C3 - LESSON 2: ANTIBODIES Flashcards
Specific proteins referred to as immunoglobulins
Antibodies
Antibodies Composition:
86%-98% polypeptide; 2%-14% carbohydrate
Considered to be the humoral branch of immune response
Antibodies
can be found in blood plasma and in many body fluids such as tears, saliva and colostrum.
Antibodies
The primary function of an antibody in body defenses is to (?), which may be enough to neutralize bacterial toxins or some viruses.
combine with antigen
A secondary interaction of an antibody molecule with another (?) (e.g., complement) is usually required to dispose of larger antigens (e.g., bacteria).
effector agent
5 Major Classes (basis: heavy chain)
IgG-
IgM-
IgA-
IgD-
IgE-
IgG- gamma
IgM- mu
IgA- alpha
IgD- delta
IgE- epsilon
Each immunoglobulin is made up of a number of regions called
domains
All immunoglobulin molecules are made up of a basic (?) (TETRAPEPTIDE) that consists of two large chains called (?) and two smaller chains called (?).
four-chain polypeptide unit
heavy or H chains
light or L chains
Tetrapeptide Structure of Immunoglobulins is held together by
noncovalent forces and disulfide interchain bridges
The basic structure of immunoglobulins was elucidated in the 1950s and 1960s by the efforts of two men:
- Gerald Edelman
- Rodney Porter
N
- Gerald Edelman
used proteolytic enzyme which cleaved IgG into 3 pieces.
- Rodney Porter
For their contributions, these men shared the Nobel Prize in physiology and medicine in 1972.
- Gerald Edelman
- Rodney Porter
They chose to work with immunoglobulin G.
- Gerald Edelman
- Rodney Porter
Tips of the heavy and light chains
Variable Region
Antigen-binding site
Variable Region
Recognizes and attaches specifically to a particular antigen
Variable Region
Number of binding sites = valence
Variable Region
Responsible for the type and antigen-antibody reaction that occurs
Constant Region
Constant region of heavy chain differs from one antibody class to the other
Constant Region
Structure serves as a basis for distinguishing the 5 different classes: IgG, IgA, IgM, IgD and IgE
Constant Region
segment of H chain located between the CH1 and CH2 regions
Hinge Region
Has high proline and hydrophobic residue
Hinge Region
Allows bending of antibody
Hinge Region
- proteolytic enzyme
Papain
used to cleave IgG into three pieces of about equal size
Papain
Carboxymethyl cellulose ion exchange chromatography separated this material into two types of fragments:
- Fc fragment (fragment crystallizable)
- Fab fragment (fragment antigen-binding)
spontaneously crystallized at 4 degrees Celsius
- Fc fragment (fragment crystallizable)
no antigen-binding ability
- Fc fragment (fragment crystallizable)
known to represent the carboxy-terminal halves of two H chains that are held together by S-S bonding
- Fc fragment (fragment crystallizable)
Important in effector functions- opsonization and complement fixation
- Fc fragment (fragment crystallizable)
Have antigen-binding capacity
- Fab fragment (fragment antigen-binding)
Each fragment represented one antigen-binding site
- Fab fragment (fragment antigen-binding)
Each Fab fragment thus consists of one L chain and one-half of an H chain, held together by disulfide bonding.
- Fab fragment (fragment antigen-binding)
- used pepsin to obtain additional evidence for the structure of immunoglobulins.
Alfred Nisonoff
- proteolytic enzyme was found to cleave IgG at the carboxy-terminal side of the interchain disulfide bonds yielding one single fragment with a molecular weight of 100,000 d and all the antigenbinding ability, known as F(ab).
Pepsin
An additional fragment called (?) was similar to FC except that it disintegrated into several smaller pieces
FC
Discovered in 1845 by Dr. Henry Bence-Jones
Bence-Jones proteins
L chains that were being secreted by malignant plasma cells
Bence-Jones proteins
60ºC- they precipitate from urine
Bence-Jones proteins
further heating to 80ºC- they re-dissolve
Bence-Jones proteins
Two main types of L chains:
- Kappa
- Lambda
each type had essentially the same sequence
Lambda
Constant region
Lambda
Variable region (amino-terminal end)
Lambda
Constant regions of the (?) are unique to each class and give each immunoglobulin type its name.
