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2.1 Molecular Immunology > Greg - Antibody Structure > Flashcards

Greg - Antibody Structure Flashcards

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1
Q

What are antibodies?

A

Glycoprotein molecules

Produced by plasma cells

They bind to antigenic targets

They are also called immunoglobubins/gamma globulins

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2
Q

What are antibodies?

A

Glycoprotein molecules

Produced by plasma cells

They bind to antigenic targets

They are also called immunoglobulins/gamma globulins

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3
Q

What is a glycoprotein?

A

A protein with a carbohydrate post translation modification

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4
Q

How can antibodies neutralise/block entry of pathogens?

A

Antibody against viral surface proteins block entry to human cell by binding to the protein - prevents it from binding to host’s cell

= neutralisation

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5
Q

Explain agglutination/precipitation
(3)

A

Antibodies can clump antigens together into an immune complex for removal

Agglutination = insoluble target

Precipitation = soluble target

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6
Q

What effector functions do antibodies have?
(3)

A

Activate complement
Aid phagocytosis via opsonisation
Mediate antibody mediated cellular cytotoxicity by NKCs

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7
Q

List the functions of antibodies
(5)

A

Block entry/neutralisation of pathogen

Agglutination/precipitation

Complement activation

Opsonisation

Aids NKCs

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8
Q

What is a myeloma?

A

Cancer of plasma cells
i.e. one plasma cell keeps producing it’s antibody continuously

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9
Q

Why were antibodies hard to study?

A

Due to their heterogenous nature i.e. they are all different

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10
Q

What research allowed us to study antibodies?

A

Studies of myelomas as these cells produce a monoclonal antibody population which is identical and can therefore be separated and studied

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11
Q

How are the different parts of an antibody examined?
(3)

A

We cleave the antibody into parts

Papain cleaves the antibody at the middle and creates three fragments - we can then make crystals of Fc because the Fc molecule is the same in every antibody (uniform) -> can examine Fc now

Pepsin cleaves antibody at hinge - gives us a F(ab’)2 with the hinge and a partial Fc fragment (pFc) - research can now be carried out on the Fab

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12
Q

What joins the light and heavy chains of an antibody?

A

Disulphide bonds between cysteine residues

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13
Q

What does mercaptoethanol do to antibodies?
(3)

A

This chemical destroys the disulfide bridges between the light and heavy chains

The four chains fall apart

This gives us 2 heavy chains and 2 light chains which can be examined further

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14
Q

What are the two types of disulphide bonds?

A

Inter-chains
Intra-chains

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15
Q

List the variable and constant regions

A

VL & CL

VH & CH

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16
Q

List the parts of an antibody
(7)

A

Fab region

Fc region

Intra-chain

Inter-chain

Variable region

Hinge region

Constant region

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17
Q

What is the hinge region?
(3)

A

Part of the molecule that can move

It gives the antibody some flexibility

It makes binding to antigens easier

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18
Q

The functions of the antibody molecule are spatially segregated, what does this mean?

A

Some functions of antibodies relate only to the Fab region while others relate only to the Fc region

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19
Q

What function is the Fab site responsible for?

A

Antigen binding

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20
Q

What functions is the Fc site responsible for?
(3)

A

Complement binding site

Binding to Fc receptors

Placental transfer

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21
Q

Write about the antibody-antigen reaction?
(2)

A

A non-covalent bond
Reversible reaction - i.e. antigen has affinity for antibody but can be pulled apart

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22
Q

What is affinity in terms of antigens?

A

The strength of the reaction between a single antigenic determinant and a single combining site

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23
Q

What is avidity in terms of antigens?

A

The overall strength of binding between antigen with many determinants and multivalent antibodies

The total attractive force = the total affinity

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24
Q

What type of antibody is first made by plasma cells?

