Antibodies and Antigens Flashcards
1
Q
What are antibodies?
A
Circulating proteins that are produced in vertebrates in response to exposure to foreign structures known as antigens. Mediators of humoral immunity against all classes of microbes
diverse and specific
2
Q
What term is often used interchangeably with Abs?
A
Immunoglobulins
3
Q
How were antibodies first discovered?
A
In serum as serum molecules that provided protection against diphtheria toxin, and thus termed antitoxins
4
Q
What are the three classes of molecules used by the adaptive immune system to bind antigens?
A
Antibodies
MHC molecules
T cell antigen receptors
5
Q
What are antibodies only synthesized by?
A
Cells of the B lymphocyte lineage
6
Q
What are the two forms antibodies exists as?
A
Membrane-bound antibodies on the surface of B lymphocytes: function as antigen receptors
Secreted antibodies: neutralize toxins, prevent entry and spread of pathogens, and eliminate microbes
7
Q
What do membrane bound antibodies on naive B cells do?
A
When the antibody recognizes antigen, it activates these lymphocytes and initiates a humoral immune response
8
Q
Where are secreted forms of antibodies present?
A
In the plasma, mucosal secretion, and the intersitial fluid of tissues
9
Q
What happens to secreted antibodies during the effector phase of humoral immunity?
A
The secreted antibodies bind to antigens and trigger several effector mechanisms that eliminate the antigens
10
Q
What are the effector properties of Abs?
A
Toxin neutralization Activation of complement system Opsonization Immunomodulation-> reduced damage to host from inflammatory response->organized T-cell response Direct antibacterial activity
11
Q
Define serum
A
Is the component that is neither a blood cell nor a clotting factor; It includes all proteins not used in blood clotting including antibodies
12
Q
When blood or plasma forms a clot, where are the antibodies?
A
They remain in the residual fluid, which is called serum
13
Q
Define antiserum
A
Any serum sample that contains detectable antibody molecules that bind to a particular antigen
14
Q
Define serology
A
The study of antibodies and their reactions with antigens
15
Q
How many antibodies does a healthy adult human produce? What is the type of the majority of antibodies produced?
A
2 to 3 g everyday
2/3 is IgA: produced in the walls of gastrointestinal and respiratory tracts
16
Q
How can plasma or serum proteins be physically separated?
A
By solubility characteristics into albumins and globulins
And may be more precisely separated by migration in electrophoresis
17
Q
Plasma or serum proteins are divided into migrating groups in electrophoresis. What are the groups? Which group are most antibodies found?
A
Albumins, alpha 1-, alpha 2- (alpha1 and 2 count as one), beta-, and gamma-globulins
Most antibodies are found in the third fastest migrating group of globulins, named gamma globulins for the third letter of the Greek alphabet
18
Q
What term is used to refer to the immunity-conferring portion of the gamma globulin fraction?
A
Immunoglobulin
19
Q
How does each antibody differ?
A
All antibody molecules share the same basic structural characteristics but display variability in the regions that bind antigens
The effector functions and common physiochemical properties are associated with the non-antigen binding portion-few variations
20
Q
An antibody molecule has a symmetric core structure composed of what?
A
Two identical light (L) chains and two identical heavy (H) chains
=four polypeptide chains
21
Q
The repeating homologous units in both the light chains and heavy chains fold independently in a globular motif called what?
A
Ig domain
22
Q
What does an Ig domain contain?
A
Two layers of beta-pleated sheets held together by a disulfide bridge
23
Q
Both H and L chains have ________ every 90 amino acid residues, which creates _________ domains, of 110 amino acids
A
Intrachain disulfide bridges
polypeptide loops
24
Q
What are the antigen-binding sites formed by?
A
Juxtaposition of VL and VH domains
25
ABS have a basic unit of how many polypeptide chains?
4
26
What are the two light (L) chains and heavy (H) chains bound together by?
Covalent disulfide bridges as well as by noncovalent interactions
27
Abs can be proteolytically cleaved to yield what?
two Fab fragments and an Fc fragment
28
What are both H and L-chains divided into?
V and C regions
29
How many domains are in the V and C regions of heavy chains?
V region is composed of one Ig domain and the C region is composed of three or four Ig domains
30
How many domains make up the V and C regions of light chains?
Each light chain is composed of one V region Ig domain and one C region Ig domain
31
What regions participate in antigen recognition and binding?
V regions
32
What regions participate in determining the fate of the Ag and mediate effector functions?
