Lectures 5 - Antibody Structure and Function Flashcards
(91 cards)
1
Q
Resting B cell contains _____ on its membrane
A
Ig (acts as receptor for bacterial antigen)
2
Q
Epitopes, a.k.a. ________, are part of what molecules?
A
Antigenic Determinants
Part of antigen (that antibody binds to)
3
Q
Most antigens have ___ epitopes, which makes them _______
A
multiple epitopes
Multivalent
4
Q
Epitopes are usually what types of molecules?
A
CHO or peptides
5
Q
Vaccine antigens molecule-type examples?
A
- bacterial proteins & polysaccharides
- virus like particles
- whole bacteria
6
Q
Epitopes recognized by antibodies are usually located…
A
on the antigens surface
7
Q
Antibodies (& B cells) can bind to ______ epitopes.
T cells can bind to ______ epitopes
A
- B cells = Linear or discontinuous
- T cells = Linear Only
8
Q
Haptens are small molecules that _______, but can bind to __________.
A
Not immunogenic themselves, but can bind to Ig’s or TCR’s
9
Q
Haptens can induce immune response when…
A
Linked to a larger protein carrier
10
Q
Example of clinical hapten
A
Penicillin
11
Q
Penicillin two mechanisms
A
- Binds to bacterial transpeptidase and inactivates it
- Modifies proteins on human RBC to create foreign epitopes
12
Q
B cells are activated by _____ and ______
A
antigen and activated TH2 cells
13
Q
Plasma cells secrete ___________ which binds to _________
A
Penicillin-specific IgG which binds to modified RBCs
14
Q
Chains of Ig molecule:
A
2 Light chains (lambda and kappa)
5 Heavy chains (mu, gamma, alpha, epsilon, delta)
15
Q
Ig domains are linked by
A
disulfide bonds
16
Q
The antigen binding part of the Ig molecule is the _____ terminus
A
N terminus
17
Q
Ig variable region, a.k.a…
A
Antigen binding site
18
Q
Effect of papain on Ig molecule
A
Proteolytically cleaves into 2 FAB and 1 FC domains
19
Q
Fab domain aka…
A
antigen binding fragment
20
Q
Fc domain aka
A
crystallizable fragment
21
Q
proteolytic cleavage of Ig by pepsin does what?
A
Creates F(ab’)2 domain and multiple fragments of the constant domain
22
Q
Disulfide bonds of Ig molecule are present at ….
A
hinge region
23
Q
Fragments (names) that comprise the antigen binding sites of Ig molecule
A
Vh + Ch1
CL + VL
24
Q
Domains (names) that make up the constant part of the Ig molecules
A
CH2 and CH3
25
Fab structure is comprised of many passes of ________ joined by \_\_\_\_\_\_
Beta strands
joined by loops
26
Hypervariable region = CDR = \_\_\_\_\_\_\_\_\_\_\_
complementarity-determining region
27
L chain is made of ______ or \_\_\_\_
Kappa or Lambda
28
Ig isotypes are the same thing as \_\_\_\_\_. Examples?
Classes
M, D, G, E, A
29
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.
30
Allotypes' differences may affect...
* half life
* subclass distribution
31
Allotype genetics display what three things?
1. **mendelian inheritance** (autosomal dominant)
2. Variations among **ethnic** groups
3. associations with **infectious diseases** and AI diseases
32
\_\_\_ and ___ domains of Ig interact with antigen
Vh and VL
33
Antigen and antibody interactions type
Non-covalent binding (Electrostatic, H bonds, VDWF, Hydrophobic)
34
Affinity definition
Strength of interaction between epitope and **one antigen binding site**
35
Noncovalent Ag-Ab binding forces are ____ forces
Short range forces
36
Avidity definition
**Sum of interactions** between antibody and antigen
37
Crossreactivity can occur between \_\_\_\_\_\_\_\_
Example?
_antigens that share epitopes_
_Influenza_ (H2N2, H2N3)
38
Two steps to make MABs
* **Immortalization** of a **single clone** of antibody-secreting cells
* **Fusion** of B cells with neoplastic plasma (**myeloma**) cells
39
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**
40
Polyclonal vs monoclonal origin
Poly = purified from serum of immunized animals (goats/rabbits)
Mono = Made from immortalized plasma cells (mouse origin)
41
Polyclonal vs Monoclonal specificity/affinity
Poly = multiple, Mono = single
42
Polyclonal vs Monoclonal supply
Poly varies from batch to batch
Mono gives an unlimited supply of identical molecules
43
-ximab suffix
Chimeric
44
-zumab suffix
humanized
45
-umab suffix
human
46
Four types of therapeutic MAB's
Mouse, chimeric, humanized, human
47
Psoriasis MAB name and target
infliximab
anti-TNF
48
7 types of immunoassays
Precipitation, Agglutination, ELISA, RIA, Western blot, Immunofluorescence
49
Most - to - least sensitive immunoassay (3)
Precipitation (least)
Agglutination
RIA and ELISA
50
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**)
51
Antigens can have several _______ that bind to antibodies.
