Immunology 3- Antibody Structure + Function Flashcards
What two forms do antibodies come in?
Soluble (antibody) and membrane bound (BCR)
What shape are antibodies typically
Y SHAPED
How many polypeptide chains comprise an antibody?
4
2 heavy chains and 2 light chains
The two heavy chains of an archetypal antibody are identical in amino acid sequence, as are the light chains. True or false?
True
How many amino acids are usually found in the heavy and light chain?
Heavy chain = 450
Light chain = 200/250
How many domains does the light chain have and what are they called?
It has two.
The variable light chain (VL)
And the constant light chain (CL)
How many domains are found in the heavy chain? What are they
4.
The variable heavy domain, and the three (usually) constant heavy domains CH1, CH2, CH3
What is found intra and intermolecularly along an antibody?
Disulfide bonds
How many genes does the light chain have? What are they? (?: KL)
It has two. Kappa and Lambda
How many genes does the heavy chain have? (?: GAME Time)
G:gamma
A:alpha
M:Mu
E: elipson
T: theta
What determines the class of an antibody?
By its heavy chain type
Each class of antibody has the same properties, true or false?
False, they each have different properties
What stabilises the heavy chain ?
Glycosylation
What links the heavy and light chains?
A disulfide bond
What two class of molecules can an antigen bind to?
- Bind antigen- tips/variable (Fv) paratope of antibody
2. Associates with host cells (I.e dendritic cells)- at the constant regions
Antibodies are used to identify and neutralise parasites and viruses? T or false?
True?
Recognition of the antigen occurs at Fc. True or false?
False it occurs at Fv
How many amino acids long is Fv?
15-22
In what two ways can an antibody neutralise it’s target antigen?
- It can tag/opsinonise for phagocytosis
2. Can block regions of the antigen vital for toxicity or survival/growth
What do BCRs do?
They facilitate the activation of the B cells they’re associated with
What interaction most commonly activates B cells?
B cells with helper T cells
What are antibodies also known as?
Immunoglobulins
What did proteolytic cleavage of antibodies allow scientist to achieve?
To assign function to the antibody structure
What 3 digests were performed on antibodies?
Papain, pepsin and Marcoptoethanol reduction
What results did antibody Papain digestion yield?
Papain cleaves at hinge regions between CH1 and CH2 above S-S bond between heavy chains , producing three molecules fragments. 2 x Fab and 1 x Fc
What results did antibody Pepsin digestion yield?
Produced 1 large molecule and several tiny fragments. Pepsin extensively digests below disulphide bond between heavy chains. Yields an F(ab’)2 + Fc fragments
What did Mercaptoethanol reduction produce?
4 fragments/chains, 2x heavy and 2 x light.
Why was Fc termed so?
Because it was a fragment (F) that could crystallise (c)
Why can Fc crystallise?
Because it has constant/conserved amino acid sequence
What did the papain digest determine?
That the fab fragment was monovalent, in that it bound 1 molecule
What did the pepsin digest determine?
That the F(ab’)2 fragment was bivalent, so it was deduced that the antibody must contain two Fab regions, arranged in a Y shape
Why is Fab termed so?
Because it is the fragment (F) that antigen (a) binds (b) to
What is the purpose of the hinge region?
To allow flexibility of the distance between binding areas.
What is the term for the hyper variable regions found at the paratope of an antibody? How many are there on each chain and at what amino acid sequence positions?
Complementary determining regions (CDRs)
There are 3;
CDR1= 25-30 CDR2= 50-60 CDR3= 80-95
There are no CDRs on the variable heavy chain domain. True or false?
False, there are 3 on each heavy chain at the variable domain, at HVR/CDRs. Each paratope contains 6 CDRs
What do CDRs do?
They determine the shape of the binding site
What is the Ig fold?
A common structure of Igs. Consists of 2 beta sheet folds, one on the CL and one on the VL. The beta sheet fold of the VL domain brings it’s 3 CDRs into close proximity. Also present on the tips of the 3 other chains
There are 6 CDRs in a Y shaped antibody overall. True or false?
False. There are 3 on each chain so 12 overall.
What is the binding cleft of an antibody?
It is the region where antigens bind and is brought about by the coming together of 3 CDRs of the light chain and 3 CDRs of the heavy. It can accommodate up to 12aa (if a peptide)
The two binding sites of an antibody aren’t identical. True or false?
False, they are identical
If an antigen has 4 epitopes what is it termed?
Multivalent
If an epitope of a protein is Multivalent and they are identical, you would need different antibodies to bind them all. True or false
True. Identical epitopes in protein antigens are rare, but common in carbohydrate antigens
If an epitope of an antigen consisted of consecutive residues what is it termed?