A

IgM

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25
What is specificity?
The ability of an antibody binding site to react with only one antigenic deteminant
26
What is cross-reactivity?
The ability of an individual antibody binding site to react with more than one antigenic determinant
27
What is an epitope?
Regions of an antigen recognized by B cell antibody and T cell receptor
28
What is the difference between T cell dependant antibody production and T cell independent antibody production?
T-dependent results in high affinity antibodies, memory B cells, longer lived plasma cells, and isotype switching of antibodies (i.e. switch from IgM) T-independent results in low affinity antibodies, short lived plasma cells and mainly IgM antibodies
29
What determines the classes or isotypes of antibodies?
Their heavy chain amino acid sequences
30
List the five antibody isotypes and their heavy chains
IgG - Gamma heavy chain IgA - alpha heavy chain IgM - mu heavy chain IgD - delta heavy chain IgE - epsilon heavy chain
31
Where is IgD found?
The surface of B cell receptors where it acts as a receptor
32
Where is IgA found?
The IgA monomer is found in blood but the dimer is found on cell surfaces
33
What determines amino acid subclasses?
Heavy chain amino acid sequences
34
How many subclasses does IgG have?
4
35
What are the four main igG subclasses?
IgG1 -> gamma 1 heavy chain IgG2 -> gamma 2 heavy chain IgG3 -> gamma 3 heavy chain IgG4 -> gamma 4 heavy chain
36
How many subclasses does IgA have?
2
37
What are the subclasses for IgA?
IgA1 -> alpha 1 heavy chain IgA2 -> alpha 2 heavy chain
38
How are the types of light chains determined?
They are determined by sequence Either kappa (K) or lambda
39
How many kappa subtypes are there?
None
40
How many lambda subtypes are there?
1-4 subtypes
41
How would IgA , subtype 1, lambda 2 be written?
IgA1(lamda2)
42
List the subtypes of IgG in order of population?
IgG1 IgG2 IgG3 = IgG4
43
How much of the gamma globulins in serum are IgG?
80%
44
What is the only class of antibody that can cross the placenta?
IgG2
45
What subtypes of IgG can fix complement?
IgG3 and IgG4
46
Which IgG subtypes act as opsonins and how?
IgG2 and IgG3 act as opsonins They can be bound by the Fcgamma receptor
47
What is responsible for the long serum half-life of IgG?
FcRn
48
What is FcRn?
The neonatal Fc receptor for IgG2
49
Describe the structure of IgA in secretions
A dimer with a J-chain and secretory component (hence why it's found in secretions)
50
Hoe much of the gamma globulins in serum are IgA
Between 10 and 15 percent
51
List some places IgA can be found (3)
Saliva Breast milk Tears
52
What is the J-chain of IgA similar to?
Similar to the J-chain of IgM
53
Write a note on the structure of IgM (5)
Pentameric in serum but a monomer on B-cell surface In pentamer - all light and heavy chains are identical Extra domain (CH4) J chain Valency of 10
54
What is the extra domain in IgM called?
CH4
55
Write about the properties of IgM (5)
3rd highest concentration in serum First Ig made by foetus and B cells Best complement fixer Agglutination IgMFc receptor -> binds Fc receptors on B cells Anchors in B cell membrane but must partner with other proteins on the surface to act as a receptor and activate the cell
56
Write about IgE structure (3)
Monomer in structure Extra CH4 domain Only survives for 2 and a half days
57
Write about the properties of IgE (6)
Very potent activity Very low serum concentrations Hypersensitivity reactions Hay fever, asthma hives etc Binds Fce receptors on basophils and mast cells (degranulation) Anti-parasitic defence
58
Explain what happens in an allergic reaction
IgE has been produced against a harmless environmental molecule When you first come across the allergen you won't have a reaction but IgE will be made and it will bind to Fc epsilon receptors on mast cells -> next time you meet the allergen the IgE will cross bind and send an activation into the mast cell which releases histamine
59
Write about the structure and properties of IgD (5)
Monomer Tail piece to be anchored in B cell membrane along with IgM 2nd lowest concentration in blood Most ancient antibody class We though it had no function but now see it is produced in the lower respiratory tract during infection
60
What do most cells express that act as receptors for the Fc portions of antbiodies
Glycoprotin
61
What do most cells express that act as receptors for the Fc portions of antbiodies
Glycoprotiens
62
Why are Fc receptors needed on cells?
Opsonisation Antibody-dependent cellular cytotoxicity Cell activation Cell differentiation
63
What is the requirement to be part of the immunoglobulin superfamily
Must have at least one domain homologous to other members e.g. Immunoglobulin-fold domain structure
64
Why might specificity be measured in the lab?
Chemical methods could not distinguish between similar proteins (e.g. Human vs. pig insulin) but antibodies can do this absolutely
65
Why might avidity be used in the lab?
Affinity dictates the effectiveness of an antibody. For example, the higher the affinity of an antibody, the less you might need for a procedure
66
Under what conditions might patient antibodies be measured?
Inflammation/infection Cancer Allergy Parasitic infection Immunodeficiency Autoimmunity
67
Why might antibodies be measured during inflammation?
Due to increase in antibody response due to immune responses
68
Why might antibodies be measured during cancer?
Cancer of B cells result in monoclonal antibody type
69
Why might antibodies be measured during allergic reaction
Increase in IgE Increase in specific IgE
70
Why might antibodies be measured during parasitic infection
Increased total IgE
71
Why might antibodies be measured in immunodeficiency
Can the patient make normal levels of all antibody classes?
72
Why might antibodies be measured during autoimmunity
What antibody is being produced against self
73
What antibodies are used in the lab?
Anti-sera = antibodies produced in animals - a mixture (cheap) Monoclonal = antibodies are identical to each other and bind the exact same epitope
74
How are anti-sera antibodies produced? (5)
We take antigen A and vaccinate a rabbit using it The animal produces different types of antibodies against it We take antibodies from rabbit Purify antibodies We now have a polyclonal mixture of antibodies which can be used to detect Antigen A
75
How do anti-immunoglobulin antibodies worj
Antibodies against antibodies Human antibody injected into animal -> provokes an immune response as it is seen as an antigen Antibodies produced can be used to detect antibody
76
How is anti sera used? (5)
ELISA test Antigen bound to well Human antibody (if present) binds to antigen Anti human antibody is added and binds to antibody Anti-human antibody is marked with a horse radish peroxidase which creates a colour
77
What is a monoclonal antibody?
An artificially created antibody from a single cell clone - binds same epitope
78
Who immortalised plasma cells?
Georges Kohler Cesar Milstein
79
How were plasma cells immortalised?
Plasma cell were fused with myeloma cell which resulted in immortal cell producing monoclonal antibodies
80
Describe the process of making monoclonal antibodies
Inject animal with antigen (multiple epitopes) Animal makes antibodies - for each epitope Kill the animal and take its spleen Take plasma cell of spleen making the antibody of interest Immortalise plasma cells by fusing them with myeloma cells Dilute wells until we have a single cell per well (you have created an eternal source of antibodies for a specific antigen)
81
Explain the relationship between HGPRT and spleen plasma cells (3)
Myeloma cells are engineered so that they are deficient in HGPRT Without HGPRT the myeloma cells will die The spleen plasma cells gain immortality by providing the myeloma cell with HGPRT
82
What is HGPRT?
Hypoxanthine-guanine phosphoribosyl transferase Enzyme involved in purine synthesis

2.1 Molecular Immunology (19 decks)

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