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BIOC2004 > 6 - Antibodies > Flashcards

6 - Antibodies Flashcards

1
Q

How does the structure of antibodies allow for their function?

A

They have two specific regions that allow linking to two objects as well as a third binding surface that can be used to bind them onto a membrane or other surface while still retaining their function.

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2
Q

What are IgA antibodies?

A

These are dimeric antibodies that line the intestines and mucosal tract as a first line of defence against pathogens.

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3
Q

What are IgD antibodies?

A

Early response antibodies, cell surface bound.

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4
Q

What are IgE antibodies?

A

Degranulation
Binding to poisons
Stimulating mast cell histamine response

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5
Q

What are IgG antibodies?

A

The primary plasma antibody, responsible for opsonisation and agglutination.

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6
Q

What is opsonisation?

A

Identification of pathogens.

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7
Q

What are IgM antibodies?

A

Early response
Expressed on B-cell surface
Can form soluble pentamers that link at their J chains

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8
Q

What is the chain structure of an antibody?

A

Two long heavy chains that combine to form two ß-sandwiches at one end and split apart at the other at a midpoint linker sequence to allow two half-length light chains to make another two ß-sandwich immunolobulin fold with either one.

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9
Q

How many amino acids make up the heavy and short chains respectively?

A

440 and 220

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10
Q

What is polyclonal?

A

An antibody sample that contains a mixture of antibodies with different variable regions.

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11
Q

Why can polyclonal antibodies not be used for structural analysis?

A

Methods such as XRD require very high purity, and the difference in the variable regions in a polyclonal mixture would prevent proper crystallisation.

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12
Q

What must antibodies be in order to have their structure determined?

A

Monoclonal, but even then the hinge region flexibility can prevent proper crystallisation.

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13
Q

How are monoclonal antibodies prepared from polyclonal mixtures?

A

By affinity chromatography using whatever the antibody is specific for.

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14
Q

How can monoclonal antibodies be prepared de novo?

A

Crossing a specific antibody producing B-cell can be crossed with a tumour cell line causing proliferation and hence large amounts of monoclonal antibody production.

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15
Q

How can the structure of immunoglobulins be determined despite the difficulty in crytallising even monoclonal preparations due to the flexibility of the molecule?

A

By cleaving them into fragments composed of their three domains.

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16
Q

What is used to cleave antibodies into their domain fragments?

A

Papain protease cleavage of the heavy chain ‘hinge’ linker sequences.

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17
Q

What fragments are produced by papain cleavage of anitbodies?

A

2 Fab fragments that are the variable domains

1 Fc fragment that was the dual heavy chain region

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18
Q

How many amino acids make up a ß-sandwich immunoglobulin fold?

A

110 residues.

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19
Q

Why is fragmentation useful for structural analysis?

A

The linker regions are the largest obstacle to whole antibody crystallisation as they allow all the regions to move with great freedom relative to one another. This allows them to have their structure analysed separately.

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20
Q

Describe the immunoglobulin fold.

A

A stable fold consisting of two ß-sandwich motifs that are packed together at a 30° angle.

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21
Q

How many immunoglobulin folds make up a single antibody?

A

12 - 14, four of them binding in pairs in each fragment region.

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22
Q

How are immunoglobulin domains named?

A

V or C depending on their constancy or variability for an antibody type, with an L or H in subscript to denote whether they are on the light or heavy chains. On the heavy chains the constant regions are notes as 1, 2, 3 and sometimes 4 starting from the variable end.

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23
Q

Which immunoglobulin types possess heavy chain C_H4 domains?

A

IgMs and IgGs, hence these are the ones that have fourteen total domains.

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24
Q

How are disulphide bonds used in antibodies?

A

Each domain is stabilised by a single disulphide bond and more are employed to join the domains at the hinge region.