An LINEAR epitope
If an epitope of an antigen consists of residues at different positions but specially close then what is it called?
A distcontinous epitope
Linear epitopes are just as common as discpntinous epitopes, T or F?
true
What is an immunogen?
It is an antibody that evokes an immune response
Polyclonal antibodies recognises multiplied epitopes of any one given antigen. True or false?
True
Polyclonal antibodies are taken from the blood of an animal (such as a mouse) after it has mounted an immune response to the introduced pathogen, true or false?
True
What mixture of antibodies will be in a polyclonal antibody sample ?
A heterogenous mixture of antibodies of differing affinities
Polyclonal antibodies are mostly IgA, true or false?
False, they are mostly IgG
What type of antigens are used to generate polyclonal antibodies, and which animals are usually used?
Peptide antigens, and animals such as goats, rabbits and mice
What 6 advantages do polyclonal antibodies have?
- Inexpensive to make
- Amplify the signal of the LOW EXPRESSED protein antigen, many antibodies bind to one molecule of antigen
- Less susceptible to chemical/physical changes in the anitgen
- Can recognise homogenous antigens
- Generally more ROBUST than monoclonal antibodies
- Less complex and time consuming
3 disadvantages about polyclonal antibodies?
- Not suitable for probing specific antigen domains
- Low specificity in general
- More background noise (especially in staining)
Monoclonal antibodies detect several types of epitopes of a given antigen, true or false?
False, they only detect one type of epitope
Monoclonal antibodies are specific to an antibody subtype e.g. IgG1, IgG2 etc. t or f?
True
Monoclonal antibodies have low specificity, true or false?
False, they have low overall affinity, but high specificity
Name 4 disadvantages to monoclonal antibodies?
- Expensive to produce
- More susceptible to changes in the antigen (desaturation)
- Technically more challenging to manufacture
- More time consuming, need to culture the antibodies
Name 6 advantages of monoclonal antibodies?
- Once made, they are a constant and renewable source
- They are good for affinity purification techniques, high specificity
- They are less likely to cross-react
- Less BACKGROUND noise
- Highly reproducible results
- Best at detecting antigen in tissue (AIDs/CANCER etc)
Name 6 applications of monoclonal antibodies?
- Diagnostics
- Drug testing
- Therapeutics, delivery of drugs, e.g radioimunnnotherapy in cancer treatment
- Capable of Classifying single strains of pathogen, high sensitivity
- Detecting and location of trace molecules in organisms
- Alleviation of ORGAN transplant rejection
Briefly explain the process of monoclonal antibody production!
- Inject chosen organism e.g mouse/rabbit with pathogen
- Take serum from spleen, and spleen cells (mostly plasma cells)
- Fuse B cells with cultured myeloma cells (done by virus or electroporation)
- Culture mixture on HAT medium (hypoxanthine-aminopterin-thymidine) Only Hybridoma cells survive
- Screen for desired antibody using SDS-PAGE and western blot analysis (probe is the epitope to the desired antibody)
- Separate using immunoflourescence or affinity chromatography
Describe the process of polyclonal antibody production in 6 words?
- Infect
- Serum
- Fuse
- Select
- Screen
- Isolate
IS FSSI HOME??!
What is the methodology behind the HAT medium in selection of hybridoma cells?
Hypoxanthine amino-pterin Thymidine (HAT) only supports cells with the enzyme HGPRT (hypoxanthine-guanine-phosphoribosyl-transferase) which is needed for salvage nucleic acid synthesis. B cells have it, but die in the medium as they have finite lifespan. Myeloma cells don’t have this enzyme and so can’t synthesis nucleic acids needed for growth and survival. Hybridoma cells have HGPRT and have infinite lifespan
Chimeric monoclonal antibodies are less likely to evoke an immune response in humans compared to humanised antibodies? True or false
False, humanised antibodies are less likely to evoke an immune response than chimeric ones
What is the difference between a chimeric antibody and a humanised antibody?
An chimeric antibody has the Fab section of a non-human antibody sequence, and the Fc section of a human.
A humanised antibody consists of all human derived amino acid sequences, except those belonging to the CDRs, which origin from a non-human species
Nowadays monoclonal humanised antibodies can be synthesised using techniques such as phage display ?. T or F?
True!
IgG has 5 subclasses, true or false?
False, it has four, (three in mice, no IgG4)
How many subclasses do IgA have?
2
To which areas are IgA antibodies most frequently found?
Mucosal associated lymphoid tissue, in respiratory and gastrointestinal tract.
IgA and IgG are not related, T or F?
