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Cellular and Molecular Immunology > Antibody Structure and Function > Flashcards

Antibody Structure and Function Flashcards

1
Q

Importance of Antibodies

A
  • Ig have the ability to recognize an almost unlimited number of targets with high afiinity and specificity.
  • Provide the first line of defense against a wide range of pathogens (viruses, bacteria, fungi, parasites) upon pathogen re-encounter.
  • Correlate of protection for most vaccines and their prodcution by long-lived plasma cells in bone marrow can confer long-term immunit
  • Secreted by B-cells.
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2
Q

Antibodies/Immunoglobulins

A

Glycoproteins produced by B-lymphocytes in response to an antigen:
* Epitope: specific site on antigen recognized by antibody
* One antigen can have many epitopes
* Can be expressed in a membrane-bound form (B cell receptor) or be secreted as soluble antibody by plasma cells.
* Hapten: Small molecule that only elicits an antibody response when attached to a larger carrier.

Found in most body fluids (plasma, mucosal secretions) with the exception of immune provoliged sites (CSF, ocular fluids).

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3
Q

Basic Antibody Structure I

A

Typical “Y” shape
Composed of 4 peptides: two heavy chains (4 Ig domains) and two light chains (2 Ig domains) connected through disulfide bonds.
Chains consist of variable (V) and constant (C) region
V region medites antigen binding
C region mediates antibody effector function
Flexible hinge region between CH1 and CH2 domains

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4
Q

Inter and intra-chain disulfide bonds

A

Disulfide bonds not only connect the light and heavy chains but also contribute to the globular structure of the Ig domains

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5
Q

Basic Antibody Structure II

A

Fab (fragment antigen binding) - SPECIFICITY
Fc (fragment crystallizable) mediates effector functions by recruiting other components of the immune system - ACTIVITY

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6
Q

Structural basis for diverse yet specific binding

A
  • Multiple genes encoding for the variable region
  • Complementarity-determining regions (CDR) forming loops
  • Light chain and heavy chain contain each 3 CDRs (CDR 1, 2, 3)
  • CDR3 contains the HIGHEST DEGREE OF VARIABILITY (V-D-J recombination)
  • Diversity of the naive antibody repertoire is estimated to be >= 10^12
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7
Q

Complementarity-determining regions (CDRs) form the majority of the paratope (antigen-binding site)

A
  • Amino acid variability results in DISTINCT 3-DIMENSIONAL structures “GROOVES” into which the antigen/epitope fits.
  • Non-covalent binding through a variety of intermolecular forces depeding on amino acid composition of CDRs.
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8
Q

Antibodies can recognize two distinct kinds of epitopes

A
  • Linear/continuous epitopes: Sequence of amino acids (~6-10 aa), primary structure
  • Confromational/discontinuous epitopes: tertiary/quaternary stucture (3D)
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9
Q

Why should you care about the kind of epitope that is recognized by your favorite antibody?

A

The kind of epitope recognized by an antibody determines its USEFULNESS for certain applications

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10
Q

Human immunoglobulin isotypes/subclasses

A

Five different isotypes (different Fc heavy chain usage) with four IgG (IgG 1-4) and two IgA (IgA1/2) subclasses.
Two different light chains: kappa (k), lambda
One antibody-secreting plasma cell is only making one heavy and one light chain

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11
Q

Human antibody isotypes and subclasses

A

Human IgG subclasses classification based on mean abundance IgG1>IgG2>IgG3>IgG4
Half-life IgG subcalsses in humans ~21 days, except IgG3 (7days).

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12
Q

Affinity versus avidity

A

Affinity = binding strength of one antibody binding site to one epitope
Avidity = overall binding strength (affected by antibody valency and epitope availability).