C regions
33
Paired VL and VH domains retain the ability to bind antigen and are called ______ fragment
Fab
| VL-CL associated with VH-CH1
34
Two identical disulfide-linked peptides, each containing the heavy chain CH2 and CH3 domains have the propensity to self-associate and to crystallize into a lattice and are called ____ fragment
Fc (fragment, crystallizable)
35
There are five different kinds of H-chains referred to as what?
γ, δ, α, μ and ε
36
What type of H-chain determines IgM?
μ
37
What type of H-chain determines IgD?
δ
38
What type of H-chain determines IgG?
γ
39
What type of H-chain determines IgE?
ε
40
What type of H-chain determines IgA?
α
41
What are the two types of L-chains that have a MW of 23 kDa each?
κ and λ
42
Each Ab unit can have only (fill in blank) but not both
Only κ or λ L-chains
43
The domains of the V and C regions are referred to as what?
VH, VL, CH1, CH2, CH3 etc
44
What do heavy chain C regions end in?
Tail pieces
45
True or False?
| The location of complement - and Fc receptor-binding sites within the heavy chain constant regions are accurate
False: they are approximations
46
Compared to a secreted IgG, membrane-bound IgM on B cells have ____ more CH domain(s) than IgG has
One
47
Membrane-bound IgM on B cells have C-terminal ________ and ______ portions that anchor the molecule in the plasma membrane
Transmembrane and cytoplasmic portions
48
When papain is used with IgG, the enzyme acts on what region?
It cleaves at the hinge region
49
What does papain digestion allow?
THe separation of two Fab fragments and complement- and Fc receptors-binding the Fc fragment
50
Where does pepsin cleave IgG?
Distal to the hinge region
51
What does pepsin generate from IgG?
A single bivalent antigen-binding fragment, F(ab')2
52
How can Abs bind to both widely spaced cell surface determinants and closely spaced cell surface determinants?
Because the hinge region allows flexibility between the CH1 and CH2 domains
53
A large group of cell surface and soluble proteins that are involved in the _____, ______, or ______ processes of cells belongs to the immunoglobulin superfamily.
recognition, binding, or adhesion
54
What are 6 examples of Ig superfamily proteins in the immune system?
```
membrane-bound IgG molecule
T cell receptor; TCR
MHC class I molecule
CD4 coreceptor of T cells
CD28: costimulatory receptor on T cells
Adhesion molecule ICAM-I
```
55
Proteins that contain domains with an Ig fold structure (two adjacent beta-pleated sheets held together by a disulfide bridge) belong to what family?
Ig superfamily
56
Most of the sequence differences and variability among different antibodies are confined to what?
Three short stretches in the V region of the heavy chain and three stretches in the V region of the light chain
57
What are segments of the greatest diversity known as?
Hypervariable regions; also called complementarity-determining regions (CDRs)
58
How many CDRs are in the Ag-binding site of T cell receptors?
Three CDRs in V-alpha and three CDRs in V beta domains
59
What types of Ag may be bound to immunoglobulins?
Proteins, lipids, polysaccharides and small chemicals
60
What types of Ag may be bound to T cell receptors?
Peptide-MHC complexes
61
What antigenic determinants are recognized by immunoglobulins?
Linear and conformational determinants of various macromolecules and chemicals
62
What antigenic determinants are recognized by T cell receptors?
Linear determinants; only 2 or 3 aa residues of a peptide bound to an MHC molecules
63
What is immunoglobulins affinity of Ag binding?
Kd 10^-7 -10^-11 M
64
what is the T cell receptor affinity of Ag binding?
Kd 10^-5 -10^-7 M
65
What is the on-rate/off-rate of immunoglobulin?
Rapid on-rate, variable off-rate
66
What is the on-rate/off-rate of T cell receptors?
Slow on-rate, slow off-rate
67
Kd stands for:
Dissociation constant
68
Which CDRs are the most variable?
The CDR3s of both the VH segment and VL segment
69
What are loops that protrude from the surface of the two Ig V domains and in combination create an Ag-binding region
They are CDRs from the H and L chain
70
Each domain is composed of two ________ strands, to form two _________ held together by a disulfide bond
antiparallel beta
| beta-pleated
71
Define immunogens
Molecules that stimulate immune responses
72
Immunologists commonly attach multiple copies of small molecules to a protein or polysaccharide before immunization. This combo is called what?
The hapten-carrier complex
Hapten: small chemical
Carrier: large molecule
73
Antibodies bind to only a portion of a macromolecule. This portion is called what?
