Chapter 5

Antibodies and Antigens

Xulong Zhang
Department of Immunology,
School of Basic Medical Science
CMU
2023.9.25
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CONTENTS

Antibody Structure,
General Features of Antibody Structure,
Structural Features of Antibody Variable Regions,
Structural Features of Antibody Constant Regions,
Monoclonal Antibodies,
Synthesis, Assembly, and Expression of Immunoglobulin Molecules,
Half-Life of Antibodies,
Antibody Binding of Antigens,
Features of Biologic Antigens,
Structural and Chemical Basis of Antigen Binding,
Structure-Function Relationships in Antibody Molecules,
Features Related to Antigen Recognition,
Features Related to Effector Functions,
Summary
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 针对抗体研究所获得的诺贝尔奖
时间 获奖人 获奖成就
1901 E.A.Behring 发现抗毒素，开创血清免疫疗法
1908 P.Ehrlich 提出体液免疫理论和抗体生成的侧链学说
1960 F.M.Burnet 提出抗体生成的克隆选择学说
1972 G.M.Edelman 阐明抗体的本质
P.R.Porter 阐明抗体的化学结构
1977 R.S.Yalow 创立放射免疫测定法
1984 G.Kohler 建立杂交瘤技术，制备单克隆抗体
C.Milstein Ig基因表达的遗传控制
1987 Tonegawa 阐明抗体多样性的遗传基础-基因重排

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General Features
Antibodies are circulating proteins
the mediators of humoral immunity against all classes of
microbes
extremely diverse and specific
Antibodies are synthesized only by B lymphocyte lineage
Exist in two forms: membrane-bound antibodies on the
surface of B lymphocytes function as antigen receptors, and
secreted antibodies function to protect against microbes.
Antibodies recognize antigens and also have effector
functions that contribute to the elimination of the antigens
可变区的作⽤
neutralize microbial toxins
prevent the entry and spread of pathogens
trigger several effector mechanisms to
eliminate the microbes interaction of
antibodies with other components of the
immune system, including molecules such
as complement proteins and cells such as
phagocytes and mast cells, NK cells:
activation of the complement system; 恒定区
的作⽤
opsonization of pathogens for enhanced
phagocytosis;
antibody-dependent cell mediated
cytotoxicity
Hypersensitivity (type I) IgE
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Ig and TCR

A healthy 70-kg adult human

produces about 2 to 3 g of
antibodies every day. Almost two-
thirds of this is a type of antibody
called immunoglobulin A (IgA), most 都有CDR识别抗原

of which is produced by
intestinal plasma cells and secreted
不能直接识别抗原
into the gut lumen. MHC结合抗原肽后才能识别

TABLE 5.1 6
Antibody Structure

General Features of Antibody Structure

In electrophoretic separations of serum or plasma, most antibodies are found in the third-fastest migrating
group of globulins, named gamma globulins for the third leter of the Greek alphabet.

albumin ⽩蛋⽩
球蛋⽩
globulin
Amounts

β γ 抗体在这⾥！
α1 α2 Serum after antigen immunization

Serum after antigen absorption

Normal serum
Protein mobility

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Structure of an antibody
All antibody molecules share the same basic structural characteristics but display remarkable
variability in the regions that bind antigens.
约 脯氨酸多，因为构
150kDa， 象可以改变
轻链25，
重链50

铰链区

⼀个球蛋⽩
结构域

FIGURE 5.1 Structure of an antibody molecule 8

 编写原则of an antibody
Structure

two identical light chains and two identical heavy chains

Ig domain：about 110 amino acid residues，a globular motif

variable (V) regions that participate in antigen recognition

carboxy-terminal constant (C) regions: the C regions of the heavy chains help mediate some of the effector
functions of antibodies.

amino acid sequences vary among antibodies made by different B cell clones.

V region of one heavy chain (VH) and the adjacent V region of one light chain (VL) form an antigen-binding
site every antibody molecule has at least two antigen-binding sites.

hinge region: which connects the CH1 domain to the CH2 region

In human IgM and IgE antibodies, the C regions contain four tandem Ig domains

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Antibody Variable Regions
Structural Features of Antibody Variable Regions

Most of the sequence differences and variability among different antibodies are confined to three short
stretches in the V region of the heavy chain and to three stretches in the V region of the light chain.

hypervariable regions V区中的超变区（HVR区）识别结合抗

原，也叫互补决定域（CDR）
V区中剩余的区域称为⻣架区

FIGURE 5.5 Hypervariable regions in immunoglobulin (Ig) molecules 10

 CDRs

⼀个完整的抗体共有12个CDRs，
可结合2种不同的抗原

Complementarity-determining regions (CDRs):

CDR1, CDR2, and CDR3.

The CDR3s of both the VH segment and the VL

segment are the most variable of the CDRs.

The most extensive contact is with the third

hypervariable region (CDR3).

