Lecture #3 Flashcards
Immunoglobulins can be _______ or ______ receptors
Membrane Bound or Soluble
What causes a resting B cell to generate antibody?
Encounter with antigen turns it into a plasma cell
What is an epitope?
An antigenic determinant The part of an antigen to which an antibody binds
T or F. Most antigens have a single epitope.
False. Most antigens ahve multiple epitopes
What are epitopes usually made of?
Carbohydrates of pepride
Difference between DTwP and DTaP
DTwP = Whole Cell Pertussis VaccineDTaP = Acellular Pertussis Vaccine
An immune response to _____ is triggered in an HPV vaccination.
Caspid
Where are epitopes recognized by antibodies typically located?
At the antigen surface. There they can be directly bound
Two different types of epitopes an Ab can bind to.
Linear and Discontinuous
Difference between linear and confirmational/discontinuous epitopes.How is this different from T Cells
- 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
What lymphocytes recognize confirmational epitope?
B Cells.NO T Cells
What lymphocytes recognize linear epitopes?
B Cells and T Cells
What are Haptens?
Small molecules that are not immunogenic alone. They can bind Igs and TCRs.
What do Haptens do?
They can induce immune responses when linked to a lager carrier.
Example of a Hapten?
Penicillin
Effect of Penicilin on bacteria
Binds bacterial transpeptidase and inactivates it
Effect of Penicillin on RBC
Modifies RBC Proteins to generate a foreign epitope
What happens to RBCs after they receive their hapten epitope?
B Cells are activated by antigen+TH2Ab binding to RBCsRBC destruction
What holds together the light and heavy chains?
disulfide bonds
Where can disulfide bonds be found?
Between light and heavy In the Hinge Region
Names of the two light chains
Lambda and Kappa
Name the five heavy chains
Mu, Gamma, Alpha, Epsilon, Delta
The specificity of binding comes from the…
Antigen binding sites/Variable Region
What do you find when Ab is cleaved by Papain?How many total fragments are generated?
Antigen-binding FragmentCrystallizable FragmentThree
What do you find when Ab is cleaved by pepsin?How many total fragments are generated?
F(ab’)2 fragmentChopped up base2 Fragments
What function might F(ab’)2 have?
Naturally, none. In labs, it can be used in binding studies.
Use of the hinge region?
Gives flexibility at antigen binding sites
Downside of the hinge region?
Susceptible to proteolysis by bacterial and host proteases
How many domains in heavy chain? Light Chain?What are they?
- 3C, 1 V2. 1C, 1V
Significance of V and C Regions?
V=Epitope BindingC = Biological Fxn of Molecule
Describe the molecular structure of the Ab domains.
Two beta sheets held together by a disulfide bond”Sandwich with a Toothpick”
Another name for hypervariable region?
Complementarity-Determining Region
What do hypervariable regions do?
They provide the actual antigen binding specificity.
How many HV regions are in an antigen binding site?
6 (3 on light and 3 on heavy)
Are the HV regions adjacent genetically?
No, hydrophobic regions come together from different parts of the genome in the process of protein folding.
Name the 5 types of Immunoglobulins
Ig G, M, D, A, E
Which immunoglobulins have no Hinge region?What else is special about them?
IgM and IgEAlso have a 4th constant domain
Problems with the unique structure of IgM and IgE?
Less Flexibility of antigen binding.
What are isotypic differences between immunoglobulins?
Like IgG vs IgA
What are allotypic differences between immunoglobulins?
Variations of immunoglobulins seen because they come from the two different genes. Usually only very small differences.
What are idiotypic differences between immunoglobulins?
Like IgGs with different epitope binding sites
Why care about allotypical differences (3)
May alter half lifeMay Affect subclass distributionAssociated with susceptibility to infectious and autoim. disease
How are allotypes inherited?
Autosomal dominant
T or F. Binding strength can be very variable between different antibody-epitope pairs.
True
What forces drive the antibody-antigen interaction?
Non-Covalent:-Electrostatic- Hydrogen Bonds- Van der Waals- Hydrophobic
What is affinity?
