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Immunology
Antibodies
102
Science
Professional
09/22/2007

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Term
What are the three main (general) properties of antibodies?
Definition
1.) They are found in a number of different bodily fliuds and excretions.
2.) Chemical and Physical
3.) Immunogenic Properties
Term
In which bodily secretions are antibodies found?

Which has teh highest concentration of Ab?
Definition
Tears, mucous, saliva, feces, urine, breast milk and serum.

Serum
Term
What type of structure do antibodies resemble?
Definition
Glycoproteins, possessing chemical and physical properties of glycoproteins
Term
What is the globulin fraction of serum?
Definition
The INSOLUBLE fraction which contains ANTIBODY activity.
Term
What is the albumin fraction og serum?
Definition
the SOLUBLE fraction of serum...NO ANTIBODY activity.
Term
Where does the term Immunoglobulin (Ig) come from, in respect to Antibodies??
Definition
The GLOBULIN fraction of serum contains antibody activity...or immunologic activity.
Term
What is the cause of difference fractions of whole serum being formed?
Definition
Serum proteins precipitate in different concentrations of AMMONIUM SULFATE.
Term
Explain the electrostatic charge of an Ab.
Definition
As proteins, Ab's are chains of amino acids that possess either a net (+) or (-) or neutral charge at a certain pH.

Electrostatic charge is the neutral charge.
Term
What is ELECTROPHORESIS?
Definition
The separation of proteins based on the net charge at certain pH's across an electrical potential.
Term
When you send a sample of serum through electrophoresis, how many fractions yield, and what are these fractions?
Definition
(+)---Albumins-alpha-beta-gamma---(-)

1 Albumin + 3 globulin factions (a,B,Y)
Term
Where is most of the antibody activity on the Ab??
Definition
In the GAMMA globulin fraction, hence the term GAMMAGLOBULIN for Ab or Ig.
Term
What is the relative molar mass/size of an Ab?
Definition
A range from 150 - 900 kDa
Term
What are the immunogenic properties of an Ab?
Definition
An Ig can act as a potent immunogen in other species and can elicit Ab production against Ig-associated epitopes.

They allow for the division of In's into 5 major classes of ISOTYPES.
Term
What are the five major Ig isotypes or classes?
Definition
1.) IgG: gamma epitopes...4 sub-isotypes in humans... IgG1,IgG2,IgG3,IgG4
2.) IgM: mu epitopes
3.) IgA: alpha epitopes...2 sub-isotypes in humans...IdA1 & IgA2.
4.) IgE: epsilon epitopes
5.) IgD: delta epitopes
Term
In what direction does electrophoresis go?
Definition
From negative to positive.
Term
Which three reagents were used to reveal the basic structure of an antibody?
Definition
Mercaptoethanol (S-S reducing agent)-break disulfide bonds.
Papain - Protease
Pepsin - Protease
Term
What did treatment with mercaptoethanol produce?
Definition
2 Heavy chains and 2 light chains[image]
Term
What did treatment of an Ab with Papain produce?
Definition
2 Fab's and 1 Fc Fab = fragment antigen binding Fc = Fragment, crystallizable, and did NOT bind to an antigen.[image]
Term
What did treatment of Ab with Pepsin result in ?
Definition
1 F(ab')2 and several Fc fragments.[image]
Term
What was the proposed model for Ig(G)Structure?
Definition
4 polypeptides (2H, 2L)
2 Ag-binding sites (each is a Fab)
Each Fab fragmen associated with 1L chain
Fc fragment forms BASE of "Y" sheped molecule
Term
What is special about the L chain molecules in Humans?
Definition
There are 2 of them present: KAPPA and LAMBDA.

