Immunology Section 2

Front 1

Antigen

Back 1

A substance that binds to an antibody - contains two identical epitopes

Front 2

Immunogen

Back 2

Something that elicits an immune response via binding to an AB -> all are antigens

Front 3

Hapten

Back 3

An antigen that doesn't cause an immune response, usually due to small size (lack of carrier) or monovalence

Front 4

Four properties of an immungen

Back 4

1)Bivalence: Two epitopes on a carrier
2)Size: over 5kMW
3)Complexity: So most are proteins, though some are polysaccharides, lipoproteins, or glycoproteins.
4)Foreign, can have self antigens, but not immunogens

Front 5

Antibody

Back 5

Immunoglobulin proteins consisiting of two heavy and two light chains that recognize a particular epitope of an antigen. Roughly 150,000 MW. Serve as surface receptors of B cells, which produce them in response to discovering their cognate antigen in order to counteract the pathogen with that antigen.

Front 6

Adjuvant

Back 6

Something that enhances the immune response
-may or may not be an antigen
-usually leads to inflammation
(AKA biological response modifiers)

Front 7

Freunds Adjuvant

Back 7

First adjuvant: dead mycobacterium, mineral oil, and lanolin

Front 8

What electrophoresis revealed about Antibodies?

Back 8

Four peaks of blood serum: albumin, alpha, beta, and gamma. Found in beta and gamma. Kabat showed that when rabbits formed AB against sugar it was found in gamma peak. Lucky b/c choose a polysaccharide = simpler.

Front 9

What did ultacentrifugation of AB teach us?

Back 9

Gave two distinct bands, 7S and 19S. (Swedburg units) Therefore all AB are not the same.

Front 10

How did Edelman and Porter determine AB structure?

Back 10

Electrophoresis on reduced 7S (150kMW) antibody band from myeloma cells. Found two peaks, one at 50kMW and one at 25kMW. Therefore, two types of polypeptide chains.

Front 11

List types of AB chains

Back 11

Light: kappa and lambda
Heavy: gamma, alpha, mu, delta, and epsilon

Front 12

Draw primary and secondary AB structure based on IgG with interchain and intrachain bonds.

Back 12

Notes from 26 September
-note where glycosylation occurs (glycoforms)
-labeled hinge region

Front 13

Paratope and its synonym

Back 13

Few amino acid region of AB that binds to epitope, in variable region.
-involves up to 6aa from each chain's variable region (Compliment determining region)
-rather like an enzymes active site

Front 14

Flexibility of hinge region of AB and how determined?

Back 14

Can go from 90º to 0º. Tested by putting dinitrophenol with Fe at ends of "barbell" antigens. Saw lines, triangles, and squares formed.
-hinge is proline rich

Front 15

Epitope

Back 15

Portion of antigen that is recognized and bound my an antigen. Can be continuous or discontinuous.

Front 16

Type of bonding between paratope and epitope

Back 16

-Van der Walls
-Weak ionic bonds

Often epitope changes shape to increase stability

Front 17

Affinity vs avidity

Back 17

Affinity: How well Ag-AB fit together (association constant from biochemistry)
Avidity: Strenghth of Ag-AB binding when multiple epitopes on an antigen interact with binding sites on an AB

Front 18

Cross reaction

Back 18

When AB reacts with a similar epitope with lower affinity
-choice of epitope will be [epitope] dependent

Front 19

Enzymatic digestion of AB

Back 19

1)Papain results in Fc region and FAB (fragment antigen binding) by cleaving at hinge. Fc is just bottom constant regions of heavy chain. FAB is rest = two monovalent pieces.

2)Pepsin results in a monovalent F(AB')2 and junk. Leaves cysteine bridges in place in AB hinge.

Front 20

Isotype

Back 20

The class of antibody determined by five heavy chains. First constant domain of HC and only constant domain of light are the determinants. Every individual of a species has the same isotypes, but a different species will form AB against them. (Therefore dominant epitope over allotype markers).

