Feb. 14th (Exam 2) Flashcards
How are the different isotypes of immunoglobulins characterized?
By their constant region
What are the three things that vary between the isotypes?
- Hinge region
- Heavy chain length
- Carb binding
What are the 5 different classes of immunoglobulins as defined by the isotypes of the heavy chains?
- IgG (γ)
- IgM (μ)
- IgD (δ)
- IgA (α)
- IgE (ε)
What are the two light chain isotypes?
- Kamma (κ)
- Lambda (λ)
What are the functional differences between the two light chain isotypes?
Can one Ig have both types of light chains?
There has not been any functional difference found, and all the isotypes of heavy chains will bind to either light chain isotype.
Each Ig can only have one type of light chain isotype.
What is an immunoglobulin domain?
This is a single stable motif of about 100-110 amino acids
*on either the light and heavy chains that folds up into a compact and exceptionally stable protein domain.
What is the variable domain?
This is the domain that is present at the amino terminal end of each light or heavy chain
What is the antigen binding site created by?
- Variable Heavy domain
- Variable Light domain
What is the constant domain of the light chain?
This is always a single domain per light chain
Describe the constant domains of the various heavy chains.
What isotypes have three constant domains?
Four?
Can consist of 3 or 4 constant domains depending on the isotype.
Three constant domains: IgG, IgD, IgA
Four constant domains: IgM, IgE
Describe a single immunological domain and how it is held together generally.
Resembles a bulging sandwich of two Beta sheets that are held together by strong hydrophobic interactions of their side chains as well as a disulfide bond.
Adjacent strands within the beta sheet are connected by loops.
What are the hypervariable regions?
These are the regions of the variable regions that have extreme amino acid variability.
What is another name for the hypervariable regions regarding the binding that will happen to an epitope?
How many are there for each domain?
What type of protein structure do they always display?
We call these areas the Complementary Determining Regions (CDRs)
There are three per variable domain.
Loops
What is the rest of the variable domain that does not display such amino acid variability called?
Framework regions
What is an epitope?
What are epitopes usually made of?
This is the part of the antigen to which the antibody binds.
Carbohydrate or protein.
What is it called when an antigen contains more than one type of epitope or more than one of the same epitope?
When this is the case, we call it multivalent.
What are the two types of protein epitopes? Describe them.
Linear - these contain successive amino acids in a sequence
Discontinuous - these have amino acids that are brought together through complex folding
What type of chemical interactions are utilized in antibody-antigen interaction?
All non-covalent
- electrostatic forces
- hydrogen bonds
- van der Waals forces
- hydrophobic interactions
Define affinity as it relates to antigen-antigen binding site interaction.
Affinity is the binding strength of one epitope to an antigen binding site (there are two per antibody)
Why would two different antibodies that recognize the same epitope have different affinities?
Because there are small differences in the shapes and chemical properties of the antibodies.
What is avidity?
In what classes of antibodies is this term relevant?
This is the overall binding strength of multiple epitopes of an antigen
IgM pentametric 10 antigen binding sites
IgA 4 antigen binding sites
Explain, in detail, how monoclonal antibodies are made, as well as what they are.
In the explanation define:
- Myeloma cell
- Monoclonal antibodies
B-cells from an animal that is immunized with a particular antigen, usually a mouse, are isolated and fused with a myeloma cell.
Myeloma cell: cancerous plasma cell
The many different hybrid cells are then grown in a drug containing medium to kill off B-cells/Myeloma cells that did not fuse.
Then the individual hybrid cells are separated and the fused cells that are making the desired antibodies are identified and selected for future propagation.
Monoclonal Antibody: the antibodies that are made from one hybrid cell line - these are identical antibodies.
How does flow cytometry work?
Explain how to read the quadrant graph.
In the process of flow cytometry, we tag specific antibodies that are made in mice, specifically for human cell surface receptors, with fluorescence and are passed through the cytometer in droplets so they are singled out.
As they pass through a laser shoots at the cell, emitting a certain wavelengths.
The quadrant graph shows data collected for each individual cell.
Horizontal axis is for one type of cell receptor and how the various cells bound to the antibody that was tagged, either not at all (bottom left) or a lot (bottom right).
The vertical axis is for the other type of cell receptor and how the various cells bound to the antibody that was tagged, either a lot (top left) or not at all (top right).
Describe a chimeric monoclonal antibody.
This is the C region of a human antibody fused with the V region of mice antibodies.
Describe a humanized monoclonal antibody.
This is a human antibody that has been genetically engineered to have specific parts of the mice’s sequences be incorporated into the light and heavy chains of the V region in a human antibody
What are gene segments?
These are fragments of genes across a chromosome that must first come together to be expressed instead of a single complete gene.
What do all the cells in the body have in reference to the gene fragments that make up different parts of an antibody?
What is unique in this regard about B cells?
- Regions of a scattered gene that code for the heavy chain
- Regions of a scattered gene that code for the light chain.
Only B cells have the ability to rearrange and assemble the functional Ig gene.