Strength of the interaction between the epitope and one antigen binding site.
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.)
Why might avidity differ from affinity.
Binding with both antigen binding sites can significantly increase the strength.
Explain how cross reactivity works.
Antiserum raised against antigen A also reacts against antigen B because they share an epitope.
Two scenarios in which cross-reactivity is very important.
Impt. for vaccines and laboratory reactions
How many Hs and Ns in Influenza?
16H and 9N
What are monoclonal antibodies.
Immortilizations of single clone of antibody-secreting cells
How are monoclonal antibodies made in the lab? (broad concept)
B Cells + Neoplastic (myeloma) cells
Steps of monoclonal antibody generation.
- Fuse B and Myeloma Cells2. Grow in drug-containing medium to select for hybrids3. Select for antigen-specific hybridoma4. Clone the winner
How are polyclonal antibodies made?
From serum of immunized animals. (goats, rabbits)
Concerns regarding polyclonal antibodies
Multiple specificities and affinitiesVariation from batch to batch
Name the four types of therapeutic monoclonal antibody.
MouseChimericHumanizedHuman
Nomenclature for Chimeric
-ximab
Nomenclature for humanized
-zumab
Nomenclature for human
-umab
Problems with therapeutic mouse antibodyWhen is it still useable
Patients develop anti-mouse antibodies Diagnostic
What mouse regions are maintained in humanized antibodies?
HVR
Can therapeutic human antibody elicit an immune response?
yes
Three types of immunoassays and level of detection.
Precipitation – 30ug/mLAgglutination – 1 ug/mLRadioimmunoassay/ELISA – 1 pg/mL
Whats precipitation all about?
Antigens form large enough complexes to see with the naked eye. Insensitive and rarely used.
What is agglutination all about?
Usually hemaglutination
What is ELISA all about?
Very sensitive. Good for studies of viruses and such
To form the largest precipitates, what proportion of antibody to antigen is required?
Equivalence
Aggregates are known to get stuck in _____ and _____
Kidneys and Joints
Larger antigens are more prone to have _______ epitopes
MoreMakes large antigens better for aggregation
Do you have to be sensitized to have anti-A or B
No – prbs cross reactions with gut microbes
Ab type used in hemagglutination
IgM
Why is a different AB blood type baby fine?
Mom’s IgMs can’t cross the placenta
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.
What type of Ig is used in Coombs?
IgG
Why don’t the IgGs agglutinate spontaneously?
They’re too small, so RBC without Ab can’t
What is Coombs test even assessing?
Rhesus factor antibody response in mother and child (i.e. Rh- mom and Rh+ kid)
Should a Rh- mother be worried about her first Rh+ child?
Probs not. Should not have Abs developed till the 2nd time
Steps in an ELISA
- Coat Wells with Antigen2. Add serum sample3. Add enzyme-labeled anti-human IgG4. Add substrate
Steps in a Western Blot
- 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
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)
Explain Flow Cytometry.
- 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.
Value of flow cytometry?
Qualitative, RapidCommon in Diagnostic Labs.
Fragments of genome seen in lambda chain? kappa chain? heavy chain?
l – VJCk – VJCh – VDJC
Describe the steps of kappa chain generation
- Germline DNA undergoes VJ combination2. B cell DNA undergoes transcription into RNA transcript3. VJC is made via RNA splicing4. Translation into kappa chain polypeptide
List the order of fragments coming together in heavy chain
DJ –> VDJ –> VDJC
Significance of alrge number of gene segements in Ig loci?
Allow for A TON of recombinatory mechanisms
What is the purpose of recombination signal sequences?
They prevent bonding of regions out of order
Significance genetically of the 12 and 23 nuc. segments
12 = 1 twist of DNA helix23 = 2 twists of DNA helix
What are the nucleotide segments involved in recombination signal sequences?
7-23-9 binds with 9-12-7
Enzyme complex that conducts recombination. Mentioned components.