H chain
Hence, IgG has an
γ H chain, IgM a μ chain, IgA an α chain, IgD a δ chain, and IgE an ε chain.
- unique amino acid sequence that is common to all immunoglobulin molecules of a given class in a given species
Isotype
- Minor variations of these sequences that are present in some individuals but not others
Allotype
- variable portions of each chain are unique to a specific antibody molecule.
Idiotype
75-80% of total serum immunoglobulins
IgG
has the longest half-life of any immunoglobulin class
IgG
- Second most efficient at binding complement
IgG1 (67%)
- largest hinge region and the largest number of interchain disulfide bond
IgG3 (7%)
most efficient at binding complement
IgG3 (7%)
have shorter hinge segments, which tend to make them poor mediators of complement activation
IgG2 (22%)
IgG4 (4%)
Major functions of IgG:
1. providing (?) because IgG can cross the placenta
2. fixing (?)
3. coating antigen for enhanced (?)
4. neutralizing (?)
5. participating in (?)
immunity for the newborn
complement
phagocytosis (opsonization)
toxins and viruses
agglutination and precipitation reactions
Macroglobulin
IgM
5-10% of total serum immunoglobulins
IgM
Half-life: 10 days
IgM
- glycoprotein with several cysteine residues which serves as linkage points and holds together the five monomeric units
J or joining chain
Starlike shape
IgM
Primary response antibody
IgM
it is the first to appear after antigenic stimulation, and it is the first to appear in the maturing infant.
IgM
most efficient of all immunoglobulins at triggering the classical complement pathway
IgM
also serves as a surface receptor for antigen
IgM
Functions of IgM:
- Complement fixation
- Agglutination
- Opsonization
- Toxin neutralization
10-15% of all circulating immunoglobulin
IgA
is synthesized in plasma cells found mainly in mucosal-associated lymphoid tissue, and it is released in dimeric form
Secretory IgA
It is not capable of fixing complement by the classical pathway, although aggregation of immune complexes may trigger the alternate complement pathway
IgA
IgA Secretory component (molecular weight of about (?))
70,000
is later attached to the FC region around the hinge portion of the a-chains
IgA
- plays an important role in neutralizing toxins produced by microorganisms and helps to prevent bacterial adherence to mucosal surfaces.
Secretory IgA
Monomer; mainly found in serum
IgA1
Dimer held together by a J chain
IgA2
predominant form in secretions at mucosal surfaces
IgA2
found as a dimer along the respiratory, urogenital, and intestinal mucosa
IgA2
IgA2
it also appears in milk, saliva, tears, and sweat
IgA2
Half-life: 2 to 3 days
IgD
Most of the IgD present is found on the surface of immunocompetent but unstimulated B lymphocytes
IgD
second type of immunoglobulin to appear
IgD
play a role in B-cell activation (regulating B-cell maturation and differentiation)
IgD
more susceptible to proteolysis than other immunoglobulins
IgD
does not bind complement
IgD
does not bind to neutrophils or macrophages
IgD
does not cross the placenta
IgD
0.0005% of total serum immunoglobulins; least abundant
IgE
most heat-labile of all immunoglobulins
IgE
does not participate in typical immunoglobulin reactions such as complement fixation, agglutination, or opsonization
IgE
it is incapable of crossing the placenta
IgE
may serve a protective role by triggering an acute inflammatory reaction that recruits neutrophils and eosinophils to the area to help destroy invading antigens that have penetrated IgA defenses
IgE
Eosinophils, especially, play a major part in the destruction of large antigens such as parasitic worms that cannot be easily phagocytized
IgE
150,000
900,000
160,000
180,000
190,000
IgG
IgM
IgA
IgD
IgE
7S
IgG
IgA
IgD
19s
IgM
8S
IgE
γ
μ
α
δ
ε
IgG
IgM
IgA
IgD
IgE
H chain subclasses: γ1, γ 2, γ 3, γ 4
IgG
H chain subclasses: None
IgM
IgD
IgE
H chain subclasses: α1, α2
IgA
H chain molecular weight: 50,000-60,000