False, they are closely related
What difference between the subclasses of IgG are most significant?
The length and flexibility of their hinge region
As a general rule, the higher the flexibility of an IgG the more susceptible it is to cleavage (thus shorter lifespan) and the more accessible it is to C1q to initiate complementation. True or false?
True. IgG3, has 62 amino acids in its hinge region and is good for complementation, yet it is often cleaved by proteolytic enzymes of pathogens
How many amino acids are in the hinge regions of IgG3 and IgG2?
- 62
2. 12
IgGs are numbered 1-4 in decreasing order of prevelance, true or false?
False, they are numbered with IgG1 the most, all the way to IgG4 the least prevalent
Which of the IgG subclasses have the most rigid hinge region?
IgG2
How many allotypes does IgG3 have? And is it the most polymorphic of any IgG?
19 and yes it is
What is alloytyping of Immunoglobulins useful for?
Diagnostics and paternal exams
How many polypeptides is C1q comprised of?
18
How many IgGs are required for compliment fixation? And how many IgM’s?
6 and just one for IgM
Which immunoglobulins are best for initiating the compliment cascade?
IgG3 and IgM, IgG1 is also good
Outline the complementation cascade, leading to the Membrane attack complex causing pathogen cell lysis?
- C1q component of C1 is activated by either binding to cluster of antibodies (6xIgG or 1x IgM) on pathogen cell surface or binding directly
- Activation of C1q leads to activation of C1r2 (Serine protease) which then cleaves and activates C1S2 (also serine protease)
- C1r2s2 combine to cleave C2 and C4, producing C2a,C2b,C4a and C4b
- C2a+C4b forms C3-convertase (C4b2a)
- C3-convertase cleaves vital C3, forming C3a and C3b
- C3b binds to C5, cleaving it into C5a and C5b
- C5b binds to pathogen surface, along with C6,7,8 and 9 to form MAC (membrane attack complex)
- MAC is inserted into outer membrane of pathogen, leading to increased osmotic pressure and eventual cell lysis (death)
What is the function of C3a?
It is a precursor to the cytokine Apidokine, attracting inflammatory cells
What is the function of C5a?
It is a chemotactic protein, attracting phagocytic cells to the area of infection
What is a Anaphylatoxin? Give examples
They cause smooth muscle relaxation, histamine release of Mast cells and increase in vascular permeability for lymphocyte migration into tissue
C5a and C3a are glycoproteins, True or false?
True
What is CK-MAC?
CELL KILLING MEMBRANE ATTACK COMPLEX. End product of complementation cascade
What other role does C3b do ?
It can act as an OPSONIN
What is the main role of compliment?
It compliments the ability of antibodies in fighting pathogens
Compliment is part of the adaptive immune system, true or false?
False, it is part of the innate Immune system
How many proteins are involved in compliment?
Around 30 protiens
What do complement proteins circulate in and what form do they usually take?
They circulate the blood, as inactive precursors called pro-proteins
Serine Proteases comprise a large group of complement proteins, true or false?
TRUE!
What cells synthesis these compliment proteins?
Macrophages and Monocytes and epithelial cells Urogenital/Gastrointestinal system
Apart from the classical compliment pathway what other pathways are there and what do all three have in common?
- The alternative pathway- C3 is hydrolysed not by C3-convertase
- The mannose-binding lectin pathway- no antibodies needed
They all involve C3!
To what region does C1q bind on the antibody?
The Fc region
What does the classical compliment pathway require?
A specific immune response: antigen:antibody interaction
Where are IgA’s mostly found?
In the gastrointestinal tract or any other mucosal site
IgM is heptametic, t or f?
False, it is pentameric
How many antibodies comprised IgM? And what are they joined by?
5, joined by intermolecular disulfide bonds
What does the M stand for in IgM?
Mu!
What domains are found on the heavy chain of IgM?
1 variable Vh and 4 constant heavy domains!
There is an S-S bond between CH2 and CH3, true or false ?
False, it is between CH3 and CH4
What is J? In regards to IgM
It is the joining chain
How many amino acids make J chain?
About 20
What does the J chain of IgM do?
It is a small peptide essential for linking each of the five antibodies
Where is J chain synthesised and amalgamated with IgM components? What happens if IgM is without J chain?
In the ER, and without it, it isn’t secreted
In what weight range is IgM in (daltons) and how many polypeptides make it?
Millions of Daltons and 21 peptides
What percentages of each of the five immunoglobulin isotopes comprise the immunoglobulin fraction of human serum?
Ig:
alpha (A): 15%- Gamma (G): 75# Delta (D): less than 1% Elipson (E): less than 1% (least abundant) Mu (M): 10%
IgG is the first antibody produced in an immune response, true or false?
