5. Antibodies Flashcards
What type of immune response are antibodies part of?
Adaptive immune response
What are the four roles of antibodies? What are names of the forms?
- Cell surface receptors - IgM/IgD
- Blood plasma - IgG/IgM
- Inflammation - IgE
- Lining intestines/mucosal tract - IgA
Which antigens are expressed on the surface of B cells?
IgM and IgB
Describe what roughly happens when the adaptive immune system is activated
- Surface IgM/IgD of B Cell binds antigen by chance (weak interaction)
- Antigen gets chewed up into little fragments which are expressed on surface
- Interacts with T cell via TCR
- Cytokines released causing B cell to copy itself
- Clonal expansion, antibody maturation, optimisation of binding and cats switching to IgG occur
- IgG then binds to antigen undergoing blocking, opsonisation and triggers complement
What is antibodies maturation?
Mutation of antibodies in order to optimise binding
What is class switching?
Switching of IgM/IgB to a different type of antibody (IgG) which is soluble and can enter blood plasma
What is opsonisation?
Binding of antibody to pathogen epitope which attracts phagocytes (which bind using Fc receptor)
Describe the antibody composition
Light chain - 220 AA long - 2 domains -VL and CL (constant and variable regions)
Heavy chain - 440AA long - 4 domains - VH, CH1, CH2, CH3
Variable Hinge region
2 heavy and 2 light chains in each antibody
Fc region, Fab arm, CDRs
Each domain has disulphide bonds - 2 between 2 heavy chains and 1 linking each heavy chain to a light chain
What are polyclonal antibodies? Do they crystallise?
Polyclonal antibodies are a mix of antibodies which bind the same antigen.
These do not crystallise as they are all different.
What are monoclonal antibodies? Do they crystallise?
Identical antibodies
No, they are flexible which prevents the formation of a regular lattice
How is it possible to crystallise antibodies?
- Cleave antibodies into 2 fragments using Papain
- FAB (fragment antigen binding) regions do not crystallise because they vary highly
- BUT Fc regions do! This is the stem bit and remains constant
How do papain and pepsin cleave antibodies in different ways?
Papain cleaves antibody above the disulphide bond into 2 FAB fragments and 1 Fc region
Pepsin cleaves antibody below the disulphide bond into 1 FAB’ fragment and 1 Fc region
Which domain of the antibody contains sugars?
The CH2 domain (bit before the hinge region)
How do IgG and IgM/IgE vary?
IgM/IgE have an extra CH4 domain
How many loci are in the antibody genome? What are their names?
Lambda light chains, Kappa light chains and Heavy chains
Describe the structure of the lambda light chain loci. How is variation generated?
- 30 variable gene (V) segments with leader sequence in front
- 4 constant gene (C) segments
- Joining (J) segment in front of each C segment
- Splicing occurs at DNA level, taking V segment and splicing to J segment and then splicing VJ to a C segment
What is the difference between kappa and lambda light chains?
No functional difference but gene loci are arranged differently
How many different variation of the lambda light chain are there?
30x4 = 120
Describe the loci of the kappa light chain. How many variations are there of it?
40 Variable genes (V), 5 J segments, 1 constant region
200 variations!!
Describe the loci of the heavy chain. How many variations are there of it?
65 Variable (V) segments, 26 diversity (D) segments, 8 joining (J) segments, 1 constant (C-mu) domain
4,212,000 variations
What is the total amount of different antibodies producible?
1.3billion
What are the 5 antibody classes? Describe each one.
IgM - early response antibody - 2 forms due to alternative splicing at mRNA, one w/ tail which sticks into membrane and produce surface receptor
IgD - Receptor
IgG - Late response, antibody the goes round killing shit
IGA - found in tears/mucus
IgE - degranulation of histidine release, insect bites - IgE goes to bite creates MAST cell which causes blood vessels to open up
How do the different antibody classes vary?
By their heavy chain.
IgM - mu IgD - delta IgG - gamma IgA - alpha IgE - epsilon
The greek stuff is the name of their heavy chains
How does class switching occur?
