Lecture #3 Flashcards
1
Q
Immunoglobulins can be _______ or ______ receptors
A
Membrane Bound or Soluble
2
Q
What causes a resting B cell to generate antibody?
A
Encounter with antigen turns it into a plasma cell
3
Q
What is an epitope?
A
An antigenic determinant The part of an antigen to which an antibody binds
4
Q
T or F. Most antigens have a single epitope.
A
False. Most antigens ahve multiple epitopes
5
Q
What are epitopes usually made of?
A
Carbohydrates of pepride
6
Q
Difference between DTwP and DTaP
A
DTwP = Whole Cell Pertussis VaccineDTaP = Acellular Pertussis Vaccine
7
Q
An immune response to _____ is triggered in an HPV vaccination.
A
Caspid
8
Q
Where are epitopes recognized by antibodies typically located?
A
At the antigen surface. There they can be directly bound
9
Q
Two different types of epitopes an Ab can bind to.
A
Linear and Discontinuous
10
Q
Difference between linear and confirmational/discontinuous epitopes.How is this different from T Cells
A
- Linear epitope binding happens to series of peptide in a row. - Discontinuous epitope binds to peptides adjacent to one another because they are disrupted and unfolded. - Ts Only Do Linear
11
Q
What lymphocytes recognize confirmational epitope?
A
B Cells.NO T Cells
12
Q
What lymphocytes recognize linear epitopes?
A
B Cells and T Cells
13
Q
What are Haptens?
A
Small molecules that are not immunogenic alone. They can bind Igs and TCRs.
14
Q
What do Haptens do?
A
They can induce immune responses when linked to a lager carrier.
15
Q
Example of a Hapten?
A
Penicillin
16
Q
Effect of Penicilin on bacteria
A
Binds bacterial transpeptidase and inactivates it
17
Q
Effect of Penicillin on RBC
A
Modifies RBC Proteins to generate a foreign epitope
18
Q
What happens to RBCs after they receive their hapten epitope?
A
B Cells are activated by antigen+TH2Ab binding to RBCsRBC destruction
19
Q
What holds together the light and heavy chains?
A
disulfide bonds
20
Q
Where can disulfide bonds be found?
A
Between light and heavy In the Hinge Region
21
Q
Names of the two light chains
A
Lambda and Kappa
22
Q
Name the five heavy chains
A
Mu, Gamma, Alpha, Epsilon, Delta
23
Q
The specificity of binding comes from the…
A
Antigen binding sites/Variable Region
24
Q
What do you find when Ab is cleaved by Papain?How many total fragments are generated?
A
Antigen-binding FragmentCrystallizable FragmentThree
25
What do you find when Ab is cleaved by pepsin?How many total fragments are generated?
F(ab')2 fragmentChopped up base2 Fragments
26
What function might F(ab')2 have?
Naturally, none. In labs, it can be used in binding studies.
27
Use of the hinge region?
Gives flexibility at antigen binding sites
28
Downside of the hinge region?
Susceptible to proteolysis by bacterial and host proteases
29
How many domains in heavy chain? Light Chain?What are they?
4. 3C, 1 V2. 1C, 1V
30
Significance of V and C Regions?
V=Epitope BindingC = Biological Fxn of Molecule
31
Describe the molecular structure of the Ab domains.
Two beta sheets held together by a disulfide bond"Sandwich with a Toothpick"
32
Another name for hypervariable region?
Complementarity-Determining Region
33
What do hypervariable regions do?
They provide the actual antigen binding specificity.
34
How many HV regions are in an antigen binding site?
6 (3 on light and 3 on heavy)
35
Are the HV regions adjacent genetically?
No, hydrophobic regions come together from different parts of the genome in the process of protein folding.
36
Name the 5 types of Immunoglobulins
Ig G, M, D, A, E
37
Which immunoglobulins have no Hinge region?What else is special about them?
IgM and IgEAlso have a 4th constant domain
38
Problems with the unique structure of IgM and IgE?
Less Flexibility of antigen binding.
39
What are isotypic differences between immunoglobulins?
Like IgG vs IgA
40
What are allotypic differences between immunoglobulins?
Variations of immunoglobulins seen because they come from the two different genes. Usually only very small differences.
41
What are idiotypic differences between immunoglobulins?
Like IgGs with different epitope binding sites
42
Why care about allotypical differences (3)
May alter half lifeMay Affect subclass distributionAssociated with susceptibility to infectious and autoim. disease
43
How are allotypes inherited?
Autosomal dominant
44
T or F. Binding strength can be very variable between different antibody-epitope pairs.
True
45
What forces drive the antibody-antigen interaction?
Non-Covalent:-Electrostatic- Hydrogen Bonds- Van der Waals- Hydrophobic
46
What is affinity?
Strength of the interaction between the epitope and one antigen binding site.
47
What is avidity?
Strength of the sum of interactions between antibody and antigen. Also any other rxns that might be invovled (i.e. TCR, etc.)
48
Why might avidity differ from affinity.