When and where does this rearrangement by the B cells occur?
Immunoglobulin-gene rearrangements occur during the development of B-cells from the B cell precursors in the bone marrow
What is the germline form / germline configuration?
The general immunoglobulin families of gene segments that are inherited in the scattered form.
Where are all the immunoglobulin genes?
- Heavy Chain locus (all varieties) - chromosome 14
- Kamma (κ) light chain locus - chromosome 2
- Lambda (λ) light chain locus - chromosome 22
How would you describe the segments that encode the various regions within the two light chain loci and the heavy chain locus?
They are distinct, and different segments encode the leader peptide (L) the variable region (V) and the constant region (C).
How are the gene segments that encode the leader peptides (L) and the constant regions (C) of the light and heavy chains similar to that of other human genes?
They have introns and exons and are ready to be transcribed, spliced and translated.
How are the variable regions (V) different than that of other human genes?
The variable regions are either composed of 2 V segments in the light chain OR 3 V segments in the heavy chain that must be selected from arrays of similar gene segments and rearranged to produce an exon that can be transcribed.
What are the two V segments in the light chain variable region (V)?
- variable (V) gene segment
- joining (J) gene segment
What are the three V segments in the heavy chain variable region (V)?
- variable (V) gene segment
- diversity (D) gene segment
- joining (J) gene segment
What is somatic recombination?
This is the process of bringing gene segments together during cell development.
Describe the basic outcome of somatic recombination that happens during B cell development, for both light and heavy variable regions (V).
How many recombinations for each chain?
During B-cell development, one gene segment from each type - (V and J) for - the variable region of the light chain and (V, D, J) for the variable region of the heavy chain.
The light chain only requires one recombination.
The heavy chain requires two recombinations.
How does the recombination of the heavy chain variable chain work?
The J(h) and the D segments are joined.
Following this, the J(h)D segment is joined to the V(h)
What determines which gene segments, from either the light or the heavy chain parts of the variable regions of the immunoglobulin, are brought together?
*whether it be the V(L) J(L) or the V(H) D J(H)
They are selected randomly.
What does the random joining of segments provide us?
How many combinations of the light chain variable region gene segments are there?
How many combinations of the heavy chain variable region gene segments are there?
Total combinations?
diversity of the variable region of immunoglobulins!
295 for light chain
5520 for heavy chain
1.6 million total combinations for the antibodies!
What are recombination signal sequences? (RSS)
Where are they in relation to the V, J, and D segments?
These are sequences that exist adjacent to the segments involved in the recombination of gene segments for the variable region of the immunoglobulin.
V segments (both light and heavy) have RSSs that flank the 3’ end.
D segments have RSSs that flank both the 3’ and the 5’ ends.
J segments (both the light and the heavy) have RSSs that flank the 5’ end
What are the variety of enzymes that are able to recombine the V,J,D gene segments?
V(D)J recombinase.
What are the two components of V(D)J recombinase that are only made in lymphocytes?
What about the other components, what are they used for?
Recombination-activation genes
(RAG1 and RAG2)
The other parts are used in all cells for activities like double stranded repair, bending of DNA, and fixing the ends of DNA
What is the 12/23 rule?
What defines what type of RSS there is?
Two segments cannot be connected directly if the RSSs that flank them are the same type of RSS
i.e if both have a 12bp space between the heptamer and nonamer
the spacer defines the RSS
What does the RAG complex bind to?
What does this result in?
It binds to the opposite RSS.
It results in RAG cleaving the DNA making a circular DNA and a connected strand that stays within the parent DNA
What are the two connections of DNA (joints) that result from RAGs work?
- Coding Joint - this is the two segments that had opposite RSSs that were connected and stay in the parent DNA
- Signal Joint - this is the ends of the removed DNA that is connected.
Describe the first part of Junctional Diversity.
What are P nucleotides?
What are hairpins?
What is one source of diversity that is often overlooked?
This is the general idea that through the process of connecting two gene segments that have opposite RSSs we generate diversity through a few mechanisms.
When the RAG complex cleaves right at the heptamer (closest to the segment) leaving hairpins on the ends of the segments. These hairpins are then opened (cut from one of the single strands) by DNA repair enzymes, leaving what are called Palindromic nucleotides.
Palindromic nucleotides meaning their complementary base pairs are the same as the opened hairpins read backwards
The opening of the hairpins can occur at a few different locations, so this increases the diversity! (overlooked)
Continue with Junctional Diversity.
What happens when once the hairpins are opened (who knows where)
What removes nucleotides?
What adds nucleotides?
What are N nucleotides?
Once the hairpins are opened, their ends are modified.
Exonucleases can remove germline DNA
terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the ends of hairpins, generating even more diversity.
N nucleotides are nucleotides that are NOT encoded in the Germline DNA
Define Junctional Diversity.
How much does it increase the immunoglobulin diversity by?
It is the contribution of P and N nucleotides to coding joints (what stays in the DNA)