Bind with epitope 表位或决定簇（是抗体结合抗原的相应位置）

Bind with epitope and MHC

more conserved framework sequences

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Hinge region and flexibility

FIGURE 5.7 Flexibility of antibody molecules 12

 Constant regions
⼆聚体（多）
单体/三聚体
Structural Features of Antibody Constant Regions

Antibody molecules can be divided into distinct

classes and subclasses on the basis of differences in
the structure of their heavy chain C regions.
基于重链的C区分类
Classes or isotypes and are named IgA, IgD, IgE, IgG,
and IgM C区是功能区

In humans, IgA and IgG isotypes can be further ⾄少两个才可以激活补体

subdivided into closely related subclasses, or 可以透过胎盘
subtypes, called IgA1 and IgA2 and IgG1, IgG2, IgG3,
and IgG4
抗体与基因对应关系
五聚体
IgA1 α1; IgA2, α2; IgD, δ; IgE, ε; IgG1, γ1; IgG2, γ2; 只需要⼀个就可以激
IgG3, γ3; IgG4, γ4; and IgM, μ. 活补体

Different isotypes and subtypes of antibodies perform

different effector functions.
TABLE 5.2 13
DNA重组

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Constant region of
light chain

There are two classes, or isotypes, of light chains, called κ and λ, that have distinct carboxy-terminal
constant (C) regions.
Each antibody molecule has either two identical κ light chains or two identical λ light chains
but never one of each.

humans, about 60% of antibody molecules have κ light chains and about 40% have λ light chains.

同型互斥：两个轻链⼀定都是κ或λ，不会出现两边轻链不同的情况

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Membrane-associated
antibodies

Secreted and membrane-associated

antibodies differ in the amino acid
sequence of the carboxy-terminal end of
the heavy chain C region.

FIGURE 5.8 Membrane and secreted forms of immunoglobulin (Ig) heavy chains 16
Proteolytic fragments of Ab

the hinge region between the CH1 and CH2 domains of

the heavy chain is the segment most susceptible to
proteolytic cleavage, because this region is unfolded and
proteolytic sites are thus easily accessible. Fragment antigen binding= Fab
Fragment constant= Fc

Papain
pepsin

pFc

FIGURE 5.3 Proteolytic fragments of an immunoglobulin G (IgG) molecule 17

immunoglobulin superfamily

FIGURE 5.4 Examples of immunoglobulin (Ig)

FIGURE 5.2 Structure of an immunoglobulin domain. superfamily proteins in the immune system.
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 Others

Multimeric IgM and IgA molecules contain an additional non-Ig 15-kD polypeptide called the
joining (J) chain, which is disulfide bonded to the tail pieces of the Ig C regions and serves to
stabilize the multimeric complexes and to transport multimers across epithelial cells from the
basolateral to the luminal end.

the sequences that differ among individuals are called allotypes.

Even in the same individual, differences among different antibodies that are
concentrated in the CDRs constitute the idiotypes of antibodies.

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Conclusion

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Monoclonal Antibodies

identical antibodies

produced by the progeny of a single B cell clone, and all the molecules therefore have
the same V region and bind to the same antigen that originally triggered that B cell.

Georges Kohler and Cesar Milstein in 1975

myeloma, or plasmacytoma

fusing B cells from an immunized animal (typically a mouse) with an immortal myeloma cell line and
growing the cells under conditions in which only the fused normal and tumor cells can survive

Hybridomas: hybrids of normal B cells and a myeloma tumor, each hybridoma makes only one Ig,
derived from one B cell from the immunized animal.

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Monoclonal Antibodies

The selection medium includes hypoxanthine,

aminopterin, and thymidine

FIGURE 5.9 The generation of monoclonal antibodies 22

Application of Ab
Application:

1. Identification of phenotypic markers unique to particular cell types.

2. Immunodiagnosis

3. Tumor identification.

4. Therapy.

5. Functional analysis of cell surface and secreted molecules.

Some examples include antibodies specific for the cytokine tumor necrosis factor (TNF) used to treat
rheumatoid arthritis and other inflammatory diseases, antibodies against CD20 for the treatment of B cell–
derived tumors and for depleting B cells in certain autoimmune disorders, antibodies specific for the T cell
regulatory molecules PD-1 and CTLA-4 used in therapy for many types of cancers, antibodies that bind
to epidermal growth factor receptors to target cancer cells, antibodies against vascular endothelial growth
factor (a cytokine that promotes angiogenesis) in patients with macular degeneration, and so on.

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Application of Ab

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Application of Ab

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 编写原则 of Ab
Application
2023上半年全球畅销药TOP10

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Application of Ab

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Limitations of monoclonal antibodies:

Make antibodies against the mouse Ig: block the function or enhance clearance
of the injected monoclonal antibody and also can cause serum sickness

Genetic engineering techniques

These are derived using phage

display methods or in mice with B
cells expressing human Ig
transgenes

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Synthesis, Assembly, and Expression of
Immunoglobulin Molecules

Synthesized on membrane-bound ribosomes in the rough endoplasmic reticulum.

The covalent association of heavy and light chains is stabilized by the formation of disulfide bonds,
which also occurs in the endoplasmic reticulum during the assembly process.