H chain molecular weight: 70,000
H chain molecular weight: 55,000 - 60,000
H chain molecular weight: 62,000
H chain molecular weight: 70,000 - 75,000
IgG
IgM
IgA
IgD
IgE
Constant domains (H chain): 3
IgG
IgA
IgD
Constant domains (H chain): 4
IgM
IgE
Percent of total immunoglobulin: 70-75
Percent of total immunoglobulin: 10
Percent of total immunoglobulin: 10-15
Percent of total immunoglobulin: <1
Percent of total immunoglobulin: 0.002
Serum concentration (mg/dL): 800-1600
Serum concentration (mg/dL): 120-150
Serum concentration (mg/dL): 70-350
Serum concentration (mg/dL): 1-3
Serum concentration (mg/dL): 0.005
Serum half-life (days): 23
Serum half-life (days): 6
Serum half-life (days): 5
Serum half-life (days): 1-3
Serum half-life (days): 2-3
Carbohydrate content (weight percent): 2-3
Carbohydrate content (weight percent): 12
Carbohydrate content (weight percent): 7-11
Carbohydrate content (weight percent): 9-14
Carbohydrate content (weight percent): 12
Electrophoretic migration: γ2–α1
Electrophoretic migration: γ 1–β12
Electrophoretic migration: γ2–β2
Electrophoretic migration: γ1
With Complement Fixation
Without Complement Fixation
Crosses placenta
The bonding of antigen to antibody is exclusively.
noncovalent
The strength of a single antigen-antibody bond (antibody affinity) is produced by the summation of the
attractive and repulsive forces.
The four types of noncovalent bonds involved in antigen-antibody reactions are:
o hydrophobic bonds
o hydrogen bonds,
o van der Waals forces
o electrostatic forces.
Attempts to explain the (?) of antibody for a particular antigen began long before the actual structure of immunoglobulins was discovered
specificity
Ehrlich postulated that certain cells had specific surface receptors for antigen that were present before contact with antigen occurred.
Ehrlich’s Side-Chain Theory
Once antigen was introduced, it would select the cell with the proper receptors, combination would take place, and then receptors would break off and enter the circulation as antibody molecules.
Ehrlich’s Side-Chain Theory
New receptors would form in place of those broken off, and this process could be repeated.
Ehrlich’s Side-Chain Theory
Ehrlich’s Side-Chain Theory Two key premises:
- the lock and-key concept of the fit of antibody for antigen
- the idea that an antigen selected cells with the built-in capacity to respond to it
1950s- (?) independently supported the idea of a clonal selection process for antibody formation
Niels Jerne and Macfarlane Burnet
Key premise- individual lymphocytes are genetically pre-programmed to produce one type of immunoglobulin and that a specific antigen finds or selects those particular cells capable of responding to it, causing them to proliferate.
Clonal Selection Theory
The receptors Ehrlich originally postulated are the surface immunoglobulins IgM and IgD, found on unstimulated B lymphocytes.
Clonal Selection Theory
Repeated contact with antigen would continually increase a specific lymphocyte pool.
Clonal Selection Theory
Such a model provides an explanation for the kinetics of the immune response
Clonal Selection Theory
Monoclonal Antibody
1975- (?) discovered a technique to produce antibody arising from a single B cell, which has revolutionized serological testing. For their pioneering research, they were awarded the Nobel Prize in 1984.
Georges Kohler and Cesar Milstein
Their technique fuses an activated B cell with a myeloma cell that can be grown indefinitely in the laboratory.
Kohler and Milstein’s
Myeloma cells are cancerous plasma cells.
Kohler and Milstein’s
Normally, plasma cells produce antibody, so a particular cell line that is not capable of producing antibody is chosen.
Kohler and Milstein’s
In addition, this cell line has a deficiency of the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) that renders it incapable of synthesizing nucleotides from hypoxanthine and thymidine, which are needed for DNA synthesis.
