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Chapter 6: Antibody Interactions
- Antibody-antigen interactions depends on 4 non covalent forces
• hydrogen bonding
• ionic bonding
• hydrophobic interactions
- water forces hydrophobic groups together

• van der Waals interactions
- electron clouds of two or more atoms interact

- Cross-Reactivity: occurs if two different antigens share same/similar epitope
- observed among polysaccharide antigens that contains similar oligosaccharide residues
• Blood types
- ex: ABO blood-group antigens are glycolipids expressed on rbc
- blood type (A, B, AB, O) is equivalent to the type of antigens on an individual’s red blood cells
- the antibodies they have will be for the antigen they DON’T have (ex: Type A person will have
anti-B antibodies) which is why they cannot take blood that is a different type

• antibodies will precipitate out of rbc if given wrong type
- Type AB has antigens A and B, so they do not have any antibodies (universal acceptor)
- Type 0 has neither antigen A nor B, so they have both anti-A/anti-B antibodies (universal donor)

- Surface plasmon resonance (SPR) - measure rate of antibody-antigen binding =
antibody’s affinity

• sensitive, convenient, rapid way to measure antibody affinity
• detects changes in reflective properties of surface of an antigen-coated sensor when it binds to
antibody

• beam of polarized light is directed through prism onto thin gold oil coated with antigen on opposite
side

• light is reflected off the gold film toward light-collecting sensor
• some light will be absorbed by the gold (light energy transformed to waves)
• dip in light intensity measured at resonant angle: will depend on several factors which SPR takes
advantage of

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- antibody-antigen binding attached to chip has strong effect to produce change in resonant
angle (measuring the rate at which the angle changed during rxn will determine rate of rxn)

• measure on sensogram -once plateau has been reached, solution containing no antibody can be
passed through the chamber (antigen-antibody complex dissociates)

• SPR can be used to do epitope mapping
- determines if different antibody binds to same/different epitope of same antigen

- if two separate plateaus shown on sensogram upon adding two antibodies, means they
react with different epitopes on the antigen

- ELISA: Enzyme-Linked Immunosorbent Assay- depends on enzyme which binds to either antibody/
antigen to determine their respective concentrations

• Indirect: primary antibody added to antigen-coated well (binds), then washed with enzyme which will
bind to antibody, add substrate to measure color (more yellow=more enzyme bound to
antibody=more antibody)

• sandwich: same as indirect, only antigen is added to antibody-coated well first (then same steps
after— enzyme will bind to antigen instead of antibody)

• competitive: antibody incubated with antigen, the mixture is then added to antigen-coated well…
more antigen present= less free antibody will bind to antigen-coated well

- Western Blotting
• identification of specific protein in a complex mixture of proteins
• protein mixtures is treated with SDS then separated by electrophoresis in gel (separates according
to molecular weight)

- Immunoprecipitation-if [antigen] low, antigen-antibody complex—> precipitate will take hours/
days/difficult for form

• allows isolation of antigen of interest for further analysis
• sensitive assay for presence of particular antigen in given cell/tissue
• extract mixed with antibody against antigen of interest to form antigen-antibody complex that will
precipitate

- Immunofluorescence
• fluorescent molecules absorb light of a wavelength (excitation) and emit light of another wavelength
(emission)

• antibodies can be tagged with fluorochrome (immune complexes containing fluorescently labeled
antibodies which can be detected by colored light emission when excited by light of a certain
wavelength)

• has been used to identify a CD4+/CD8+ subpopulations

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• Direct: cells gained with anti-mAg antibody that is labeled with fluorochrome
• Indirect: cells incubated with unlabeled anti-mAg antibody then stained with fluorochrome-labeled
secondary reagent that binds to primary antibody

- Flow Cytometry and Fluorescence
• gives quantitative data-designed to automate the analysis and separation of cells gained with
fluorescent antibody

• laser beam and light detector counts single intact cells in suspicion
- when cell passes the laser beam, light is deflected from detector and interruption of laser signal is
recorded