The maturation of B cells from bone marrow progenitors is accompanied by specific changes in Ig gene
expression, resulting in the production of Ig molecules in different forms

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Synthesis, Assembly, and Expression of
Immunoglobulin Molecules
Mature B cells express membrane forms of IgM and IgD (the μ and δ heavy chains associated with κ or λ light
chains).

recognize antigens and

initiate the process of B
cell activation

Affinity maturation: introduction of new amino acid substitutions into the variable domains of the
antibody heavy and light chains to create high affinity antibodies.

Isotype (or class) switching: the expression of Ig heavy chain isotypes other than IgM and IgD, by a
process called heavy chain isotype (or class) switching.
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Affinity Maturation and
Isotype Switching
Tight binding is achieved by high-affinity and high avidity interactions.

Affinity maturation: Subtle changes in the

structure of the V regions of antibodies
during humoral immune responses to
protein antigens: a process of somatic
mutation in antigen-stimulated B
lymphocytes that generates new V domain
structures, some of which bind the antigen
with greater affinity than the original V
domains.

FIGURE 5.16 Changes in antibody structure during humoral immune responses. 31

Half-Life of Antibodies

IgE: 2 days in the circulation (cell-bound IgE

associated with the high-affinity IgE receptor on mast
cells has a very long half-life);
Circulating IgA: 3 days (although most IgA is produced
at mucosal sites and is secreted directly into the lumen
of the gut or airway);
IgM: 4 days;
IgG:21 to 28 days.

The long half-life of IgG: its ability to bind to a specific Fc

receptor called the neonatal Fc receptor (FcRn), IgG
remains bound to FcRn in the acidic environment of the
endosomes. Recycles it to the cell surface and releases it
in the neutral pH of the blood, returning the IgG to the 增⻓半衰期
circulation
IgG3 is relatively short-lived because it binds poorly
to FcRn. IgG1, IgG2, and IgG4 are long-lived.

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FIGURE 5.11 Neonatal Fc receptor (FcRn) contributes to the long half-life of immunoglobulin G (IgG) molecules.
Application

The long half-life of IgG has been used to provide a therapeutic advantage for certain injected proteins by
producing fusion proteins containing the biologically active part of the protein and the Fc portion of IgG.

FIGURE 5.12 A monoclonal antibody and a cytokine

receptor–immunoglobulin G (IgG) Fc fusion
protein, both used therapeutically

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Antibody Binding of Antigens

Features of Biologic Antigens

An antigen is any substance that may be specifically bound by an antibody molecule or TCR.

Not all antigens recognized by specific lymphocytes or by secreted antibodies are capable of
activating lymphocytes.

抗原：免疫原性（能诱导免
免疫原 疫应答）+免疫反应性（能
Molecules that stimulate immune responses are called immunogens. 在体内外与抗体结合）
可以出现没有免疫原性，但是有免
疫反应性的物质，如⼀些⼩分⼦，
⼜叫半抗原 若要产⽣抗体，需要活化B细胞和Th细胞，但是半抗原不能活化Th细胞
the small chemical is called a hapten, and the large molecule to which it is conjugated is
called a carrier.

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 Antigen
Macromolecules, such as proteins, polysaccharides, and nucleic acids, are usually much larger than
the antigen-binding region of an antibody molecule. Therefore, any antibody binds to only a portion
of the macromolecule, which is called a determinant or an epitope.

polyvalency or
multivalency

The spatial arrangement of different

epitopes on a single protein
molecule may influence the binding
of antibodies in several ways

FIGURE 5.13 The nature of antigenic determinants. 35

 Ag-Ab

Structural and Chemical Basis of Antigen Binding

The recognition of antigens by antibodies involves

noncovalent, reversible binding.

The strength of the binding between a single

combining site of an antibody and an epitope of an
antigen is
called the affinity of the antibody.

This overall strength of attachment is called the avidity

and is much greater than the affinity of any one
antigen-binding site.

FIGURE 5.14 Valency and avidity of antibody antigen interactions. 36

 Ag-Ab

At the correct concentration, called a zone of equivalence, antibody and antigen form an extensively
cross-linked network of attached molecules such that most or all of the antigen and antibody molecules
are complexed into large masses.

If a zone of equivalence is reached in vivo,

large immune complexes can form in the
circulation. Immune complexes that are
trapped or formed in the walls of blood
vessels can initiate an inflammatory reaction,
resulting in immune complex diseases.

inflammation

immune complex diseases

FIGURE 5.15 Antigen-antibody complexes. 37
Structure-Function Relationships in
Antibody Molecules
V regions:
cross-reaction: Antibodies that are produced in response to a microbial antigen
Specificity sometimes cross-react with self antigens, and this may be the basis of certain
immunologic diseases

Diversity antibody repertoire

C regions:
Many of the effector functions of antibodies are mediated by the Fc portions of the molecules, and Ig
isotypes that differ in these Fc regions perform distinct functions. isotype (or class) switching
Opsonization
ADCC
IgA can be secreted efficiently across mucosal epithelia and is the major class of antibody in
mucosal secretions and milk.
Neonates are protected from infections by IgG antibodies they acquire from their mothers through
the placenta during gestation.
IgE binds to mast cells and triggers their degranulation.
activation of the classical pathway of the complement system: C1q to the Fc portions of antigen-
complexed IgG or IgM
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THANKS