Antibody Structure and Function Flashcards
Importance of Antibodies
- 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.
Antibodies/Immunoglobulins
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).
Basic Antibody Structure I
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
Inter and intra-chain disulfide bonds
Disulfide bonds not only connect the light and heavy chains but also contribute to the globular structure of the Ig domains
Basic Antibody Structure II
Fab (fragment antigen binding) - SPECIFICITY
Fc (fragment crystallizable) mediates effector functions by recruiting other components of the immune system - ACTIVITY
Structural basis for diverse yet specific binding
- 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
Complementarity-determining regions (CDRs) form the majority of the paratope (antigen-binding site)
- 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.
Antibodies can recognize two distinct kinds of epitopes
- Linear/continuous epitopes: Sequence of amino acids (~6-10 aa), primary structure
- Confromational/discontinuous epitopes: tertiary/quaternary stucture (3D)
Why should you care about the kind of epitope that is recognized by your favorite antibody?
The kind of epitope recognized by an antibody determines its USEFULNESS for certain applications
Human immunoglobulin isotypes/subclasses
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
Human antibody isotypes and subclasses
Human IgG subclasses classification based on mean abundance IgG1>IgG2>IgG3>IgG4
Half-life IgG subcalsses in humans ~21 days, except IgG3 (7days).
Affinity versus avidity
Affinity = binding strength of one antibody binding site to one epitope
Avidity = overall binding strength (affected by antibody valency and epitope availability).
Antibody Functions
Antibody Neutralization of Microbes and Toxins
Fc-Independent
- “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)
Antibody-medaited Opsonization and Phagocytosis of Microbes
Fc receptor-dependent
Antibody-dependent Cellular Cytotoxicity (ADCC)
Fc receptor-dependent
Complement Activation by Antibodies
Complement-dependent
Solubel Hepatic Proteins complement cascade by potease cleavage leads to: chemotaxis (C5a), inflammation and increased capillary permeability (C3a, C5a), opsonization (C3b), and cell lysis.
Why we have many flavours of istopes and subclasses?
Beacause it allows for functional diversification.
Isotype specific functions
- 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.
Fc region determines antibody effector functions
Human and mouse IgG subclasses are not equivalent
- Human IgG1 ~ mouse IgG2a/c (best ADCC)
- Human IgG4 ~ mouse IgG1 (poorest ADCC)
Monoclonal antibodies
- 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
Polyclonal Antibodies
- 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.
Advantages/disadvantages of monoclonal antibodies
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)
Advantages/disadvantages of polyclonal antibodies
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.
Generation of monoclonal antibodies (mAbs)
- 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.
Recombinant generation of monoclonal antibodies
- 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.
Summary
Take home message(s)
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