- cells with fluorescently-tagged antibodies on cell surface antigens are excited/emit light
• analysis: use to determine how many members of a cell pop
...
imitates formation of MAC (membrane-attack complex)
- Alternative Pathway
• C3 hydrolyzes spontaneously
• C3b fragment attaches to foreign surface
• Factor B binds to fragment, Factor D comes in and cleaves Factor B so Ba comes off
• C3bBb generated (left on foreign surface)

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• Properdin binds and stabilizes C3b = C5 convertase
• Convertase generates C3b (then some binds to C3 convertase = C5 convertase = imitation of MAC)
• Lectin Pathway: same as classical but beings with MBL (mannose-binding lectin instead of C1)
• membrane-attack complex: terminal sequence of complement activation involving C5b, C6-C9,
which interact sequentially

- complex forms large channel through membrane of target cell, enabling ions and small molecules
to diffuse freely across the membrane

• end result of activating any pathway is production of active C5 convertase (extremely labile and
becomes inactive unless stabilized by C6)

• Regulation
- C1 inhibitor (classical pathway) serine protease inhibitor causes C1r and C1s to dissociate from
C1q

- Factor H (alternative pathway) blocks formation of C3 convertase by binding C3b (can’t bind with
Factor D, Factor B or Properdin)

- S Protein (all pathways/terminal) binds soluble C5bC67 and prevents its insertion into cell
membrane —stops MAC from generating

• various smaller fragments generated during MAC formation play role in development of effective
inflammatory response

- smaller fragments, C3a and C5a bind (anaphylatoxins) help promote inflammation by binding to
receptors on mast cells/blood basophils to induce degranulation…release histamine etc
...
interference with binding of complement to antibody-antigen complexes
- 2
...
incorporation of cellular complement regulators in the virion
• long polysaccharide chains (gram-negative) in cell wall of microbes, side chains will prevent
insertion of MAC into the membrane

• peptidoglycan layer (gram-positive) prevents insertion of MAC into membrane
• proteins can mimic complement regulatory proteins that inhibits the complement cascade
• Deficiencies
- because rbc have fewer receptors than macrophages, the latter can strip the complexes from the
rbc as they pass through the liver/spleen (can lead to renal damage because of accumulation of
immune complexes)

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Chapter 8: Major Histocompatibility Complex + Antigen Presentation
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MHC’s

- presents peptides of self (critical in self/non-self recognition)
- restricted to host
- MHC’s very complicated, many expressed for various reasons
• certain organs can be donated more/less easily depending on expression of MHC I’s those
cell types usually express

- ex: muscle and liver cells have low MHC I expression, and donor has better chance of
working if lower levels of MHC I… kidneys have high MHC I expression so a donated
kidney wouldn’t last forever

- liver transplants typically not rejected for this reason (also liver can self regenerate)
- peptides in MHC I vs MHC II: very distinct abilities to present different types of antigens to
immature T and B-cells

- MHC’s given different designations in various organisms (ex: mice vs humans)
• lots of different types
• number of different copies of class II MHC genes in an individual person…causes
enormous amount of variety of genes that can be expressed

- HLA complex: is polygenetic (hundreds of different copies), human leukocyte antigenhelps the immune system distinguish the body's own proteins from proteins made by
foreign invaders such as viruses and bacteria; human version of MHC

- get multiple copies from each parent- expands repertoire of different MHC complexes
you can express

- genes don’t have shuffling ability like T and B-cell receptors on antibodies
- MHC genes tend to cluster: haplotype
• certain versions tend to go along with other versions
• crossing over of genes can occur (hybrids) functionally limitless number of combinations in
HLA

- basic structures of class I and II are similar: main difference is that second class of
polypeptides in class I are smaller

- non-covalent interaction, but don’t dissociate (strong interactions)

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- Class I MHC
• alpha chain
• beta 2 microglobulin (interacts w/ alpha 3 domain)
• a1 and b2 are very similar in structure, which is similar to Fc region of antibody
- both beta sheets that resemble constant portions of antibodies
- both in immunoglobulin superfamily
- (motifs repeat because are useful)
• Peptide-binding cleft: separates a1 from a2, gets its shape because 8 beta strands which
form beta sheet, two antiparallel alpha helices on top which form ridges on either side of
peptide-binding cleft = pocket for antigen to fit into