Kohler and Milstein’s
The greatest impact of MAbs in immunology has been on the analysis of (?).
cell membrane antigens
MAbs have multiple clinical applications, including the following:
- Identifying and quantifying hormones
- Typing tissue and blood and delivering immunotherapy
- Identifying infectious agents
- Identifying clusters of differentiation for the classification of leukemias and lymphomas and follow-up therapy
- Identifying tumor antigens and autoantibodies
- fusion of two different types of cells
Hybridoma
cancerous plasma cells
Myeloma cells
Myeloma cells are deficiency of the enzyme:
hypoxanthine- guanine phosphoribosyl transferase (HGPRT)
Cannot make their own DNA
Myeloma cells
They will die out unless they are fused to a plasma cell
Myeloma cells
HYBRIDOMA PRODUCTION
1. (?) in immunized with a certain antigen
2. (?) are harvested
3. Spleen cells are combined with myeloma cells in the presence of (?) (surfactant)
4. (?) brings about fusion of plasma cells with myeloma cells producing a hybridoma
5. After fusion, cells are placed in culture using a selective medium containing (?).
6. Myeloma cells are normally able to grow indefinitely in tissue culture, but in this case they cannot, because both pathways for the synthesis of (?) are blocked.
7. This leaves only the (?), which have the ability (acquired from the myeloma cell) to reproduce indefinitely in culture and the ability (acquired from the normal B cell) to synthesize nucleotides by the HGPRT and thymidine kinase pathway.
Mouse
Spleen cells
PEG
PEG
hypoxanthine, aminopterin, and thymidine (HAT)
nucleotides
fused hybridoma cells
Culture in this medium is used to separate the hybridoma cells by allowing them to grow selectively.
hypoxanthine, aminopterin, and thymidine (HAT)
One pathway, which builds DNA from degradation of old nucleic acids, is blocked, because the myeloma cell line employed is deficient in the required enzymes (?)
HGPRT and thymidine kinase
The other pathway, which makes DNA from new nucleotides, is blocked by the presence of (?)
aminopterin
Consequently, the (?) die out.
myeloma cells
(?) cannot be maintained continuously in cell culture, so these die out as well
Normal B cells
is the ability of a substance to induce an immune reaction.
Immunogenicity
(?) is the ability of a substance to react with a preformed antibody or receptor.
antigenicity
(?) must be somewhat complex, with an appreciable molecular weight, and must be considered foreign by the immune system.
Immunogens
The relatively small area of the antigen that reacts with an antibody or receptor is called a (?).
hapten
(?) are too small to be immunogens on their own right but may become immunogenic when associated with a larger molecule.
Haptens
The (?) may act as haptens.
penicillin
Two terms, affinity and avidity, are used to describe the strength of the (?) between antigen and antibody.
noncovalent, reversible interactions
describes the strength of association between one epitope and one idiotype.
Affinity
describes the strength of association between multiple binding sites such as those formed by the polyvalent IgM and its antigens.
Avidity
can thus have a high avidity if multiple binding sites are engaged simultaneously.
A low- affinity antibody
are compounds that nonspecifically increase the immune response to immunogens.
Adjuvants
Immunoglobulins are V-shaped proteins having two identical light(L) and two identical heavy(H) chains, which are held together by (?).
disulfide bridges
Both the low-molecular weight L and the high molecular-weight H chains have (?).
constant and variable regions
These regions are subdivided into segments called (?).
domains
L chains have (?).
one variable and one constant domain
H chains have (?) and there or (?).
one variable
four constant domains
The (?) are responsible for antigen binding, and the (?) are responsible for various other functions.
variable domains
constant chains
Amino-acid differences in the constant region divide the L chains into either a
Kappa or a lambda type
There are five immunoglobulin classes (lgG, IgM, IgA, IgE, and IgD), which are identified by differences in their
heavy chains
The gamma H chain is expressed on IgG, the alpha on IgA, the mu on IgM, the epsilon in IgE, and the delta on on IgD.