Determinant or epitope
74
Define polyvalency/multivalency
The presence of multiple identical determinants in an antigen
75
Explain conformational determinant
Conformational determinants are formed by amino acid residues that are not in a sequence but become spatially juxtaposed in the folded protein. If the protein is denatured, then the determinant is lost
76
Explain linear determinant
Epitopes formed by several adjacent amino acid residues.
If linear determinants appear on the external surface of a native folded protein, then they may be accessible to antibodies. Also accessible to Igs in denatured protein
The linear determinant may be inaccessible if folded in the protein. Ig can bind to determinant in denatured protein only
77
Proteins may be subjected to modifications such as glycosylation, phosphorylation, ubiquitination, acetylation, and proteolysis. These modifications, by altering the structure of the protein, can produce new epitopes called what?
neoantigenic determinants
78
The strength of the binding between a single combining site of an antibody and an epitope of an antigen is called:
affinity of the antibody
79
The tightness of Ag-Ab binding is called:
affinity
80
Different Abs vary in their _____ to the same Ag determinant
affinity
81
What indicates how easy it is to separate an antigen-antibody complex into its constituents?
Dissociation constant (Kd)
82
The higher the binding constant the (more/less) likely the Ab is to dissociate from Ag?
Less
83
The smaller the Kd the (stronger/weaker) the affinity interaction
Stronger
84
Abs formed in the primary response soon after the injection of Antigens are generally of (higher/lower) affinity?
Lower
85
Abs produced by a memory response have (higher/lower) affinity than those in a primary response
higher
86
The association constant of memory Abs are _____ times higher
1000
87
When is the affinity of an Ab critical?
When the Ag is a toxin or virus and must be neutralized rapidly
88
The valence of an Ab is:
The maximum number of antigenic determinants with which it can react
89
What is the valence of IgG?
Contains two Fab regions and can bind two molecules of Ag or two identical sites on the same particle, thus have a valence of two
90
What is important for binding affinity because it can dramatically increase the tightness of binding of the Ab to Ags?
valence
91
What is the valence of IgM?
10
92
Define advidity
The combined effect that results from synergy of the binding strengths of each binding site
93
What can cause an Ab with a low affinity for Ag determinant to be extremely effective in neutralizing a microorganism?
IgM for example has a low affinity but has a large number of binding sites (larger valence) and thus can produce a high-adviity interaction
94
Give an example of a monovalent, bivalent, and polyvalent Ab
mono- one Fab (IgG binding to only one antigen determinant)
Bivalent-IgG
Polyvalent - IgM
95
The earliest cell in the B lymphocyte lineage that produces Ig polypeptides synthesizes the membrane form of the u heavy chain. This cell is called:
pre-B cell
96
What stage of B cells produce κ or λ light chains, which associate with u proteins to form IgM molecules?
Immature and mature B cells
97
What B cells express membrane forms of IgM and IgD?
Mature
98
What stage of B cells have increased production of the secreted form of Ig relative to the membrane form and have expression of Ig heavy chain isotypes other than IgM and IgD, called heavy chain isotype switching?
Activated B cells
99
What process results in an increase in the average binding affinity of antibodies for an antigen as a humoral immune response evolves?
Affinity maturation
100
What are the three types of affinity maturation?
Mutation in V regions: changes in fine specificity without changes in C region
Switch from membrane bound to secreted antibodies
Isotype switching: C regions change without changes in the V region
101
Different ______ have different biological properties and can _____ the body through different routes such as _________.
Microbes
enter
Ex. skin, GI tract, respiratory tract, and genitourinary tract
102
The five different Ab classes and subclasses may have likely evolved to do what?
Facilitate protection against microbes entering at the different sites of entry
103
IgA is most common for what role?
mucosal secretions
104
IgM is mainly found where?
The plasma
105
Where is IgE absorbed?
on the surface of mast cells
106
Which antibody penetrates deeper into the tissue?
IgG
107
Where are IgG found?
In both vascular and extravascular spaces as well as in secretions
108
What is the MW of IgG?
150 kDa
109
What is the most abundant immunoglobulin in the blood?
IgG
110
What Ig proves the bulk of immunity to most bloodborne pathogens?
IgG
111
What is the only Ab class to cross the placenta to provide passive humoral immunity to the developing fetus and to the infant on its birth?
IgG
112
What chains do IgG have?
Two heavy γ chains with either two κ or two λ light chains
113
What are the four subclasses of IgG?
IgG1, IgG2, IgG3, IgG4
114
How do subclasses of IgG differ?
Slightly different sequences in their H-chains that results in differences in their functional activities
115
What epithelial cell surface receptor is present in the placenta?
