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Year 3 - Biotechnology > 8 - Antibody Biology > Flashcards

8 - Antibody Biology Flashcards

1
Q

Describe innate immunity

A
  • Pathogen recognized by receptor encoded in the germline – pattern recognition receptors
  • Receptors have broad specificity; ex: recognize many related molecular structures like PAMPs (pathogen-associated molecular patterns)
  • Immediate response
  • No memory of prior exposure
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2
Q

Describe adaptive immunity

A
  • Pathogen recognized by receptors generated randomly – B-cell and T-cell receptor for antigen
  • Receptors have very narrow specificity (recognize a particular epitope after processing)
  • Slow (3-5 days) response b/c of the need for clones of responding cells to develop
  • Memory of prior exposure
  • Divided into humoral and cell-mediated
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3
Q

What is the antigen in humoral immunity vs. cell-mediated immunity?

A
  • Humoral = extracellular bacteria
  • Cell-mediated = intracellular microbes in macrophage or intracellular microbes (ex: viruses) replicating w/in infected cell
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4
Q

What is the responding lymphocyte in humoral immunity vs. cell-mediated immunity?

A
  • Humoral = B lymphocyte

- Cell-mediated = T lymphocyte

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5
Q

What is the effector mechanism in humoral immunity vs. cell-mediated immunity?

A
  • Humoral = secreted Ab and elimination of bacteria

- Cell-mediated = activation of macrophage to initiate microbial killing, or lysis of infected cell

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6
Q

What is used for transfer in humoral vs. cell-mediated immunity?

A
  • Humoral = serum (antibodies)

- Cell-mediated = lymphocytes

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7
Q

Describe humoral immunity

A
  • Mediated by B lymphocytes
  • B lymphocytes synthesize and secrete immunoglobulin (Ig)
  • 5 classes of antibodies, w/ differences in structure and function
    • Differences are in heavy-chain constant region
  • Sequencing of several immunoglobulins revealed
    • Minor differences led to sub-classes of IgA and IgG
    • Each class can have either k or gamma light chains
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8
Q

Describe the antibody structure

A
  • IgG are 150 kDa glycoproteins
    • 2 identical heavy chains (50-55 kDa)
    • 2 identical light chains (25 kDa)
    • Intra-molecular disulfide bridges
  • Antigen-binding fragment Fab, a constant part and variable domain Fv
  • Constant region Fc
  • First 100 AA of N-terminal of both H and L chain (Fv) are variable and referred to as VL and VH
  • CDR (complementarity determining regions about 10 AA) are where antibody binds antigen
  • Remaining regions are very similar w/in same class – repeating regions and hinge region
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9
Q

Describe repeating regions of antibodies

A
  • Each region is about 110 aa

- Intra-chain disulfide bonds w/in each domain

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10
Q

Describe hinge regions of antibodies. Which immunoglobulins have a hinge region? What is present in those w/o a hinge region?

A
  • Rich in proline residues (flexible)
    • Proline residues are target for proteolytic digestion (papain and pepsin)
  • Rich in cysteine residues (disulfide bonds)
  • Hinge found in IgG, IgA, and IgD
  • IgM and IgE lack hinge region, instead have extra CH4 domain
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11
Q

Describe the enzymatic digestion of antibody

A
  • Papain digestion –> 3 fragments, 2 identical Fab and 1 Fc
  • Pepsin digestion –> F(ab’)2, no Fc
  • Mercaptoethanol reduction –> eliminates disulfide bonds
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12
Q

Describe the 2-gene model of Dryer and Bennett

A
  • 2 separate genes encode a single Ig H or L chain, one gene for the V region and the other for the C region
  • Genes are rearranged in the course of B-cell differentiation
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13
Q

Gamma-chain multigene family in human

A
  • V region = 30 V-gamma segments and 4 J-gamma segments
  • C region = 4 C-gamma segments
  • 480 combinations (3044) of VJC may occur
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14
Q

K-chain multigene family in humans

A
  • V region = 40 V-k segments and 5 J-k segments
  • C region = 1 C-k segments
  • 200 combinations of VJC may occur
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15
Q

H-chain multigene family in humans

A
  • V region = 51 V-H segments, 27 D-H segments, and 6 J-H segments
  • C region = 9 C-H segments
  • 5,382 combinations of VDJC may occur
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16
Q

Describe gene rearrangements

A
  • H-chain V-region genes rearrange first and then the L-chain V-region genes
  • Rearrangement occurs in an ordered sequence, but they are random events
  • Rearrangement of Ig and TCR genes are the only known site-specific DNA rearrangements in vertebrates
17
Q

H-chain DNA rearrangements

A
  • First – D gene joins J gene
  • Second – V gene joins DJ
  • Third – C gene joins VDJ
  • Depending on which C gene joins VDJ, determines which class of antibody it will be
18
Q

L-chain DNA rearrangements

A
  • First – L-V gene joins J gene
  • Second – C gene joins LVJ
  • C gene represents different type of light chain
19
Q

Heavy chain composition of the 5 immunoglobulin classes in humans

A
  • IgG = gamma
  • IgM = mu
  • IgA = alpha
  • IgD = delta
  • IgE = epsilon (looks like uppercase E)
20
Q

Describe IgG – Which is most present in serum? Which can cross the placenta? Which have affinity for Fcr? What does it bind?

