Immunology Chapter 7

Antibody Structure

• 2 heavy chains

• 2 light chains

• heavy chains linked to 2 disulfide bonds

• each chain composed of immunoglobulin domain

Immunoglobulin Domain

• each domain has 2 layers of β-pleated sheets with 3-4 antiparallel strands

• 2 layers held together by disulfide bonds

Variable & Constant Regions

• immunoglobulin domains named according to variable or constant region

• ends of heavy chain and light chain contains hypervariable regions and framework regions

• combination of hypervariable regions from heavy and light chain = antigen binding site

• hypervariable region AKA CDR because confirmation must be complementary to antigen for binding

• between CH1 & CH2 = hinge region

  • flexible for binding

• cleavage of antibody

  • 1.) papain = cleaves Ab above disulfide bridges linking heavy chains

    • result = 1 Fc region and 2 Fab fragments

  • 2.) pepsin = cleaves Ab below disulfide bonds holding 2 heavy chains together

    • result = 1 F(ab’)2

Antibody Isotope

• IgA = α

• IgG = γ

• IgD = δ

• IgE = ε

• IgM = μ

• no hinge region = ε and μ

• 1 disulfide bond = α and δ

• 2 disulfide bonds = γ

• 2 immunoglobulin regions = α, γ, and δ

• 3 immunoglobulin regions = ε and μ

• tail pieces = α, δ, and μ

  • α and μ tail pieces bind to each other and to J chain to form multimers

• no tail pieces = γ and ε

IgM

• produced first

• location = blood

• function = activates complement

IgG

• main isotope

• location = blood and extracellular fluid

• function = complement activation and opsonization

IgA

• primary component secreted onto mucus membranes (AKA secretory antibody)

• function = neutralization

IgE

• found in low levels in blood and extracellular fluid

• location = close to mast cells just beneath the skin and mucus membranes

• mast cells become activated when IgE binds antigen

• mast cells produce cytokines

IgD

• in small concentration in blood

Differences in Light Chain

• 2 types:

  • κ

  • λ

• more κ than λ in humans

• more λ than κ in mice

• no functional difference

• difference = framework regions of the variable and constant regions

Polyclonal vs. Monoclonal Antibodies

• antibody recognizes epitope

• antigen injected into animal → animal produces antibodies against every epitope of antigen

• monoclonal = specific (one kind)

• polyclonal = not specific (multiple kinds)

• Monoclonal steps:

  • monoclonal antibodies = inject antigen → polyclonal antibodies

  • spleen isolated and activated B cells from spleen are grown in tissue culture

  • spleen cells fused with myeloma cells → become immortalized

  • immortalized spleen cells → hybridomas = are diluted so that a single cell is present in each well of a tissue culture plate

  • one B cell can only produce antibody specific for one epitope, each well produces different antibodies for different epitopes

Antibody binding to Antigen

Epitope Structure

1.) Linear Epitope

• amino acid adjacent to each other in primary protein

• small (6 amino acids)

• to be recognized by antibody, it must be present on surface of protein or protein must be denatured

2.) Conformational Epitope (discontinuous epitope)

• recognizes shaped formed by folded protein

• actual amino acid in forming this shaped could be far apart in primary protein sequence

Affinity

• measurement of strength for antibody binding in ONE site

• affinity = equilibrium constant

• repeated antigen = increase in affinity → tighter antigen binding → affinity maturation

Avidity

• antibodies have multiple antigen binding sites

• each binding site can bind to an epitope

• avidity = measurement of strength for antibody binding in ALL sites

• example = IgM has low affinity than IgG but high avidity

• after affinity maturation, IgG increases to where it is equivalent to IgM

Antibody Functions

1.) Neutralize bacterial toxins

• exotoxins produced and secreted by bacteria

• toxin binds to receptor on target cell → internalized and active portion of toxin exerts its effect on cell

• antibodies can bind to receptor binding portion of toxin → blocks binding of toxin to its receptor → doesn’t get internalized and cannot carry out toxin function

• neutralizing antibodies = IgG in tissue or IgA on mucosal surface

2.) Prevent infection by virus

• viral attachment protein must bind to specific receptor to infect cell

• antibodies bind to viral attachment proteins = blocks virus from infecting cell

• virus neutralization = IgG and IgA

3.) Block attachment by bacteria

• bacteria attach to cell surface and colonize surface during infection

• antibodies bind to adhesins = block attachment

4.) Activation of complement vis classical pathway

5.) Antibody Dependent Cell Cytotoxicity (ADCC)

6.) Antibody Mediated Opsonization

• aggregation of immunoglobulin = cross-linking of Fc receptors → activation of macrophage, phagocytosis, and destruction of bacteria

• free immunoglobulin = no cross-linking of Fc receptors → no destruction of bacteria

• Opsonization by antibody and complement can occur at the same time = increase effectiveness

  • antibody binds to Fc receptor and C3b binds to Cr1 → macrophage membrane fuse → phagosome → lysosome in phagosome → enzyme degrades bacteria

7.) Degranulation of eosinophils and other phagocytes

• cross-linking Fc receptor on parasite too big for phagocytosis → lysosome from phagocyte fuses with cell membrane releasing contents on surface

• eosinophils = primary cells in response to parasite infection

• cross-linking receptors = IgE and FcεRI

8.) Activation and degranulation of mast cells

• mast cells have FcεRI on surface that binds to IgE

• cross-link IgE = mast cell activation

• activated mast cell = degranulation, releasing contents of granules that contains histamine

  • can produce prostaglandin D2, leukotriene C4, and TNF α = inflammation

• activated mast cell = allergic reaction

3 ways fighting infection:

• recruit leukocytes to infection area by increasing vascular permeability and inflammation

• increase flow of lymph to lymph nodes where antigen can be captured for processing and presentation

• trigger muscle contraction (coughing) or increase peristalsis in intestine (pooping)