- Heavy chain locus contains a series of alternative constant domains
- Switch sites are present in front of every constant domain
- Switch sites come together in DNA in middle is looped out and removed
- this process is uni-directional
What are the three types of Fc receptors? What cells are they present on?What do they do?
FcgR - binds IgG, present on neutrophils/macrophages/monocytes and triggers phagocytosis (of the IgG coated pathogens)
FceR- binds IgE, present on eosinophils/basophils/mast cells & triggers histamine release
FcaR- binds IgA, present on neutrophils/monocytes/macrophages/ eosinophils and responsible for phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), release of superoxide and inflammatory mediators
How do IgM and IgA form multimers?
IgM forms a pentamer with 4 other IgM molecules attached via a J chain (not related to J segment).
IgA forms a dimer in the same way
What does C1q do?
- c1q binds to IgM and IgG
- binds to at least two Fc’s to become active
- c1q then drills hole in the cell like a big pneumatic drill!!!
woah steve that’s sick
Describe the protein structure of the Fab fragment arm
- each domain has immunoglobulin fold
- Ig fold consists of a beta sandwich of 110AA with the beta sheets inclined at 30deg. to each other (superfold - stable
- disulphide bridges link the 2 sheets and stabilise the beta sandwich - stable framework to retain shape when constantly changing CDRs
- VH/VL chains can move with respect to one another (by 60deg.) - this means the binding site at the end can change considerably
Between which domains of the antibody do interactions occur?
VL and VH
CH1 and CL
CH3 and CH3
CH2 DOES NOT INTERACT
How may you imagine an antibody with the aid of a few LSD tabs?
AS A BODY - SHOULDERS AND ELBOWS AND HANDS THEY ALL CAN MOVE HALP
How do the VL and VH domains interact?
Vie 5 strand sheets to form a barrel shape
What molecules sit between the CH2 fragments? What do they do?
Sugars - dis is odd
dunno
What are the elbow angles of an antibody?
The angle between the variable and constant domains
What molecules may antibodies interact with?
Proteins, peptides, carbs, lipids, small molecules
What did Wu and Kabat do?
undertook sequence analysis, Invented the variability plot (plot each position in a sequence and how variable it is), found hyper variable regions (CDRs) of antibodies
Also found that framework regions of antibodies are well conserved (even between humans/mice)
Something about CDRH3 and CDRL3 sitting in middle of binding site
:)
What was Chothias analysis?
Collected antibody structures and found that the structures of the hyper variable loops were actually well conserved in shape - all the loops would be similar shape (even if had different AA)
Found the difference to be in CDR length and difference in certain key residues
What are the key residues in CDR-L1?
I, A, VIL, LM, YF
What may antibodies be used for in vitro?
- on immunoaffinity chromatography columns
- diagnostic agents (measure presence of stuff in blood e.g. ELIZA assay for testing HIV presence
- therapeutic agents
- ## protein purification/labelling - western blots
What may antibodies be used for in vivo?
- magic bullets (targeted drugs - paul ehrlich)
- imaging - gamma-imaging, PET, guiding surgery
- therapeutic - immune activation, direct effects
- targeting - radio, direct, indirect (ADEPT)
- catalytic -
What is ADEPT?
Antibody directed enzyme pro-drug therapy
Prodrug is a drug which is activated by metabolism
basically pro-drug injected into body directed by antibodies and then activated
What are the various treatments offered by antibodies? What do they treat?
Herceptin - cancer
Reopro - binds to platelet fibrinogen receptors to stop platelet clumping and artery clogging
Synagis - introduces passive immunity by binding to respiratory syncytial virus (stops REV infections)
How does Radioimmuno guided surgery work?
Patient injected with radio-labelled antibody before surgery
What is radioimmuno-scintography?
Radio labelled Fab fragments can be imaged used to detect bowel cancer (anti-CEA antibodies used)
What are abzymes?
An antibody which has enzymatic activity
What are abzymes used for?
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