But only 1 type of L chain is found per Ig molecule. Either κ or λ, NOT BOTH!!
In Humans, 60% are kappa and 40% are lambda.
Term
Serum contains a ______ mixture of Ig...explain
Definition
Heterogeneous...the Ig's have similar structure, BUT with a wide range of specificities available (variability).
Term
The variability of Ig's in serum ia part of the ____ structure of Ig.
Definition
Primary structure
Term
What part of the Ig was examined to see HOW variability is achieved.
Definition
Examining amino acid sequences on H & L chains.
Term
How many regions or domains can the Light chain be divided into?
Definition
2: 50% constant(CL) and 50% variable (VL) region
Term
Describe the CONSTANT region of the LIGHT chain.
Definition
The Light chain can be either Kappa or Lambda. All light chains have the SAME CONSTANT region. It is near the C-Terminal...COOH.
Term
Describe the VARIABLE region of the LIGHT chain.
Definition
The VARIABLE region changes, its variable from L chain to L chain and from kappa chain to lambda chain. It is near the N-terminal, 50% of L chain sequence.
Term
Howmany regions or domains are HEAVY chain sequences usually divided into?
Definition
4-5 for EACH ISOTYPE
Term
Describe the CONSTANT region of the HEAVY chain.
Definition
It is constant for EACH ISOTPYE (so all ε chains of IgE have the same constant region, but a diff. constant region thatn IgG)
Term
Describe the VARIABLE region of teh HEAVY chain.
Definition
Variable from H chain to H chain...N-terminal is 20-205% of H chian sequence.
Term
VL and VH possess WHAT two general features??
Definition
1) Complementary Determining Region(CDR)

2) Framework Regions
Term
What are CDR's???
Definition
Complementary Determining Regions: Located on variable regions of H&L chains. Here, variability is essentially LIMITED to specific regions composed of amino acid sequences whose chemical groups directly interact with an EPITOPE on an Ag molecule - hypervariable regions.
Term
What are CDR's???
Definition
Complementary Determining Regions: Located on VARIABLE regions of H&L chains. Here, variability is essentially LIMITED to specific regions composed of amino acid sequences whose chemical groups directly interact with an EPITOPE on an Ag molecule - hypervariable regions.

Basically....CDRs are the antigen-binding regions of antibodies.
Term
What are hypervariable regions?
Definition
CDRs
Term
How many CDRs are there on each variable region?
Definition
There are THREE CDRs on each VARIABLE region of both L and H chain.
Term
What are FRAMEWORK regions?
Definition
Relatively constant sequences w/in variable regions.
Important in aligning CRDs in correct spatial orientation w/in Ag-binding site (ABS).
Term
What is the main structural characteristic of Constant regions? CH and CL?
Definition
H chain constant regions and L chain constant regions contain "repeated" sequences of amino acids termed DOMAINS!!...forming a structural motif known as the.....immunoglobulin-fold (Ig-fold).
Term
How are the VL & VH similar to the constant regions?
Definition
REMEMBER: The VL & VH sharea similar Ig-fold structural motif.
Term
Describe the domains that are present among the different isotypes.
Definition
IgG,IgA & IgD have 3 CH domains(CH1,CH2,CH3).

IgE & IgM have 4 CH domains (CH1,CH2,CH3,CH4)
Term
What makes up the antigen binding site for each Fab?
Definition
VH and VL domains pair to form the Ab-binding site.
Term
How do the domains pair in a simple IgG antibody
Definition
CH1 - CL

CH2 - CH2
CH3 - CH3
CH4 - CH4 in IgE & IgM
Term
What is the Hinge region?
Definition
Fab fragments ("arms") of some Ig isotypes exhibit free range of movement about the Fc fragment("base") of the molecule... appear HINGED

Connects the Fab arms to Fc
Term
Which isotypes have SUB isotypes?
Definition
IgG and IgA
Term
Which isotypes have a hinge region?
Definition
each IgG subisotype, IgS subisotype, adn IgD
Term
Which Isotypes do not have a hinge region?
Definition
IgE and IgM
Term
Does the 3-dimensional structure of Ig's change between isotypes?
Definition
No, the overall 3D structure of an Ig monomer is fairly consistent among different isotypes.
Term
What provides variability to the ABS?
Definition
CDRs w/in V-regions of both H & L chains.
Term
Describe the 3D regions of a typical Ig.
Definition
A typical Ig has 3 globular regions...2 Fab and 1 Fc.
Term
Describe the Fab of a normal Ig.
Definition
It contains the: ABS (VL-VH) and CL-Ch pairing.