• b2 microglobulin not found in HLA complex, found on different chromosome, but is
transcribed at same time and is inserted into the complex

• span membrane of mostly all cells in an organism
Class II MHCs

- alpha and beta, contain same basic regions
- held together non-covalently, but strong associations
- a2 and b2 are structurally similar to a3 and b2 in class I (like Fc region on
antibodies)

- restricted to cells of the immune system (dendritic, macrophage, B-cells…)
• Class I and II peptide binding completely different
- Class I , binding site is on one peptide
• binds 8-10 amino acids (bowl-closed at both ends of peptide-binding cleft)
• peptides tend to be hydrophobic
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- Class II, alpha-region from one side, beta-region from the other
• binds 13-18 amino acids (hot dog bun-open at both ends of peptide-binding cleft)
• scattered peptides

• peptide itself takes on particular shape when binds to MHC I or II
• alpha helices on class I interact with each other, creating a “wall” on each side so not as
many peptides can fit in

• orientation of peptides differs because of the alpha and beta interactions
- class I: amino acid regions found at position 2 and 9 mostly hydrophobic (of peptide-binding cleft)
- average of 9-AA long, a little too big to fit into binding cleft of MHC I
• can’t sit perfectly flat if there are 9 AA

• internal amino acids (in the center), form a bulge when there are 9 amino acids
• the longer the peptide, the bigger the bulge
• huge amount of variation for the orientation the amino acids are in

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- MHC proteins are polygenetic
• all cells express MHC I molecules at the same time (have many from each parent)
• class II expression only activated when antigen-presenting cells are there
• side-note: neurons express low/no MHC I: immune privilege organs (meaning they are able to
tolerate the introduction of antigens without eliciting an inflammatory immune response
...
beta OR delta/gamma expressed
- joining region between variable and constant regions, angle is more severe in alpha/beta
version of T-cell receptor

- also do fairly different things
• alpha/beta version, possibility of rearrangement is great
• gamma/delta version, possibility of rearrangement is smaller
- most cells, neither express CD4+ nor CD8+ = no accessory to become activated
- can bind to completely intact proteins—NOT MHC restricted
- can be activated readily (by phospholipids)
• most T-cells found are alpha/beta
- cells are split unevenly (most CD4+, MHC II associated)
- alpha/betas are MHC restricted
• has to be presented on MHC molecule
- recognize peptide fragments
• tells that gamma/delta T-cells can bind to bacteria/foreign things (don’t require
processing)

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- will be at site of injury first before alpha/beta T-cells
- behave like innate immune system things
• have VDJC regions like antibodies
• Leader sequence
- unique: on chromosome 14 of mice, have alpha and delta chain on same segment
• delta chain sits in the center of alpha chain segments
...
=additional level of complexity in T-cells that is not in antibodies
• Junctional Flexibility: variable regions sliced at different places
• N-nucleotide addition
• P-nucleotide addition
• further shuffling
- alpha/beta have short non-catalytic domains so need CD3
• needs accessory proteins in order to signal= CD3
- 6 polypeptides: gamma epsilon and delta epsilon heterodimers, zeta chains
- when bound, expose positive residues in transmembrane segments that attracts other receptors,
then are pulled together

- ITAM: immunoreceptor tyrosine-based activation motif
• conserved regions able to activate tyrosine kinases-cluster together which causes signaling

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- T-cell-antigen binding is weak
• need accessory molecules to strengthen interaction
• CD4 and CD8: job is to recognize conserved regions on MHC molecules to strengthen
• complementary molecules: know that there are adhesion molecules
• Class II: has p56lock which takes CD4 and pulls it into the complex, CD4 binds to conserved
residues on class II MHCs to keep it together long enough (stabilize)

- Tissue rejection
• direct allorecognition
- take tissue, will have foreign antigen-presenting cells…shouldn’t be able to activate
resonant T-cells
...
how dd you get a receptor simultaneously able to recognize a self
MHC receptor vs a peptide that is self/non-self? and make sure it binds the correct way?