Front 21

Allotype

Back 21

Allelic variations within an AB class, so variations in aa sequence (markers) in Fc region. 25 known gamma markers for IgG and others for kappa. AB will form against these in different members of the same species.

Front 22

Idiotype

Back 22

Variation in variable region aa sequence. Found by comparing individuals with the same allotypes.

Front 23

Haplotype

Back 23

A cluster of linked genes. AB are a classic example inherited as a group.

Front 24

IgG characteristics

Back 24

Monomer
Half life of 23 days
3 Ch domains: can fix complement, leads to ADCC, 4 subtypes (1-4)

Front 25

IgM

Back 25

First produced, either a monomer or pentamer
Pentamer is pentavalent (not 10 valence)
J chain in the middle
4 Ch domains: Longest Fc region
Can also initiate complement

Front 26

IgA

Back 26

Forms dimer with J chain
Secreted to prevent pathogen entry, when secreted has secretory (T) piece added in the epithelial cell. This is actually part of the poly-Ig receptor therefore secretion is one way
3 Ch domains
Produce 3.5g / day in gut

Front 27

IgD

Back 27

Similar to IgG, no known purpose, only expressed fetally

Front 28

IgE

Back 28

Same size at IgM (4 C heavy domains)
Attaches to basophils and mast cells to trigger degranulation

Front 29

Bence-Joyce Proteins

Back 29

Homogenous light chains secreted by myelomas

Front 30

AB class switching sequence

Back 30

IgM -> IgG -> IgA

Protect blood, then tissue, then prevent entry

Front 31

Kohler and Millsein's method to make monoclonal AB

Back 31

1)Innoculate mouse with an immunoge
2)Extract B cells from spleen
3)Add HAT sensitive, thymidine deficient myeloma cells
4) Fuse with PEG or sendai virus
5)Select for succesfull half myeloma half murine products: s-s are killed by time, my-my killed by media w/o thymidine
6)Put cells in wells at a dilution of once cell per well
7)Select AB producing clones by checking for agglutination or genetic markers
8)Humanize antibody for certain uses / studies

Result is an immortal AB producing factory (a hybridoma)
Superseded by transfectomas now made by transfecting yeast with human AB -> cuts out need to humanize AB

Front 32

Techniques to make hybrid AB?

Back 32

1)Quadroma: Fuse two hybridomas with PEG or sendai virus. Get some that produce mixed AB.
2)Reduce AB with beta-ME and let reform

Front 33

Dromadarian AB

Back 33

They make nanoantibodies, monovalent with just the variable region.

Front 34

Prevalent blood types by world region

Back 34

Mostly A and O in USA, mostly B in east Asia.

Front 35

Types of transfusion reactions

Back 35

1)Immediate, means its against preexisting AB. That is from other blood types seen on bacteria
2)Delayed: Against the allotypic antigens. Means it is best to change donors with each transfusion.

Front 36

Types of blood cross-match

Back 36

1)Major: Test donor cells with recipient serum to test for preexisting AB. If agglutination, then no transfusion

2)Minor: Donor serum with recipient cells. If it agglutinates, just centrifuge to get rid of AB in donor serum.

Front 37

Rho blood types

Back 37

Rho positive and neg (an amorph). Positive is a cluster of three linked genes.

Leads to erythroblastosis in multiparis (3+ births) women causing stillbirth when untreated.

RhoGam prevents mother sensitzation.