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25
What post translational modifications are present on antibodies?
The two hinge adjacent immunoglobulin domains (C_H2) in the heavy chain Fb fragment are linked by a pair of carbohydrate molecules.
26
How are the structure of the variable domains denoted?
The end half of the Fab fragments are known as the Fv fragments - the variable regions. These are tipped by hypervariable antigen binding sites on each chain called complementarity determining regions.
27
How are the antigen binding sites themselves formed?
The Fv pair of immunoglobulin domains come together to form a barrel that binds the antigen.
28
What is a paratrope?
The variable antigen binding tip of the Fv region.
29
What is an epitope?
The antigen to which a specific paratrope binds.
30
Which loci code for the heavy chain?
Just the Heavy Chain Locus
31
Which loci code for the light chains?
The lambda and kappa loci.
32
How was it realised that antibody genetics was employing an unusual mechanism?
Because we can produce billions of different antibodies, more than we have sufficient base pairs to code for in our entire genome.
33
Where are antibodies produced?
In B-cells.
34
How are so many antibodies produced from such little genetic material?
Each different region of the antibody is coded for by a wide variety of available versions which can be combined in any way to produce a huge number of different structures.
35
What is unique about antibody genetics?
The different DNA sections are spliced together at the DNA level, each B-cell becoming specific to producing a single antibody by excising and degrading the rest of the DNA.
36
How does splicing occur at the lambda locus?
Four regions to choose from in two pairs. L and V regions always selected together (ie. uses L1 and V1 or L4V4) J and C regions paired similarly.
37
How many LV pairs are present at the lambda locus?
30
38
What do the L and V sections code for?
The Light and Variable chains respectively.
39
How many JC pairs are present at the lamda locus?
4
40
What to the J and C sections code for?
The Joining and Constant regions respecitvely.
41
How many different combinations of light chains can be produced at the lambda locus?
30 (LV pairs) x 4 (JC pairs) = 120 combinations at the lambda locus.
42
What sections are coded for by the kappa locus?
LV pairs, as in the lambda locus. | J and C sections, now spliced independently from one another.
43
How many LV pairs are present at the kappa locus?
40
44
How many different J sections are present at the kappa locus?
5
45
How many different C sections are present at the kappa locus?
Only one.
46
How many different combinations of light chains can be produced at the kappa locus?
40 (LV pairs) x 5 (J) x 1 (C) = 200
47
What sections are coded for at the heavy chain locus?
LV pairs D (diversity) segments J chains C regions
48
How many LV pairs are found at the heavy chain locus?
65
49
How many D sections are found at the heavy chain locus?
27
50
How many J sections are found at the heavy chain locus?
8
51
How many C sections are found at the heavy chain locus?
Many, but these do not contribute to the antigen specificity at this locus.
52
Which locus is responsible for the greatest amount of diversity in antibody structure?
The heavy chain locus, by far.
53
What two features of heavy chain locus splicing give it such huge diversity?
Possession of an extra section, D, that is not present at either of the light chain loci. The heavy chain locus shows imprecise recombinance. Can be read from multiple reading frames.
54
By what factor does the imprecise recombinance and multiple reading frames increase the number of different combinations possible at the heavy chain locus?
300x
55
How many different combinations of heavy chains can be produced at the heavy chain locus?
65 (LV) x 27 (D) x 8 (J) x 300 (IR) = 4,212,000
56
How many total codable antibody variants can be made by the combined diversities of the lambda, kappa and heavy chain loci?
(140 [L] x 4,212,000 [H]) + (200 [K] + 4,212,000 [H]) = 1,347,840,000, nearly 1.5 billion. Note that the light and kappa chains are mutually exclusive.
57
How is even greater antibody variation produced outside of the diversity of the loci?
All of them are in a hypermutateable region, so controlled somatic mutation can be used to increase the variation.
58
Which locus section determines which class (A, G, E etc) of antibody is produced?
The heavy chain locus C section.
59
How are the heavy chain locus C sections named?
α, δ, ε, γ & μ according to whether they code for the | A, D, E, G or M antibody classes respectively.
60
What consequence on adaptability does the unique DNA splicing antibody genetics have?
Because the DNA not used is excised and degraded the specific splicing selection is unidirectional.
61
What is the exception to the irreversibility of antibody type selection for a B-cell?
Heavy chain C regions are left in the DNA, and preceded by switch sites that allow B-cells to produce multiple classes of antibody all with the same variable regions and hence specificity.
62
What functions do antibodies serve upon binding to the pathogenic antigen (epitope)?
Agglutination due to the binding of two pathogens per antibody, and activation of/recognition by the immune system.
63
How so antibodies activate the immune response?
Immune effector cells express Fc receptors that bind to the Fc fragment end of the antibody when it is boud to a pathogen. Complementarity with different Fc receptors can stimulate a different effect.
64
What Fc receptors recognise IgG?
FcgR - Fc G Receptor
65
What does FcgR receptor stimulation by IgG cause?
Acts on neutrophils, macrophages and monocytes to stimulate phagocytosis of the antibody covered pathogen.
66
What Fc receptors recognise IgE, and which cells express them?
FceR - Fc E Receptor Recognised by eosinophils, basophils and mast cells.
67
What does FceR stimulation be IgE cause?
Histamine release form intracellular granules.
68
What Fc receptors recognise IgA, and which cells express them?
FcaR - Fc A Receptor Recognised by neutrophils, eosinophils, monocytes and macrophages.
69