False, IgM is!
What does Secretatory IgA require for its transport across epithelia?
Poly-Ig receptor
IgM is a monomer in membrane/receptor form….true or false?
True
Of the Ig isotopes, which are monomer, dimers etc
IgG, E and D = monomers
IgA= dimer
IgM= pentameric
Binding at the CDRs to the epitope of antigens is covalent, true or false?
False, they are non-covalent bonds (h-bonds, van der waals, hydrophobic interactions)
How many binding sites does IgM have? Are they all identical?
10 and yes
IgM is very inefficient at binding bacteria, T or F?
False it is very efficient
What two notable conformations can IgM take?
The star - 2D
The staple - like a crab claw
How can IgM take on this staple conformation
Due to flexibility between CH2 and CH3
IgA is found in secretions of the gut lumen and functions to protect the mucosal surface, t or f ?
True
IgA is essentially 2 IgG’s linked by a secretatory component, t or f?
True
What does the secretatory component of IgA do?
It binds to the poly-Ig receptor for transportation across epithelia
Stabilises and protects the immunoglobulin from digestion
The human immune system has evoked to combat proteolytic cleavage of IgA by developing what vital component?
The secretatory (S) chain/component
IgE is a major component of the immune system, t or f?
False it is a minor component found in trace amounts in human serum
To what does IgE bind for stabilisation?
To Fc receptors
If a Fc receptor specifically binds immunoglobulin gamma (g) what is it termed?
FcY(gamma)
To which immune cells are FcE receptors predominantly found?
Mast cells and basophils
What occurs when IgE binds mast cells and basophils?
- On first exposure/interaction there is little to no release of allergic causing components of mast cells and basophils, but IgE can cause up regulation of FcE receptors and associated memory B cells, the response is primed for a more intense secondary exposure reaction
- On second exposure to an allergic causing antigen, immune cells are primed with FcE receptors and larger quantities of IgE, causing degranulation of histamine and inflammatory mediators causing muscle contraction and vasodilation causing typical allergic symptoms
What specifically does a basophils release on binding of IgE?
IL-4, IL-13 and TYPE 2 cytokines
What is atopy define as and what is it caused by?
Hypersensitivity- an excessive IgE reaction and concentration in the blood serum leading to long-lived interaction with FcE receptors of mast/basophils causing overstimulation
What type of pathogen do Mast cells typically fight? And what is a bi-product of their reactions?
Extracellular parasites and a bi-product is an allergic reaction
Allergies are going down globally, true or false
False, they are in fact rising
Each Fc receptor compliments certain antibodies functions, MANY are inhibitory and some are activators , t or f?
False, they do compliment, but most Fc receptors are activatory and some are inhibitory
Different cells have different Fc receptors, true or false?
True
What is a functional immune response controlled by?
Through an exquisitely controlled process, integrating signals from activating or inhibitory Fc receptors on the immune cell surface
What do ITAMs and ITIMs stand for
ITAMs: immunreceptor-tyrosine-activating-motifs
ITIMs: immunreceptor-tyrosine-inhibitory-motif
What does the quality and magnitude of an immune response rely on?
Depends on two short, loosely conserved motifs found in various Intracellular domains of signalling proteins (Fc receptors)
What do ITAMs and ITIMs act as (?: ?)
Docking sites for Tyrosine kinases for signalling cascade, leading to either inhibitory and activatory cellular responses
What part of an immunoglobulin is vital for maintaining its membrane bound form?
The C-terminus, which is embedded in the membrane
What occurs when the BCR interacts with the epitope of an antigen?
Signals for differentiation into a plasma cell and expresses its immunoglobulin in secretatory form
In what timeframe/volume and longevity are antibodies produced during the first exposure of a new antigen?
Time: 1-2 weeks
Volume: not high
Longevity: short half life of antibodies but creation of memory B cells
In what timeframe/volume and longevity are antibodies produced during the secondary exposure of an antigen? And what allows for this more intense response?
Time frame: hours to days
Volume: high
CSAE: clonal selection and expansion, expands the population of plasma cells for a given antigen
The affinity of the antibody produced in a secondary response is of lower affinity than the first, true or false?
False, it is higher, due to affinity maturation
During the first exposure IgM is made first but is soon overtaken in volume by IgG which has a higher affinity for the antigen. True or false?
True
What is the term when antibody concentration within the body is at a minimum?
Basal levels
On the secondary exposure, IgG is made is lower concentrations but in higher affinity and speed, true or false?
False, they are made in faster times and have a higher affinity, however they are also made in greater volumes