The variable regions of H and L chain combine to form a deep cleft lined w/ amino acids of CDRs to compose teh ABS.
Term
What pairs together in the Fc?
Definition
CH2 - CH2 via carbohydrate I think.
CH3 - CH3
CH4 - CH4 (in IgE and IgM)
Term
Which Ig Isotype is the most abundant in the serum?
Definition
IgG = 80% of total serum immunoglobulin.
Term
What does an IgG isotype consist of?
Definition
2 γ HEAVY chains
2 λ or κ LIGHT chains
Term
How many subisotypes are there for IgG, and why are they numbered as so?
Definition
FOUR IgG sub-classes.

Distinguished by differences in gamma-chain constant region sequenced .

They are numbered according to their decreasing average serum concentrations. i.e. IgG1 in in the highest concentration in Serum.

Vary based on size of hinge region and the no. and position of the interchain disulfide bond betweern heavy chains.
Term
Why are the subtle differences in amino acid sequences between IgG sub-isotypes important?
Definition
They affect the biological activity of the molecule.
Term
Describe the biological activity of IgG1, 3, and 4.
Definition
They readily cross teh placenta and play an important role in protecting the developing fetus.
Term
Describe the role of IgG sub-types in terms of activating the complement system.
Definition
IgG-3 is the most effective complement activator, followed by IgG-1.

IgG-2 is less efficient, and IgG-4 is not able to activate complement at all!

Complement activator: IgG 3 > 1 > 2 > 4
Term
Describe the role of IgG sub-types towards opsonization.
Definition
IgG-1 & 3 bing with HIGH affinity to Fc receptors on PHAGOCYTIC cells and this promote opsonization.

IgG-4 has an intermediate affinity for Fc receptors.

IgG-2 has an extremely low affinity.

Opsonin: IgG 1=3 > 4 > 2
Term
I what 2 forms are IgG's found?
Definition
Membrane and Serum form.
Term
Where are IgG's found when in MEMBRANE form?

What are their functions?
Definition
They are found present on some MEMORY B CELLS. An Additional transmembrane domain anchors into the membrane.

Functions as an Ag-component of BCR.
Term
What are the functions of IgG's while in SERUM form?
Definition
1) They are able to move in&out of serum and tissues and can cross placenta providing protection for infant early in life.

2) Major effector molecule (Ab) of LATE PRIMARY AND SECONDARY HUMORAL responses.

3) Major OPSONINS

4) Neutralize Ag (i.e. toxins)

5) Activate (fix) complement system, primarily IgG1 and 3.
Term
What does each monoer of IgM consist of?
Definition
2 LIGHT chains (κ or λ)
2 HEAVY chains (μ)
Term
Which IgG is the 3rd highest in Ig concentration in the human SERUM?
Definition
IgM
Term
What are the two forms that an IgM can be found in?
Definition
Serum form as a PENTAMER

Membrane form as a MONOMERIC IgM
Term
How are the 5 monomers held together in the IgM pentamer?
Definition
Held by interchain DISULFIDE BONDS (S-S).

One end of the chain is attached to the other by a J-chain polypeptide - Forming a circle. Each side is S-S bonded to the J chain.
Term
How many ABS's are present PER monomer of an Ig generally?
Definition
2
Term
How many Antigen molecules deos a typical PENTAMER IgM bind to? and WHY?
Definition
The pentamer binds to 5-10 Ag, not: 2x5=10...

because of STEARIC hindrance.
Term
What are the main functions of IgM?
Definition
1.) 1st Ab produced during teh primary humoral response.
2.) Early neutralization and agglutination of Antigen.
3.) Very efficient at activating ("fixing") complement
Term
What is a benefit of the large size of the pentamer IgM?
Definition
Its large size limits its movement out of serum but can undergo MUCOSAL TRANSPORT.
Term
In what form is the IgM when it is a pentamer?
Definition
Serum form....