What does FcaR receptor stimulation cause?
Antibody Dependent Cell-mediated Cytotoxicity (ADCC) - the destruction of infected cells through superoxide release. Also triggers release of inflammatory mediators.
70
What non-Fc named receptor also allows antibody Fc regions to activate an immune response?
C1q
71
Which antibody Fc regions bind to C1q, and what effect does this have?
IgM and IgG bind C1q to activate the classical complement pathway.
72
What is different about IgM and IgG binding to C1q compared to other Fc receptors?
Two Fc regions must bind to the C1q, causing a conformational change in the Fc themselves. This also means that this activation is more concentration dependent.
73
How many distal parts of the chains come together to form the binding area?
Six, three from each chain labelled H1-3 and L1-3
74
What part of the binding end is responsible for the most variability?
A loop that can vary in length and composition greatly which is the more proper definition of the CDR.
75
Which gene segments code for the third hypervariable regions?
The J and D sections
76
What kind of molecules can antibodies bind to with specificity?
Proteins/peptides ssDNA Carbohydrates/haptens
77
What part of the antibody do proteins bind to?
Onto a flat surface.
78
What part of the antibody does ssDNA bind to?
A narrow groove.
79
What part of the antibody do short peptides bind to?
A groove.
80
What part of the antibody do carbohydrates and haptens bind to?
A pocket.
81
What are haptens?
Small molecules added to protein or other large carrier that causes it to elicit an immune response.
82
What are the primary uses of antibodies in the lab?
Purification Labelling Western blots
83
Why are antibodies useful for measuring very low concentrations of things, and what is this used in?
Their high specificity means that they can be used to detect pathogens and measure the micro-concentration of oestrogen and progesterone in HRT.
84
What is radioimmunoscintigraphy?
Antibodies can be used with PET scanning by attaching the positron emitter to the end of an antibody specific to the tumour. This reduced the gamma dose elsewhere.
85
How can antibodies be used in chemotherapy treatments?
They can be used to direct damaging elements such as radioactive heavy metals to the tumour, thus limiting the effective dose elsewhere.
86
How are antibodies used in surgical techniques?
Visibly (or live camera visible) fluorescent antibodies can be raised to a target tissue and applied to the area in order to visualise specific targets.
87
What is HER2?
A potent variant of breast cancer that is characterised by overexpression of EGF receptors that accelerate tumour growth.
88
How can antibodies be used to treat HER2 breast cancer?
By using monoclonal antibodies (mAbs) that act as an antagonist to the EGFR (binds to and inactivates) the added risk of the overexpressed growth factor receptors is partially nullified.
89
How can antibodies be used to treat RSV?
Respiratory Syncytial Virus is often fatal in infants as their immune systems have yet to develop. Using antibodies raised to act upon these as an adults would immune function can be boosted to help fight the infection.
90
To what ends are antibodies used to modulate the immune system?
Reducing immune response to reduce rejection of transplanted tissues. Stimulating greater immune response to pathogens or tumours.
91
How are antibodies used to prevent thrombosis?
Used to ineffectually bind platelet fibrinogen receptors to prevent reclotting after angioplasty treatment (blood vessel widening to treat atherosclerosis etc).
92
How are abzymes usually produced?
By raising an antibody against the analogue of the transition state found in the desired reaction.
93
What is the most common use of abzymes?
Replacement therapy for enzymes that a person is deficient in. Generally only for well studies enzymes.
94
What was the first commercially available abzyme?
An enantioselective aldolase mimic.
95
What is ADEPT?
Antibody Directed Enzyme Prodrug Therapy - targeting drug delivery by administering an inactive drug precursor (prodrug) and an antibody specific to the target tissue and linked to an enzyme that converts the prodrug to the potent drug.
96
What is the major drawback of ADEPT?
Low dose production.
97
What is an scFv?
A single-chain variable fragment - a fusion protein produced by connecting the heavy and light chain Fv fragment by a linker peptide to make it one small protein consisting of just the binding part of an antibody - retaining original specificity.
98
What are dsFvs?
A kind of scFv where instead of using a peptide linker to join the end domains it is done using disulphide bonds - hence DiSulphide Fv.
99
What is the composition of the linker sequence used in scFvs?
Rich in glycine for flexibility and serine and threonine for solubility
100
What is a bivalent diabody?
Two scFvs attached directly to each other by a linker sequence, foregoing the other half of the Fab fragment and the Fc region. These are often more stable than truncated antibodies that have had their C_H groups truncated.
101
How were antibodies for use in medicine first produced, and what was the drawback of this?
By incecting the epitope into mice and harvesting the antibodies they produced in response to this. Howeer use of these in humans caused an immune response and production of human-anti-mouse antibodies (HAMAs).
102
What are chimeric antibodies?
Antibodies whose CDRs are of mouse origin but have been fused with human Fc regions and possible parts of the Fab region too.
103
What is the drawback with chimeric antibodies?
They are often unable to strongly bind as the supporting regions of the antibody often effect the complementarity of the CDR.
104
What does humanising antibodies involve?
Using molecular biology to edit specific residues in a mouse (or other) antibody to make it more human and hence not cause immune response, while still preserving the binding function.
105
What is total humanisation of mouse antibodies called?
Resurfacing. This much modification leaves only 10% of the antibody mousy in origin.

BIOC2004 (10 decks)

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