Binding with both antigen binding sites can significantly increase the strength.
49
Explain how cross reactivity works.
Antiserum raised against antigen A also reacts against antigen B because they share an epitope.
50
Two scenarios in which cross-reactivity is very important.
Impt. for vaccines and laboratory reactions
51
How many Hs and Ns in Influenza?
16H and 9N
52
What are monoclonal antibodies.
Immortilizations of single clone of antibody-secreting cells
53
How are monoclonal antibodies made in the lab? (broad concept)
B Cells + Neoplastic (myeloma) cells
54
Steps of monoclonal antibody generation.
1. Fuse B and Myeloma Cells2. Grow in drug-containing medium to select for hybrids3. Select for antigen-specific hybridoma4. Clone the winner
55
How are polyclonal antibodies made?
From serum of immunized animals. (goats, rabbits)
56
Concerns regarding polyclonal antibodies
Multiple specificities and affinitiesVariation from batch to batch
57
Name the four types of therapeutic monoclonal antibody.
MouseChimericHumanizedHuman
58
Nomenclature for Chimeric
-ximab
59
Nomenclature for humanized
-zumab
60
Nomenclature for human
-umab
61
Problems with therapeutic mouse antibodyWhen is it still useable
Patients develop anti-mouse antibodies Diagnostic
62
What mouse regions are maintained in humanized antibodies?
HVR
63
Can therapeutic human antibody elicit an immune response?
yes
64
Three types of immunoassays and level of detection.
Precipitation -- 30ug/mLAgglutination -- 1 ug/mLRadioimmunoassay/ELISA -- 1 pg/mL
65
Whats precipitation all about?
Antigens form large enough complexes to see with the naked eye. Insensitive and rarely used.
66
What is agglutination all about?
Usually hemaglutination
67
What is ELISA all about?
Very sensitive. Good for studies of viruses and such
68
To form the largest precipitates, what proportion of antibody to antigen is required?
Equivalence
69
Aggregates are known to get stuck in _____ and _____
Kidneys and Joints
70
Larger antigens are more prone to have _______ epitopes
MoreMakes large antigens better for aggregation
71
Do you have to be sensitized to have anti-A or B
No -- prbs cross reactions with gut microbes
72
Ab type used in hemagglutination
IgM
73
Why is a different AB blood type baby fine?
Mom's IgMs can't cross the placenta
74
Difference between Direct and Indirect Coombs Test?
Direct: anti-human Ig Abs (Coomb's reagent) + fetal RBCsIndirect: Mom's Serum+Rh+ RBCsAfter either, add anti-human antibody and assess aglut.
75
What type of Ig is used in Coombs?
IgG
76
Why don't the IgGs agglutinate spontaneously?
They're too small, so RBC without Ab can't
77
What is Coombs test even assessing?
Rhesus factor antibody response in mother and child (i.e. Rh- mom and Rh+ kid)
78
Should a Rh- mother be worried about her first Rh+ child?
Probs not. Should not have Abs developed till the 2nd time
79
Steps in an ELISA
1. Coat Wells with Antigen2. Add serum sample3. Add enzyme-labeled anti-human IgG4. Add substrate
80
Steps in a Western Blot
1. Dissociate proteins in SDS2. Run SDS-Page to separate by molecular weight3. Transfer to nitrocellulose3. Overlay with anti-serum4. Detect antibody with enzyme linked anti-IgG
81
Tell me about immunoflouresence studies.
Put an antibody on with a fluorophore attached, send exciting light, pick up emitted light on microscope. Use to identify structures (esp. in dev. bio)
82
Explain Flow Cytometry.
1. Incubate samples with Ab2. Each monoclonal Ab had differently colored labels3. Run through a small stream with a laser that can count based on the diff colors.
83
Value of flow cytometry?
Qualitative, RapidCommon in Diagnostic Labs.
84
Fragments of genome seen in lambda chain? kappa chain? heavy chain?
l -- VJCk -- VJCh -- VDJC
85
Describe the steps of kappa chain generation
1. Germline DNA undergoes VJ combination2. B cell DNA undergoes transcription into RNA transcript3. VJC is made via RNA splicing4. Translation into kappa chain polypeptide
86
List the order of fragments coming together in heavy chain
DJ --> VDJ --> VDJC
87
Significance of alrge number of gene segements in Ig loci?
Allow for A TON of recombinatory mechanisms
88
What is the purpose of recombination signal sequences?
They prevent bonding of regions out of order
89
Significance genetically of the 12 and 23 nuc. segments
12 = 1 twist of DNA helix23 = 2 twists of DNA helix
90
What are the nucleotide segments involved in recombination signal sequences?
7-23-9 binds with 9-12-7
91
Enzyme complex that conducts recombination. Mentioned components.