Pentamer is in serum!
Term
In what form is an IgM when it is membrane-bound?

Describe IgM in its membrane form.
Definition
Monomeric form.

Possesses additional transmembrane region arrached to Cμ4 domain (on heavy chain) that anchors it in membrane of NAIVE mature B cells.
Term
What is the 2nd most abundant isotype in human Serum??
Definition
IgA
Term
What are the 2 sub-isotypes of IgA??

What chains are IgA's composed of?
Definition
IgA1 and IgA2

Each IgA monomer compoesed of 2 light chains (κ or λ) and 2 heavy chains (α1 or α2)
Term
What forms are IgA's found in?
Definition
1: membrane form (as a monomer)
2: serum form (mostly as a monomer, but sometimes as a dimer or tetramer)
3: SECRETORY form (as a DIMER: 2IgA monomers joined by S-Sbonding to a J-chain, same as J-chain from IgM pentamer)
Term
Where are membraneous IgA's found?
Definition
on SOME memory B cells as part of the BCR.
Term
Explain the process of how IgA's are secreted?
Definition
Secreted by plasma cells as a DIMER, in submucosa. They bind to the BASAL side of the mucosal epithelial cell, to a POLY-Ig RECEPTOR. Dimeric IgA:poly-IgReceptor binding promotes endocytic transport to lumenal side of epithelial cell where poly-Ig receptor is cleaved at its base.

Part stays w/IgA and forms the SECRETORY COMPONENT of secretory IgA(SIgA).
Term
What components make up the secretory IgA (SIgA)?
Definition
Part of the poly-Ig receptor from the basal side of the mucosal epithelial cell, and the whole dimeric IgA.
Term
Extra facts about secretion of Ig's
Definition
- The endocytosis involved in transporting IgA from the plasma cell is RECEPTOR-MEDIATED endocytosis.
- The poly-Ig receptor is enzymatically cleaved adn becomes the secretory component.
- The Secretory IgA is released into the mucous secretions.
- The secretory component masks sites susceptible to protease cleaveage in the hinge region of secretory IgA, allowing the polymeric molecule to live linger in the protease-rich mucous.
Term
Extra facts about secretion of Ig's
Definition
- Pentameric IgM is also transported into mucous secretions by the same mechanism as IgA.
- However, secretion of IgM accounts for a much lower % of Ab in the mucous secretions than does IgA.
- The poly-Ig receptor interacts with the J-chain of both polymeric IgA and IgM antibodies.
Term
What is the purpose of the secretory component of secreted IgA??
Definition
To mask the site that are vulnerable to protease cleavage in the hinge region of secretory IgA. This allows the poymeric IgA to exist longer in the protease-rich environment of the mucous.
Term
What are the main functions of IgA's?
Definition
1) Major Isotype in bodily secretions-mucis, tears, saliva, breast milk, GI.
2) Blocks adherance of microbes to receptors on host cell.
3) agglutinates microbes facilitating their shedding w/in mucus.
4) In BREAST MILK, provides important GI IMMUNITY FOR INFANT.
Term
Which two isotypes are found in very low concentrations in Human Serum?
Definition
IgE and IgD
Term
What chains comprise the IgE isotype?
Definition
2 light chains (kappa & lambda)
2 heavy chains (epsilon)

possesses a 4th CH domain!!
Term
How many forms is IgE found?
Definition
2: membrane and serum form
Term
What is special about IgE in its serum form?
Definition
It is mainly found in tissues on MAST CELLS or BASOPHILS, rather than free in serum. Explains why IgE has such low concentrations in the serum.
Term
What are the main functions of IgE??
Definition
1) defense against certani prasites...URT & GI immunity.

