Lectures 5 - Antibody Structure and Function Flashcards
Resting B cell contains _____ on its membrane
Ig (acts as receptor for bacterial antigen)
Epitopes, a.k.a. ________, are part of what molecules?
Antigenic Determinants
Part of antigen (that antibody binds to)
Most antigens have ___ epitopes, which makes them _______
multiple epitopes
Multivalent
Epitopes are usually what types of molecules?
CHO or peptides
Vaccine antigens molecule-type examples?
- bacterial proteins & polysaccharides
- virus like particles
- whole bacteria
Epitopes recognized by antibodies are usually located…
on the antigens surface
Antibodies (& B cells) can bind to ______ epitopes.
T cells can bind to ______ epitopes
- B cells = Linear or discontinuous
- T cells = Linear Only
Haptens are small molecules that _______, but can bind to __________.
Not immunogenic themselves, but can bind to Ig’s or TCR’s
Haptens can induce immune response when…
Linked to a larger protein carrier
Example of clinical hapten
Penicillin
Penicillin two mechanisms
- Binds to bacterial transpeptidase and inactivates it
- Modifies proteins on human RBC to create foreign epitopes
B cells are activated by _____ and ______
antigen and activated TH2 cells
Plasma cells secrete ___________ which binds to _________
Penicillin-specific IgG which binds to modified RBCs
Chains of Ig molecule:
2 Light chains (lambda and kappa)
5 Heavy chains (mu, gamma, alpha, epsilon, delta)
Ig domains are linked by
disulfide bonds
The antigen binding part of the Ig molecule is the _____ terminus
N terminus
Ig variable region, a.k.a…
Antigen binding site
Effect of papain on Ig molecule
Proteolytically cleaves into 2 FAB and 1 FC domains
Fab domain aka…
antigen binding fragment
Fc domain aka
crystallizable fragment
proteolytic cleavage of Ig by pepsin does what?
Creates F(ab’)2 domain and multiple fragments of the constant domain
Disulfide bonds of Ig molecule are present at ….
hinge region
Fragments (names) that comprise the antigen binding sites of Ig molecule
Vh + Ch1
CL + VL
Domains (names) that make up the constant part of the Ig molecules
CH2 and CH3
Fab structure is comprised of many passes of ________ joined by ______
Beta strands
joined by loops
Hypervariable region = CDR = ___________
complementarity-determining region
L chain is made of ______ or ____
Kappa or Lambda
Ig isotypes are the same thing as _____. Examples?
Classes
M, D, G, E, A
Ig Isotypes, allotypes, and Idiotypes differ in their….
Isotypes = differ in heavy chain (Mu, Gamma, Delta, Epsilon, Alpha)
Allotypes may have variations in amino acids in constant regions of heavy or light chain.
Idiotypes differ in hypervariable region. Recognize different epitopes.
Allotypes’ differences may affect…
- half life
- subclass distribution
Allotype genetics display what three things?
- mendelian inheritance (autosomal dominant)
- Variations among ethnic groups
- associations with infectious diseases and AI diseases
___ and ___ domains of Ig interact with antigen
Vh and VL
Antigen and antibody interactions type
Non-covalent binding (Electrostatic, H bonds, VDWF, Hydrophobic)
Affinity definition
Strength of interaction between epitope and one antigen binding site
Noncovalent Ag-Ab binding forces are ____ forces
Short range forces
Avidity definition
Sum of interactions between antibody and antigen
Crossreactivity can occur between ________
Example?
antigens that share epitopes
Influenza (H2N2, H2N3)
Two steps to make MABs
- Immortalization of a single clone of antibody-secreting cells
- Fusion of B cells with neoplastic plasma (myeloma) cells
In making MAB’s, after growing in drug-containing medium, only __________ survive.
What do you then select for?
hybrid cells
Select for antigen-specific hybridoma and clone them
Polyclonal vs monoclonal origin
Poly = purified from serum of immunized animals (goats/rabbits)
Mono = Made from immortalized plasma cells (mouse origin)
Polyclonal vs Monoclonal specificity/affinity
Poly = multiple, Mono = single
Polyclonal vs Monoclonal supply
Poly varies from batch to batch
Mono gives an unlimited supply of identical molecules
-ximab suffix
Chimeric
-zumab suffix
humanized
-umab suffix
human
Four types of therapeutic MAB’s
Mouse, chimeric, humanized, human
Psoriasis MAB name and target
infliximab
anti-TNF
7 types of immunoassays
Precipitation, Agglutination, ELISA, RIA, Western blot, Immunofluorescence
Most - to - least sensitive immunoassay (3)
Precipitation (least)
Agglutination
RIA and ELISA
Aggretates are formed between the interaction of ______ and ________.
What does the precipitation curve look like between these two things?
multivalent antibodies and macromolecular antigens
If antibody or antigen in excess, there is a lower precipitation rate (Precipitation peaks when they are equal)
Antigens can have several _______ that bind to antibodies.
This is separate from the __________, which tells us how many antibodies will bind
epitopes
antigen valence
____ can form between RBC and antibodies. This is called _______
Immune complexes
Hemagglutination
A ___ type RBC will show no agglutination with any antibody molecule
Type O
Hemagglutination occurs with what type of Antibody?