VDJ RecombinaseRAGs, TdT, Artemis Nuclease
What are RAGs
Recombination Activity GenesLoop out intervening DNA to delete it
Steps from cleaving out the loop to recombined DNA (6)
- 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
Three ways recombination can inadvertently promote diversity.
- TdT adds in nucleotides2. Imprecise Joining3. Unpaired nucleotide removal can lead to frameshift
Initially, what heavy chain is made
IgM
Why aren’t all Abs IgMs?
Isotype switching can happen later
Naive mature B cells express what isotypes?
IgM and IgD
What is addedd to the Ig following the C chain being splied in -
AAA
Why are there two kinds of naive generated?
RNA contains both IgM and IgD componentsThey can be splicaed differently
T or F. Alternative splicing = isotype switching.
False.
Since B cell receptors lack the AA for intracellular signalling, they associate with…
Ig alpha and beta heterodimer
Vaugely descripe B cell signalling
B cell binds, conformational change, activation of heterodimer, tyrosine kinase intracellular signalling
What happens to B cells after activation?
Ab SecretionSomatic Mutation (more diversity)Isotype switching
How does the change from Membrane to secreted Ig happen?
Alternative RNA processing
T or F. Somatic hypermutation will always lead to stronger antigen binding.
F. Can increase or decrease specificity or have a neutral effect.
What mediates somatic hypermutation activity of CDR 1/2?What does it do?
Activation-induced Cytidine deaminaseCytosine->Uracil. Changes base pairs
Outcome of isotype switching?
Igs with different C regions, but identical antigen specificity.
Steps of Isotype Switching.
- 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
What is AID?
Activation-Induced Cytosine DeaminaseImportant in Somatic hypermutation AND isotype switching
After isotype switching, can you go back?
No. DNA is spliced out.
Effector fxns of Abs?
Neutralization (bind to prevent receptor binding)OpsonizationComplement Activation
Whats so cool about IgM?
Pentamer StructureCan be transported in mucosal secretions
IgM have _____ Affinity and _____ Avidity
Low Affinity and High Avidity
Who is the first Ig synthesized in immune response.
IgM
Roles of IgM
Activates complementAgglutination
___ stabilizes the IgM pentamer
J chain
Which Ab has the highest conc. in the serum?
IgG
How many IgG subclasses?
4
Complement activation of the 4 subtypes?
IgG3 > IgG1 > IgG2. Ig4 doesn’t do shit.
Roles of IgG
Binds Fc receptors of neutrophils, macrophages, NKsComplement Activation
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
Whats unique about IgG4
Functionally Monovalent
What does functionally monovalent mean?
Can dissociate at hinge region and recombineForms bivalent Ab molecules w/ 2 binding sites
End effects of functional monovalence?
Less effective
Secretory AgA is a….
Dimer with secretory component
Subclasses of IgA?
IgA1 and 2
Is IgA1 usually a monomer or dimer?
Monomer
Is IgA2 usually a monomer or dimer?
Dimer
Where do you tend to see IgA?
Mucosal Secretions
Primary role of IgA? Response to this?
IgA does neutralizationMany bacteria produce IgA proteases to fuck it up
IgA sucks at…
activating complementHelps not kill epithelial barriers of mucosa in infection
Which Abs have no hinge?
ME
IgE is normally at a ___ concentration
Low
What patient would you expect heightened IgE in?
Helminth infections or Allergies
What does IgE bind?>
Fc receptor of Mast/Baso
Where is IgD found?
URT
Lowest half life?
IgE
Relative frequency?
IgGMiddle IgA, MLowest ED
Which Igs primarily do neutralization?
G,A
Which Igs primarily do opsinization?
G1,G3
Which Igs primarily do NK killing?
G1, G3
Which Igs primarily do Mast cells?
E
Which Igs primarily do complement system?
M, G1, G3
Which Igs primarily do transport across epithelium?
A
Which Igs primarily do transport across placenta?
G134
Which Igs primarily do extravascular diffusion
G,A
List the seven events of a B cell’s life
- 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
Which two (of the seven event in a B cells life) are reversible?
Transcription of IgM and DSynthesis changes from membrane to secreted antibdy.