2) Key role in type I hypersensitivities (ALERGIES)...UNIQUE sites in Fc region BIND STRONGLY to HIGH AFFINITY RECEPTORS ON MAST CELLS AND BASOPHILS.
Term
What chains is IgD composed of ?
Definition
2 light (kappa or lambda) chains
2 heavy (DELTA) chains
Term
Why is IgD susceptibleto protase digestion??
Definition
Because it has a hinge region like IgG and IgA. IgA when secreted has secretory component which prevents digestion though.
Term
What is special about IgD in its membrane form??
Definition
It is a monomer found on the membranes of MATURE B CELLS----

It acts as an Ag receptor w/ mIgM on NAIVE MATURE Ag-reactive B CELLs.

Remember: IgM is the 1st Ab produced during the primary HUMORAL response!!
Term
Isotypes reflect differences between _________.
Definition
Species.

ISOTYPES ARE COMMON TO ALL MEMBERS OF A SPECIES.
Term
Where are isotype epitopes located on an Ig and what are they called?
Definition
Located within teh CONSTANT,HEAVY... and CONSTANT, LIGHT regions and are called ISOTYPIC DETERMINANTS.
Term
What are subisotype?
Definition
They are encoded by different genes...reflect gene amplifications and mutations,...create additional epitopes associated with CH or CL domains.

Common to all members of a particular species.
Term
Allotypes reflect differences between ________.
Definition
Members of the same species.
Term
What is an allotype?
Definition
A group of potential epitopes that are located w/in the CH or CL (allotypic determinants).

They are expressed in some but not all individuals of the SAME species.

Genetically determined (ALLELIC) VARIATIONS in genes encoding C regions of L&H chains.

Different allotypic determinants represent differences in 1-4 amino acids that are encoded by different alleles of the same gene.
Term
What happens when you inject the antibodies of 1 person into another member of the SAME SPECIES?
Definition
Antibody to allotypic determinants can be made because person B carries different allotypic determinants than person A.
Term
People have 2 copies of each of their genes, leading to.......
Definition
HOMOzygous individuals - single allotype for each Ig isotype or sub-isotype

HETEROzygous individuals - 2 possible allotypes for each Ib(sub)Isotype; so theoretically, 1/2 of any Ig (sub)isotype will be of 1 allotype and 1/2 will be of the other.
Term
What are idiotypes?
Definition
Differences between epitope-specific Ig within the same individual producing them. All idiotypes are the same within the SAME IG...but different when you switch to another IG that is still in the same person.
Term
Idiotypes are immunogenic within the _____ the Ig.
Definition
person
Term
Where are idiotypes located?
Definition
They are unique amino acid sequences of the VARIABLE heavy and light domains of a given antibody.

They can be associated with ABS sometimes combining to be the actual antigen binding site. Some comprise variable-region sequences that are OUTSIDE of the binding site.
Term
Remember this about idiotypes:
Definition
B/c the antibodies produced by individual B cells derived from the same clone have identical variable-region sequences, they all have the SAME IDIOTYPE.
Term
Explain what the Ig SUPERFAMILY is?
Definition
Ig H & L chains have structural features found in several other membrane proteins that play some role in the immune responses suggesting they evolved form a common ancestral gene.
Term
What are some common feature that are shared among the IG superfamily?
Definition
1) They have 1 or more Ig-FOLD STRUCTURAL DOMAINS.
2) Proteins that posses Ig-fold domains are members of the Ig superfamily eventhough they have diff. functions and are not genetically linked.
Term
What members are included in the Ig Superfamily??
Definition
Ig-Heavy Chain
Ig-Light Chain
Ig-alpha/Ig-beta of BCR
TCR
CD4
CD8
CD3 subunits
MHC I
MHC II
poly-Ig Receptor
Other cell adhesion molecules (VCAM-1, ICAM-1 /-2)
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