigen specificity.

Variable (V) region

Hypervariable region (HVR)complementarity-determining region(CDR) Within the variable regions of both heavy and light

chains, some polypeptide segments show exceptional variability and are termed Hypervariable regions or complementarity-determining regions(CDRs)

There are 3 complementarity-determining regions(CDRs) on both L and H chains.

Most of the differences among Abs with different specificity fall within CDRs

CDRs on both L and H chains form the antigen-binding site of the antibody molecules

Constant (C) region

The regions of relatively constant sequence beyond the V regions

The Ig constant region domains take part in various biological functions that are determined by the amino acid sequence of each domain.

In human, There are five distinct classes of antibody /immunoglobulin molecule according to the sequence of constant region of H chain

The class and subclass of immunoglobulinThe class and subclass of immunoglobulin

Ig IgM IgG IgA IgD IgE

H chain

The H chain of a given antibody molecule determine the class of that antibody.

General structure of the five major classes of secreted antibody

Size: The length of the C region: ,,: ~330 aa; , : ~440 aa Charge Amino acid sequence Carbohydrate content

IgG, IgM, IgA, IgE and IgD differ in:

Minor differences in the amino acid sequences and differences in position and numbers of disulfide bond of the and heavy chains led to further classification of the heavy chains that determine the subclass of antibody molecule they constitute.

heavy chains: 1 — IgA1 2 — IgA2

heavy chains: 1 — IgG1 2 — IgG2 3 — IgG3 4 — IgG4

In humans, 60% of light chains are kappa and 40% are lambda; in mice 95% are kappa and 5% are lambda. Comparison of λλ chain sequences revealed minor differences that could be used to classify λ λ chains into subtypes - 3 in mice and 4 in humans

Each antibody has two identical heavy and light chains, A normal antibody molecule contains only one light- chain type, either κ or λ, never both.

2 light chain types: 2 light chain types: κκ or or λλ

5. Hinge region

IgG IgD and IgAIs rich in proline residuesGive IgG IgD IgA segmental flexibility

The and heavy chain contain an extended peptide sequence between the CH1 and CH2 domains that has no homology with the other domains.

6. Accessorial structure Joining (J) chain: facilitate the

polymerization of IgA and IgM

secretory piece (SP or SC): is responsible for IgA

transporting through mucous membrane epithelial cells

Basolateral surface

luminal surface

1. Dimeric IgA bind with Poly-Ig receptor

2. Be internalized by receptor mediated endocytosis.

3. Transport to luminal surface

4. Poly-Ig is cleaved, Releasing the SP bound to the dimeric IgA

5. SP masks sites susceptible to protease cleavage, allowing the polymeric molecule to exist longer in the protease-rich mucosal environment.

3. The domains of antibody

Heavy and light chains are folded into domains, each containing about 110 amino acid residues and an intrachain disulfide bond that forms a loop of 60 amino acid

Constant Domain: CH1, CH2, CH3, CH4 ( IgM and IgE).Variable Domain: VH, VL

CH1, CH2, CH3 ( IgA, IgD and IgG).

VL VH: antigen binding site

CL CH1: Facilitate interaction with antigen and increasing the maximum rotation of the Fab arms.

Function of domains

Hinge region: flexibility, enabling the 2 arms to move relatively freely

CH2 of IgG/ CH3 of IgM: activate the complement cascade.

cross the placental barrier. (IgG)

IgA, IgD, IgG IgE, IgM

CH1HingeCH2CH3

CH1CH2CH3CH4

Secreted immunoglobulin: binding to cells by Fc receptors.

Membrane-bound immunoglobulin （ BCR ） :

An extracellular hydrophilic “spacer” sequence composed of 26 amino acid residues. A hydrophobic transmembrane sequence A short cytoplasmic tail

CH3 of IgG/ CH4 of IgE and IgM: bind with the cells

4. Enzymatic Digestion Fragments of lgG4. Enzymatic Digestion Fragments of lgG

Fc: Fragment, crystallizableFab: Fragment, antigen binding

Proteolytic enzyme

General structure of the five major classes of secreted antibody

3 major categories: Isotypic determinants Allotypic determinants Idiotypic determinants

Antigenic Determinants on lmmunoglobulins

Definition - Isotypes are antigenic determinants that characterize classes and subclasses of antibody.

Location - isotypes are found in the constant region of the heavy chain and light

Isotype

Different species inherit different constant-region genes and therefore express different isotypes

When an antibody from one species is injected into another species, the isotypic determinants will be recognized as foreign , inducing an antibody response to the isotypic determinants on the foreign antibody.