- too well, gets activated/immune system activated
• many things that have to happen to make a T-cell that is “just right”
- once precursor T-cell moves into thymus, called thymocytes
• have to go through process by which they get all components necessary for them to be functional/
mature waiting to be activated (need to get CD4+ or CD8+)

- Steps:
• first come into thymus, no CD4/CD8 (“double negative” stages) or alpha/beta chains of T-cell
receptor

• while cells are double negative, express proteins:
- c-kit receptor for stem cell profactor…
- cells have to be told to stay alive
- CD44: adhesion molecule
- CD25 portion of receptor for IO2 (cytokine, necessary for maturation of T-cells)
- RAG-1/2 get turned on, T-cell makes beta chain
• 99% cell rearranges beta chain by T-cell receptor
• surface molecules get removed after rearrangement
- T-cell receptor beta chain associates with pre-T alpha chain (structurally similar to alpha chain)
• pre T-cell receptor selection process for cells that have correctly folded beta chain
• believed to interact with indigenous ligand found in walls of thymus
...
likely that there is a blend of the two
- once a functional T-cell receptor has bound to antigen, T-cell starts to alter gene expression
• immediate genes: transcription factors that are turned on right away, cell starts making many
different proteins

• early genes: half hour-hour, cyclin (cell cycle protein) IL-2/IL-2 receptor
• late genes: up to a day, cytokines
- all happens through T-cell receptor
- MHC + T-cell receptor
• transmembrane domain (usually 10 C in phospholipids-will cluster together), but T-cell receptor has
longer than average ones

- lipid raft (long chain/congregation of phospholipids) —> Lipid rafts is a blanket term used to
describe distinct areas in the plasma membrane rich in certain lipids and proteins and which are
thought to perform diverse functions
...
made heavy chain that can properly recognize

• hypothesized that there is a ligand that binds to Vpre-B
- make surrogate light chains (membrane M chain is associated with surrogate light chain to
form light-chain-like structure)

• pre-B-cell receptor
- then stop making light chain, choose kappa or lambda
- Immature B-cells—> Naive B-cells (“naive” means they have not encountered antigen)
• kappa/lambda
• fully mature and function IgM antibody

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- any interacting of antibodies by binding to anything in bone marrow will lead to
apoptosis (90% of pre B-cells die)

- if variable regions don’t recognize proteins in bone marrow, cell left alone and leaves
to be activated

- (sidenote: alpha/beta T-cells recognize peptides, gamma/delta recognize phospholipids)
- remember: IgM/IgD are markers for mature B-cells, found in lymphoid organs
- pre-B-cells have pre-BCR (salient feature during this stage) which is made of 2 heavy chains and
2 surrogate light chains (each light chain contains (2) gamma 5 + VpreB)

- B-1 B-cells are more analogous to gamma/delta T-cells
• not variable in antigen recognition
• lungs/gut linings
• make copies of themselves (proliferate)
• variable region is not very diverse, limited because want to recognize general patterns
• no somatic hypermutation
• don’t require T-cells to be activated
• high levels of IgM (no IgD)
• recognized carbohydrates*
• no memory/memory cells because constantly self-renewing
• throwback to innate immunity
- Activation, proliferation, and differentiation of B-cells occur in the periphery in response to antigen
- B-cells capable reacting to different types of antigens (Table 11-2)
• Thymus independent: 2 types (generally don’t change Ig class because attached to the thing
it needs to kill, no Helper T-cell needed)

- Type 1: on bacterial cell wall
- Type 2: on polymeric protein antigens; capsular polysaccharides
• Thymus dependent: small soluble peptide will activate, can switch Ig type, can undergo
somatic hypermutation (every round of replication will cause higher affinity for antigen)

- B-cell and helper T-cell interaction**
• helper T-cell needed to activate, which expresses CD40L (binds to CD40 on B-cell)
= second signal to B-cell to proliferate

- simple interactions between TCR + MHC
• ability to recognize repeated cellular components/motifs (Ex: LPS, polysaccharides, capsules,
etc

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