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13
Q

Antibody Functions

A
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14
Q

Antibody Neutralization of Microbes and Toxins
Fc-Independent

A
  • “Gold” standard for vaccine-induced antibodies (preventing infection in the first place)
  • Only depends on the binding of the antibody to the target
  • Abrogates crucial function of target (prevent binding to host entry receptors or confromational changes involved in infections)
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15
Q

Antibody-medaited Opsonization and Phagocytosis of Microbes
Fc receptor-dependent

A
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16
Q

Antibody-dependent Cellular Cytotoxicity (ADCC)
Fc receptor-dependent

A
17
Q

Complement Activation by Antibodies
Complement-dependent

A

Solubel Hepatic Proteins complement cascade by potease cleavage leads to: chemotaxis (C5a), inflammation and increased capillary permeability (C3a, C5a), opsonization (C3b), and cell lysis.

18
Q

Why we have many flavours of istopes and subclasses?

A

Beacause it allows for functional diversification.

19
Q

Isotype specific functions

A
  • IgD = Differantation - Differentation to B cells
  • IgE = AlergiEES - Allergy response, mast cell degranulation, and T1 hypersensitivity
  • IgM = You go to your Mom 1st when you are sick - Pentamer, activates the classical pathway of complement, predominates in the primary immune response, and antigen receptor of naive B cells.
  • IgA = Two BreAst - Dimer, main mediator of mucosal immunity, IgA1 - Human serum and secretions, IgA2 - GI tract and colon.
  • IgG = Greatest - Opsonization of antigens Fc(gamma) R - mediated phagocytosis by macrophages and neutrophils, ADCC by NK cells, activation of the classical pathway of complement and IgG subclasses dramatically vary in their Fc-mediated functions.
20
Q

Fc region determines antibody effector functions

A
21
Q

Human and mouse IgG subclasses are not equivalent

A
  • Human IgG1 ~ mouse IgG2a/c (best ADCC)
  • Human IgG4 ~ mouse IgG1 (poorest ADCC)
22
Q

Monoclonal antibodies

A
  • One antibody-secreting plasma cell clone is making one heavy and one light chain.
  • Are identical antibodies that are produced by a single clone of B-cells or plasma cellsT
  • These antibodies are specific to SINGLE EPITOPE
23
Q

Polyclonal Antibodies

A
  • Immune responses in vivo result in polyclonal antiobdy response
  • Are a mixture of antibodies produced by different B-cell clones in response to an antigen. These antibodies recognize and bind to multiple epitopes on the same antigen.
  • Further diversification through somatic hypermutation, isotype switching.
24
Q

Advantages/disadvantages of monoclonal antibodies

A

From a single B cell clone:
=+ Specific to one epitope, defined affinity and isotype
=+ Can produce unlimeted quantities of a defined product

=- Technically challenging (isolation of singel cells, immortalization or cloning)

25
Q

Advantages/disadvantages of polyclonal antibodies

A

From multiple B cell clones:
=+ Targeting of multiple epitopes results in higher signals in staining applications.
=+ Ease of prodcution (vaccinate your favorite animal model, collect serum and use it or alternatively perform purification of antigen-specific antibodies).

=- Relatively undefined prodcut, reproducibility issues.

26
Q

Generation of monoclonal antibodies (mAbs)

A
  • Traditional and well established approach
  • Labor intensive screening of secreted antibody from individual clones to find antigen-reactive clones.
  • Mostly limited to the generation of mouse mAbs
  • Even hybridomas can suffer from genetice drift/loss of antibody production over years/decades.
27
Q

Recombinant generation of monoclonal antibodies

A
  • Sorting of antigen-specific cells reduces screening labor
  • Generation of mAbs from humans
  • Obtained DNA sequences of variable regions allow expression as preferred isotype in virtually any expression system.
  • Safe long-term storage in DNA form or even just as digital sequence.
28
Q

Summary
Take home message(s)

A

Antibodies provide the first line of defense against pathogen re-encounter
Variable CDRs in Fab mediate the majority of antigen specificity/binding.
Fc region mediates a wide range of effector functions (other than neutralization)
Avidity is the overall binding strength of an antibody (dependent on antibody valency and epitope abundance).
Ig isotypes/subclasses can differ substantially in their Fc-medaited effector functions
Human is not same as mouse IgG subclass nomenclature
Monoclonal and polyclonal antibodies have their own advantages/disadvantages

Cellular and Molecular Immunology (15 decks)

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