Front 38

7 Outcomes of Ag-AB reactions

Back 38

1)Precipitation
2)Agglutination
3)Neutralization
4)Opsonization
5)ADCC [AB dependent cellular cytotoxicity]
6)Immune exclusion
7)Complement activation - classic pathway

Front 39

AB precipitations

Back 39

-Only of soluble antigens with bivalence in an ionic environment
-Used in lab in single radial, double diffusion, and IEP
-Makes easier to opsonize or excrete Ag

Front 40

Single radial vs double diffusion

Back 40

Add AB to agar with anti-AB to quantify amount of AB by size of ring

Add AB and anti-AB in drops next to each other and lood for line. Can add anti-AB class all around AB to test which class it is

Front 41

Immunoelectrophoresis [IEP]

Back 41

Use electrophoresis on a sample then add AB in a trough down the middle to identify whatever that AB targets (can be other AB)

Front 42

Agglutination

Back 42

When AB clump several insoluble Ag
-Used in lab to determine "titer"
-More sensitive than precipitation

Front 43

Titer

Back 43

Reciprocal endpoint of agglutination = 1/final dilution at which agglutination was observed

Front 44

Indirect agglutination

Back 44

When put AB on surface of something insoluble (like latex balls) to agglutinate soluble Ag.

Front 45

Neutralization

Back 45

When AB binds to the active site of pathogens preventing activity of pathogen
-can cause infertility and basis of tetnus vaccine

Front 46

Opsonization

Back 46

Any AB that enhances phagocytosis, could also agglutinate or neutralize at the same time
-Recruits cells with Fc receptors: neutrophils, monocytes, and eosinophils
**B cells can't phagocytize

Front 47

Two ADCC methods

Back 47

1)Inducing apoptosis: Programmed cell death that protects the environment. B cells, NK, or mø can do this.
2)Lytic enzymes: Breakdown cell surface, issue of the bystander effect. Eosinophils and mø do this.

**Only mø do both with great efficiency.

Front 48

Immune exclusion

Back 48

Prevention of Ag from going somewhere, mainly IgA in gut preventing Ag entry. **Very important

Front 49

Three pathways of the complement system

Back 49

1)Classical -> AB dependen
2)Alternate
3)Lectin

Front 50

Steps of Classical pathway -> draw and check with 9 Oct notes

Back 50

1)Recognition: Cl q r s
-C1q bind to heavy chain constant domain 2. Must bind to two IgG or one IgM pentamer. Affects C1r which activates C1s
or
C1q recognizes sugars on bacteria and proceeds same

2)Activation: C1s, C4C2C3
-C1s cleaves C4 and C2, C4bC2a bind to cell surface and cleave C3
-C3b binds to C4bC2a on cell surface

3)Membrane Attack: C5C6C7C8polyC9
-C4bC2aC3b cleaves C5
-C5b pierces cell surface
-C6, C7, C8 add next to C5b
-add 10-15 C9 form a ring around to make a pore

Front 51

Alternate complement pathway draw!

Back 51

C3 is always spontaneously hydrolyzing into C3a and C3b. C3b binds to nearby cells, but falls off it [sialic acid] is high like human cells
-D properdin cleaves B and Bb stabilizes C3b on bacteria to extend its half-life to continue as in classical pathway

Front 52

Other complement dependent activities

Back 52

1)Chemotaxis = C5a and C3a attract WBC
2)Opsoniztion = C3a and C5a increase phagocytic activity
3)Immune adheraence = C3a increases stickiness of WBC
4)Anaphylatoxin generators = C5a and C3a increase inflammation -> bind to mast cells and tell to release histamines

Front 53

Two types of techniques with AB

Back 53

1) Direct: Apply florescent anti-whatever AB - searches for epitope of AB
2) Indirect: Add and AB (via serum or known one) then at florescent anti-AB AB (secondary AB)
*Used to diagnose humoral autoimmunity -> add

Front 54

Three main purposes of AB lab techniques

Back 54

1)Cell probes: location in dead cells or on live cells
2)Diagnostics: whether it is there
3)Quantify: how much

Front 55

Six ways to label AB

Back 55

1)Florescence = most common
2)Chemiluminescence - luciferase, horseradish peroxidase, or luminol -> extremely sensitive
3)Ferritin for electron microscopy
4)Microspheres - pointless nowadays
5)Magnetic beads -> used to pull bacteria out of food
6)Radiation