This is separate from the \_\_\_\_\_\_\_\_\_\_, which tells us how many antibodies will bind
epitopes
antigen valence
52
\_\_\_\_ can form between RBC and antibodies. This is called \_\_\_\_\_\_\_
Immune complexes
Hemagglutination
53
A ___ type RBC will show no agglutination with any antibody molecule
Type O
54
Hemagglutination occurs with what type of Antibody?
IgM
55
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)
56
With a direct Coomb's test, you must add a _________ to induce agglutination
secondary reagent
57
Why can't IgG's complex in a coomb's test?
The negative surface charge would prevent complexing with an RBC
58
Describe the three steps of the _indirect Coomb's_ _test_
1. Incubate test serum with RBC
2. Wash RBC
3. Add anti-human Ig antibodies
59
Direct coomb's test is done \_\_\_\_\_, and indirect coomb's is done \_\_\_\_\_\_\_
Direct = In vivo
Indirect = in vitro
60
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
61
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
62
4 steps in ELISA
1. Coat wells with **antigen**
2. Add **serum** sample
3. Add enzyme-labeled anti-human **IgG** (labeled with color-producing enzyme)
4. Add **substrate** (for the enzyme)
63
5 steps of Western blot (HIV example)
1. Dissociate the virus in **SDS**
2. Run particles on **SDS-PAGE** gel
3. Transfer to **Nitrocellulose **
4. Overlay with **antiserum**
5. Use **enzyme-linked anti-IgG** to detect the bound antibody
64
Immunofluorescence works because the ______ is linked to \_\_\_\_\_\_.
**Antibody** is linked to a **fluorochrome** that emits light
65
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
66
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** (++, +/-, --)
67
AIDS asymptomatic phase begins with what event?
Seroconversion
68
What are the segments in heavy and light chain loci?
V = Variable
J = Joining
C = Constant
D = Diversity
69
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
70
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
71
The light chain contains what gene segments?
VL and CL
72
Heavy chain contains what segments?
VH, CH1, CH2, CH3
73
Heavy chain C segments are joined by \_\_\_\_\_
RNA splicing
74
Heavy chain contains \_\_\_\_\_, (not present in the light chain)
Diversity (D) segment
75
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)
76
When does somatic recombination occur?
When the cell differentiates into a B cell?
77
Which gene segment has the greatest amount of variability in the human genome?
**Variable (V)** segments
78
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**
79
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
80
Recombination is mediated by \_\_\_\_\_\_\_.
\_\_\_\_\_ is part of this enzyme complex
**V(D)J Recombinase**
**RAG**
81
RAG binds to \_\_\_\_\_, and facilitates excision of a DNA segment, creating a \_\_\_\_\_and a \_\_\_\_\_\_
**RSS sequences**
Creates a **Signal Joint** and a **Coding joint**
82
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
83
Function of TdT in junctional diversity?
it adds **N-nucleotides** in a random manner
84
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
85
Equation for calculation of diversity of a chain? (K, L, or Heavy chains)
For light chain (e.g. Kappa)
#**V** x #**J ** = #**Vκ**
For Heavy chain:
#**V x** #**J **x #**D** = **#VH**
This shows us that D segments yield a huge amount of diversity
86
Two other factors (other than the VDJ variants) that contribute to increased diversity?
* Imprecise joining
* Addition of N nucleotides by TdT
87
(E.g. heavy chains) VDJ is the ___ domain, while rearrangement of many C gene segments makes up the ____ domain
VDJ = Variable
C = Heavy chain
88
Heavy chain gene rearrangement (C segments) determine...
the isotype of the Ig molecule
89
Naive B cells co-express ____ and \_\_\_\_.
This is due to \_\_\_\_\_\_\_\_\_\_\_
**IgM** and **IgD**
**RNA splicing** (NOT DNA REARRANGEMENTS)
90
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
91
The B cell receptor is composed of what (three) things?
Ig molecule
Igß and Ig@