IgM
What is Coomb’s reagent?
What is its purpose?
Anti-human immunoglobulin antibodies
Add it to RBC cells to test for agglutination (will occur if the RBC has antibodies on its coat)
With a direct Coomb’s test, you must add a _________ to induce agglutination
secondary reagent
Why can’t IgG’s complex in a coomb’s test?
The negative surface charge would prevent complexing with an RBC
Describe the three steps of the indirect Coomb’s test
- Incubate test serum with RBC
- Wash RBC
- Add anti-human Ig antibodies
Direct coomb’s test is done _____, and indirect coomb’s is done _______
Direct = In vivo
Indirect = in vitro
Rhesus factor is a problem when mom/baby are…
When mom is Rh-, and baby is Rh+
Mom makes antibodies against baby’s Positive rhesus factor
Direct vs indirect Coomb’s in Rh+/- situation
Direct = add antibodies to washed RBC
Indirect = Add RBC’s to maternal serum, then wash away unbound antibody
4 steps in ELISA
- Coat wells with antigen
- Add serum sample
- Add enzyme-labeled anti-human IgG (labeled with color-producing enzyme)
- Add substrate (for the enzyme)
5 steps of Western blot (HIV example)
- Dissociate the virus in SDS
- Run particles on SDS-PAGE gel
- Transfer to **Nitrocellulose **
- Overlay with antiserum
- Use enzyme-linked anti-IgG to detect the bound antibody
Immunofluorescence works because the ______ is linked to ______.
Antibody is linked to a fluorochrome that emits light
Briefly sum up Flow cytometry process.
What does this allow us to see?
- A stream of fluid drops containing antibody-labeled cells are detected by a “laser”
- It allows us to quantify populations of cells based on which antibody (and fluroescence molecule) is bound to its surface
Example of use of flow cytommetry (for cell populations)
CD4 and CD8 T cells (expression of one, both, or neither will differentiate)
CD3 & CD19 B cells (++, +/-, –)
AIDS asymptomatic phase begins with what event?
Seroconversion
What are the segments in heavy and light chain loci?
V = Variable
J = Joining
C = Constant
D = Diversity
The first two gene segments of the light chain to join are the _____ and __ segments. This is called _______.
This process takes place at the level of _______.
V, J
Somatic recombination
DNA
How is the light chain C region joined to the V(J) region exon?
RNA splicing gets rid of the introns between L/V and J/C, creating a
The light chain contains what gene segments?
VL and CL
Heavy chain contains what segments?
VH, CH1, CH2, CH3
Heavy chain C segments are joined by _____
RNA splicing
Heavy chain contains _____, (not present in the light chain)
Diversity (D) segment
Which segments are the first to join in the heavy chain?
What is this process?
D/J join first
Somatic recombination (of DNA)
(V–>DJ happens in second step)
When does somatic recombination occur?
When the cell differentiates into a B cell?
Which gene segment has the greatest amount of variability in the human genome?
Variable (V) segments
L, K, and Heavy chain genes contain _____ and ______ sequences, which allow for specificity by…
- Recombination Signal Sequences (RSS)
- Spacer Sequences
….allow for the spacers to only recombine 12 & 23
For spacer sequences in the heavy chain, ___ can only be combined to ___, not ___.
This happens d/t ________ sequences located between the V and J segments
12+23
23 can’t bind 23
Recombination is mediated by _______.
_____ is part of this enzyme complex
V(D)J Recombinase
RAG
RAG binds to _____, and facilitates excision of a DNA segment, creating a _____and a ______
RSS sequences
Creates a Signal Joint and a Coding joint
RAG cleaves a ______ from the D and J segments to make ______
RAG forms a _________ by nicking one of the sequences of DNA
heptamer (RSSs), DNA hairpins
pallindromic P nucleotides
Function of TdT in junctional diversity?
it adds N-nucleotides in a random manner
What happens after the D and J strands are paired during junctional diversity?
What is the function of this?
Exonuclease cleaves out unpaired nucleotides
This generates further diversity
Equation for calculation of diversity of a chain? (K, L, or Heavy chains)
V x #J ** = #Vκ**
For light chain (e.g. Kappa)
For Heavy chain:
This shows us that D segments yield a huge amount of diversity
Two other factors (other than the VDJ variants) that contribute to increased diversity?
- Imprecise joining
- Addition of N nucleotides by TdT
(E.g. heavy chains) VDJ is the ___ domain, while rearrangement of many C gene segments makes up the ____ domain
VDJ = Variable
C = Heavy chain
Heavy chain gene rearrangement (C segments) determine…
the isotype of the Ig molecule
Naive B cells co-express ____ and ____.
This is due to ___________
IgM and IgD
RNA splicing (NOT DNA REARRANGEMENTS)
Number of Constant (C) and VDJ domains in IgM and IgD heavy chains
IgM = 4 C regions
IgD = 3 C regions
Both have one VDJ region
The B cell receptor is composed of what (three) things?
Ig molecule
Igß and Ig@