VDJ RecombinaseRAGs, TdT, Artemis Nuclease
92
What are RAGs
Recombination Activity GenesLoop out intervening DNA to delete it
93
Steps from cleaving out the loop to recombined DNA (6)
1. RAG leaves DNA hairpins2. RAG nicks hairpins, generates palindromic P-nucleotides3. TdT adds in nucleotides at joining region4. Strand pairing5. Unpaired nucleotides are removes by exonuclease6. Gaps caused by DNA synth and ligation
94
Three ways recombination can inadvertently promote diversity.
1. TdT adds in nucleotides2. Imprecise Joining3. Unpaired nucleotide removal can lead to frameshift
95
Initially, what heavy chain is made
IgM
96
Why aren't all Abs IgMs?
Isotype switching can happen later
97
Naive mature B cells express what isotypes?
IgM and IgD
98
What is addedd to the Ig following the C chain being splied in -
AAA
99
Why are there two kinds of naive generated?
RNA contains both IgM and IgD componentsThey can be splicaed differently
100
T or F. Alternative splicing = isotype switching.
False.
101
Since B cell receptors lack the AA for intracellular signalling, they associate with...
Ig alpha and beta heterodimer
102
Vaugely descripe B cell signalling
B cell binds, conformational change, activation of heterodimer, tyrosine kinase intracellular signalling
103
What happens to B cells after activation?
Ab SecretionSomatic Mutation (more diversity)Isotype switching
104
How does the change from Membrane to secreted Ig happen?
Alternative RNA processing
105
T or F. Somatic hypermutation will always lead to stronger antigen binding.
F. Can increase or decrease specificity or have a neutral effect.
106
What mediates somatic hypermutation activity of CDR 1/2?What does it do?
Activation-induced Cytidine deaminaseCytosine->Uracil. Changes base pairs
107
Outcome of isotype switching?
Igs with different C regions, but identical antigen specificity.
108
Steps of Isotype Switching.
1. IgM and IgD are produced. 2. AID selectively targets S(mu) and S(gamma1) switch3. DNA of both switch regions is nicked4. DNA btw nicks is looped out5. IgG1 is produced
109
What is AID?
Activation-Induced Cytosine DeaminaseImportant in Somatic hypermutation AND isotype switching
110
After isotype switching, can you go back?
No. DNA is spliced out.
111
Effector fxns of Abs?
Neutralization (bind to prevent receptor binding)OpsonizationComplement Activation
112
Whats so cool about IgM?
Pentamer StructureCan be transported in mucosal secretions
113
IgM have _____ Affinity and _____ Avidity
Low Affinity and High Avidity
114
Who is the first Ig synthesized in immune response.
IgM
115
Roles of IgM
Activates complementAgglutination
116
___ stabilizes the IgM pentamer
J chain
117
Which Ab has the highest conc. in the serum?
IgG
118
How many IgG subclasses?
4
119
Complement activation of the 4 subtypes?
IgG3 > IgG1 > IgG2. Ig4 doesn't do shit.
120
Roles of IgG
Binds Fc receptors of neutrophils, macrophages, NKsComplement Activation
121
Difference between the 4 IgGs?Which is especially prevalent?
Hinge Region StructureIgG3 has a freakin huge hinge -- very susceptible to proteases and shorter half life
122
Whats unique about IgG4
Functionally Monovalent
123
What does functionally monovalent mean?
Can dissociate at hinge region and recombineForms bivalent Ab molecules w/ 2 binding sites
124
End effects of functional monovalence?
Less effective
125
Secretory AgA is a....
Dimer with secretory component
126
Subclasses of IgA?
IgA1 and 2
127
Is IgA1 usually a monomer or dimer?
Monomer
128
Is IgA2 usually a monomer or dimer?
Dimer
129
Where do you tend to see IgA?
Mucosal Secretions
130
Primary role of IgA? Response to this?
IgA does neutralizationMany bacteria produce IgA proteases to fuck it up
131
IgA sucks at...
activating complementHelps not kill epithelial barriers of mucosa in infection
132
Which Abs have no hinge?
ME
133
IgE is normally at a ___ concentration
Low
134
What patient would you expect heightened IgE in?
Helminth infections or Allergies
135
What does IgE bind?>
Fc receptor of Mast/Baso
136
Where is IgD found?
URT
137
Lowest half life?
IgE
138
Relative frequency?
IgGMiddle IgA, MLowest ED
139
Which Igs primarily do neutralization?
G,A
140
Which Igs primarily do opsinization?
G1,G3
141
Which Igs primarily do NK killing?
G1, G3
142
Which Igs primarily do Mast cells?
E
143
Which Igs primarily do complement system?
M, G1, G3
144
Which Igs primarily do transport across epithelium?
A
145
Which Igs primarily do transport across placenta?
G134
146
Which Igs primarily do extravascular diffusion
G,A
147
List the seven events of a B cell's life
1. V region assembly2. Generation of Junctional Diversity3. Assembly og Transcriptional Controlling elements4. Trans. activated with IgM and D5. Synthesis changes to secreted6. somatic hypermutation7. Isotype Switch
148
Which two (of the seven event in a B cells life) are reversible?
Transcription of IgM and DSynthesis changes from membrane to secreted antibdy.