Front 56

Diagram ELISA
-direct, sandwich, and competitive

Back 56

[Enzyme linked immunosorbent assay] p661 in book for diagram

-Key is AB is tagged with an enzyme to convert a substrate that can be detected (usually color)

Front 57

Quartz crystal microbalance

Back 57

Measures very small [] by bending quartz electrodes in a specific way

Front 58

3 uses of radioactive AB labels

Back 58

1)Measure lysis: Add chromium to a solution, all cells take it up, then treat with an agent and killed cells will release the chromium, centrifuge, and quantify chromium in supernatant
2)As tag on AB
3)Autoradiography

Front 59

Diagram a western blot

Back 59

p664 of textbook

1)Electrophoresis
2)Electrotransfer
3)Wash and treat with AB (and secondary AB if needed)

Front 60

The dilemma for AB genes

Back 60

-Generate 10^9 specificities
-Generate multiple classes
-Increase affinity with infection

Front 61

Natural selection theory

Back 61

Developed by Paul Erlich in early 20th century. Antigen selects for selector by choosing among the many specificities on each B cell

Front 62

Clonal Selection theory

Back 62

Burnet-Jerne invented in 1950's. Said Ag selected for one particular lymphocyte that had one specificity and it multiplied.
+removes need of antigen to enter cell
-seems to require more than 50% of genome to generate that many specificities.

Front 63

Current paradigm for B cell specificity variety generation = Advanced clonal selection theory

Back 63

1)Germline variation = different alleles of HC and LC exist in population -> allows evolution of resistance on a population level (think smallpox)
2)Minigene shuffling = combination of subsets of HC and LC by translocation while an embryo
3)Somatic Mutations = controlled, targeted mutations in CDR fetally
4)When infected those B cells whose AB match epitope on Ag multiply
5)Somatic Mutation during infection = memory cell CDR become less stable, allowing for clonal selection of even better fitting paratopes

Front 64

Experiment to discover minigene shuffling

Back 64

Tonegawa placed adult mRNA on a matrix and added fetal DNA. Saw huge loops, even bigger than what alternate splicing causes

Front 65

Diagram minigene shuffling of LC DNA

Back 65

Get one V with one or more J by looping out a certain number of V's and J's. Each J comes with an L region containing the promoter.
48 K v's
30 lamda v's
5 J's

Front 66

Mechanism of minigene shuffling

Back 66

Recombination Signal Sequence (RSS) on each side of the exon is palindromic and allows the Recombination Activating Gene [RAG] to loop the DNA and cut it there. Ligase recombines the chromosome and the loop into a circle. The circular DNA is destroyed. All in fetus. Only multiple upstream Vs left and downstream constant regions.

*SCID mice form no functioning lymphocytes b/c they lack RAG

Front 67

Diagram Heavy Chain fetal development

Back 67

16 Oct notes.

Same as LC w/ looping excision to choose 1 V region, 1 or more D regions and 1 or more J regions

Roughly 44 V's, 23 D's, and 6 J's.

Front 68

Origins of diversity in B cell specificity [CDR variability]

Back 68

1)Germline Variation
2)Minigene shuffling = combining different V with diff J and/ or D
3)Different Light chain - heavy chain combos
4)Junctional diversity = loss of nucleotides when joining the minigenes
5)Nucleotide addition at P or N sites in V region - TdT enzyme
6)Somatic mutation at three sites in V region

Front 69

Heavy chain class switching - Diagram

Back 69

18 Oct notes
Variation in cytokines [], mix, or type lead to activation of different switch region in front of constant region

-Also palindromic and acts like RSS to lead to looping and excision of certain constant regions
-Always produce the one right after end of last J region
-Mu is always first produced
-Mu and delta not separated by switch, but delta only produced in embryo by alternate splicing. (Stop codon between mu and delta is suppressed embryonically).

Front 70

Atopic individuals

Back 70

Jump right to producing IgE from IgM in response to certain polysaccharide Ags, commonly have bad allergies