Antibodies to isotypes are used for quantitation of Ig classes and subclasses in research or clinic.

Occurrence - Isotypes are found in ALL NORMAL individuals in the species.

Definition- are additional antigenic features of Ig that vary among individuals of the same species and are under genetic control.

Location - the allotypic differences are localized to the constant region of the heavy and light chains

Antibody to allotypic determinants can be produced by injecting antibodies from one member of a species into another member of the same species who carries different allotypic determinants.

Allotypes represent slight differences in the amino acid sequences in the heavy or light chains of different individuals

Occurrence - Individual allotypes are found in individual members of a species. Any individual Ig molecule will only have one allotype.

Definition - Unique antigenic determinants present on individual antibody molecules or on molecules of identical specificity.

Location - Idiotypes are localized on the variable region of the Ig molecules

idiotypic determinants of the antibody can recognized by self immune system to generate anti-idiotype Abs.

The unique amino acid sequence of the VH and VL domains of a given antibody can function not only as an antigen binding site but also as a set of antigenic determinants.

Each individual antigenic determinant of the variable region is refers to as an idiotype 。 Each antibody will present multiple idiotypes.

1. Neutralization Antibodies bind specifically with the exotoxin or

virus to neutralize their toxicity or infectivity.

Function of Antibody

Neutralize the infectivity of Virus

2. Activate the complement

3. Across epithelial layers and across placenta

IgA (major)

IgM

Mucosal surface of the respiratory, gastrointestinal and urogenital tracts; breast milk

IgM and most of IgG

IgG fetus

4. Antibodies bind to cell surface receptors on macrophage, NK cells, mast cells and basophils

① IgG Fc —Mφ opsonization

② IgG Fc —NK ADCC

③ IgE Fc —mast cell Type I hypesensitivity

opsonization

Antibody can promote phagocytosis of antigens by macrophage and neutrophils, is important factors in antibacterial defenses.

ADCC ( Antibody-dependent cell-mediated cytotoxicity)

The linking of Ab bound to target cells with the FcRs of a number of cell types,particularly NK cells, can direct the cytotoxic activities of the effector cell against the target cell. This process called ADCC

Perforingranzyme

Mediate Type I hypesensitivity

Unique to IgE

Inappropriate and damaging immune response that result in serious disease, tissue injury, or even death

Hypesensitivity:

The characteristics and function of The characteristics and function of each Ab classeseach Ab classes

Is the most major Ig in serum and in extra vascular space. constitutes about 75~80% of the total serum Ig.There are 4 human IgG subclasses.

IgG is the only class of Ig that crosses the placenta and play an important role in protecting the developing fetus. Not all subclasses cross equally; IgG2 does not cross well, IgG4 does not fix complement Not all subclasses activate complement equally well, IgG3 is the most effective complement activator, followed by IgG1, IgG2 is less efficient, and IgG4 dose not activate complement.

IgG1 and IgG3 bind with high affinity to Fc receptors on the phagocytic cells and thus mediate opsonization. IgG4 has an intermediate affinity for Fc receptors and IgG2 has an extremely low affinity.

IgM accounts for 5~10% of the total serum Ig and is the 3rd most common serum Ig. Monomeric IgM is expressed on the surface of B cell, act as BCR. IgM secreted by plasma cells is pentamer .IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen IgM is more efficient than IgG at activating complement. IgM is “natural” antibodies to RBC group

IgM

Constitute 10-15% of the total Ig in Serum

Predominant Ig class In secretions Breast milk, tears , saliva, mucus of digestive tract, bronchial. Most serum IgA is monomer

Secretory IgA is dimer

J Chain (Joining) and secretory component

IgA

Play important protective effect in mucosal immunity

Transport of IgA into Gut Lumen

0.03 mg/ml, 0.2% of the total Ig in Serum

Together with IgM ,is the major membrane-bound Ig expressed by mature B cellsits role in serum is uncertain.

Immunoglobulin D (IgD)

IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen. Only 0.0003 mg/ml in serum Mediate allergic reactions. IgE is involved in allergic reactions. Binding of the allergen to the IgE on the basophils an mast cells results in the release of various pharmacological mediators that result in allergic symptoms.

Immunoglobulin E (IgE)

Polyclonal Antibody(pAb) /Antisera

Monoclonal Antibody(mAb) Antibodies that are specific for one antigen epitope and produced by one B cell clone, and is produced by a B cell hybridoma

1975 Georges Kohler and Cesar Milstein

Antibodies that are derived from different B cell clones. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.

10. The preparation of Antibody