The receptor for Fc regions (FCRn) of maternal IgG
116
What transfers maternal IgG to the fetal circulation?
FCRn
117
What provides important protection prior to the generation of immunocompetence in the fetus?
Maternal IgG transferred to fetus
118
How long does the Maternal IgG protect the fetus? Why?
It provides protection beyond the fetal stage to neonate. It takes some weeks following birth before the transferred IgG is eventually all catabolized
119
What Ig is the major Ig present in external secretions such as colostrum, milk, and saliva where it exists as a 420 kDa dimer?
IgA
120
What chains do secreted IgA have?
κ or λ L-chains and the H α chain
| Two other polypeptide chains (1) secretory component (SC) and (2) J-chain (joining chain)
121
What part of IgA is part of the poly-Ig receptor involved in the transepithelial transport of exocrine IgA and stabilizes IgA against proteolytic degradation?
SC
122
What are the two Ig units composing secretory IgA held together by?
The J-chain through disulfide bridges
123
Where are most IgA synthesized?
Locally by plasma cells in mammary and salivary glands and along the respiratory, GI and GU tracts
124
What is the first line of defense against microbial invaders at mucosal surfaces?
IgA
125
What is the structure and MW of IgA in serum?
Exists as a 170 kDa, four polypeptide (two L and two H) chain protein
126
What is the first Ab produced by a B cell?
IgM
127
What initially acts as a BCR for B cells?
IgM
128
How is IgM on B cells expressed as?
Four-chain unit - two μ H-chains and two L- chains
129
What is blood IgM composed of?
four-chain units held together by disulfide bridges
130
Involving IgM, what initiates the polymerization of the subunits during secretion from a plasma cell?
J-chain
131
IgM is found primarily where and why?
In the bloodstream because it is a big molecule (900 kDa)
132
What are of particular importance BEFORE sufficient quantities of IgG have been synthesized?
IgM
133
The overall avidity of an IgM is quite (high/low)
High
134
What Ig is present in low quantities in the circulation (0.3 mg/ml in adult serum)?
IgD
135
What Ig has the primary function as an Ag receptor on B lymphocytes, but is probably also involved in regulating B cell function when it encounters Ag?
IgD
136
What Igs can be both expressed on B cells and are both specific for the same Ag?
IgM and IgD
137
When IgM and IgD expressed on a B cell interact with an Ag for which they are specific, what happens to the Ag?
The Ag is internalized, and processed and presented to T helper cells
138
What happens to B cells activated by B cell receptors?
The B cell proliferate and differentiate into plasma cells, thus initiating the development of a humoral immune response
139
What Ig is present in the serum at very low levels and plays a significant role in enhancing acute inflammation?
IgE
140
What Ig aids in protection from infection by worms and in allergic reactions?
IgE
141
Ab-mediated allergy is predominantly associated with what Ig?
IgE
142
What receptors bind to IgE that are specific for the Fc region of IgE?
The receptors on mast cells
143
When an antigen is reintroduced to a "sensitized" mast cell, what follows?
The antigen binds to the IgE molecule on the mast cell and triggers the release of mediators
144
What Ig is an important component of immediate hypersensitivity syndromes such as hay fever and asthma?
IgE
145
How are hybrid cells created?
(1) splenocytes from a mouse that has been immunized with a known Ag are isolated.
(2) With the help of polyethylene glycol, isolated cells are fused with myeloma cells to form hybrid cells
146
What are myeloma cells?
A cancer of plasma cells: they have immortalized properties but DO NOT secrete its own Ig
147
What do myeloma cells lack?
HGPRT used for purine salvage pathway in most cells.
148
Why do myeloma cells die in HAT medium?
Because the aminopterin (A) blocks de novo purine synthesis. Myeloma cells lack the HGPRT to use the alternative salvage pathway and therefore die bc they depend solely on de nova.
149
Why do B cells in HAT medium survive?
Because even though the de novo pathway is blocked, the B cells have the enzyme HGPRT and use the salvage pathway
150
Why do B cells medium eventually die?
Because B cells will die because of there inherent nature in 7-10 days
151
What happens after the hybrid cells are created?
They are placed in a selection medium (HAT) that permits the survival of only immortalized hybrids. The myeloma cells and the B cells do not survive and only the hybrid cells survive in HAT medium
152
What does the HAT medium include?
Hypoxanthine, aminopterin, and thymidine
153
What does HGPRT stand for?
hypoxanthine-guanine phosphoribosyl transferase