A
  • Most abundant immunoglobulin, 80% of serum Ig
  • IgG1, G2, G3, G4 (decreasing serum concentration)
  • IgG1, G3, and G4 cross placenta
  • IgG1 and IgG3 high affinity for FcR on phagocytic cells, good for opsonization
  • Secondary antibody for most response to pathogens, binds macrophage and other phagocytic cells by Fc-gamma-R
21
Q

Describe IgM – Where is it expressed? What version is secreted? When is it secreted? Advantage to IgG?

A
  • 5-10% of serum Ig
  • Monomeric IgM expressed on B-cells as B cell receptor (BCR)
  • Pentameric version is secreted
  • First Ig of primary immune response (activates classical complement pathway), some binding to polymeric Ig receptor, some binding to phagocytes
  • More efficient than IgG in complement activation
22
Q

Describe IgA – Where is it found? What does it bind to? What version is secreted?

A
  • 10-15% of serum Ig
  • Predominant Ig in secretions (milk, saliva, tears, mucous); 5-15 g of IgA released in secretions
  • Binds to polymeric Ig receptor
  • Serum mainly monomeric, polymers possible but uncommon
23
Q

IgE – What is its function? What does it bind? What does binding cause?

A
  • Very low serum concentration
  • Participate in immediate hypersensitivity reactions (ex: asthma, anaphylaxis, hives)
  • Binds mast cells and blood basophils through Fc-epsilon-R
  • Binding causes degranulation (histamine release)
24
Q

IgD – Where is it expressed and with what?

A
  • Expressed on B-cell surface
  • IgM and IgD co-expressed on B-cell surface
  • Don’t know any other biological effector activity
  • Low serum concentrations
25
Q

Antibody functions

A
  • Antigen binds to antibody on antigen binding site
  • Activates B lymphocytes
  • Acts as opsonins
  • Causes antigen clumping and inactivation of bacterial toxins
  • Activates antibody-dependent cellular activity
  • Triggers mast cell degranulation
  • Activates complement
26
Q

What are the 3 categories of antigenic determinants on antibodies?

A

1) Isotypic
2) Allotypic
3) Idiotypic

27
Q

Describe the isotypic determinant on antibodies

A
  • Constant region of antibody is the immunogen
  • Often occur in the antibody from different species
  • Antibody from the same species will not generate isotypic reaction
28
Q

Describe the allotypic determinant on antibodies

A
  • Small differences about 1-4 AA in the constant region of antibody from the same species can generate allotypic reactions
  • These differences can arise due to polymorphism
  • Allotypic reaction has been observed in pregnancy and blood transfusions
29
Q

Describe the idiotypic determinant on antibodies

A
  • Since Fv region can bind antigen and antibody from the same species, could also behave as immunogen
  • Even inject such monoclonal antibody into a genetically identical recipient, idiotypic reaction can occur
  • Such antibody won’t generate isotypic or allotypic reactions
30
Q

Describe applications of antibodies

A
  • Diagnostic tests – Abs are capable of detecting tiny amounts (pg/mL) of molecules (ex: pregnancy hormones)
  • Diagnostic imaging – mAbs that recognize tumor antigens are radiolabeled w/ iodine I-131
31
Q

Describe indirect antibody activites

A
  • Toxins -> ricin-A and calchaemicin
  • Radioisotopes – low dose, gamma emitters or high dose alpha or beta emitters
  • Cytokines – tumor necrosis factor alpha and interleukin-2
  • Bispecific antibodies -> tumor cells and cytotoxic T-cells, granulocytes or neutrophils
32
Q

Describe direct antibody biological activities

A
  • Blocking Ag-Ab interaction
  • Ab-dependent cellular cytotoxicity (ADCC)
  • Complement-dependent cytotoxicity (CDC)
  • Apoptosis
33
Q

Which immunoglobulins are monomer, dimer, and pentamer?

A
  • Monomer = IgG, IgE, IgD
  • Dimer = IgA
  • Pentamer = IgM
34
Q

Which IgG is the most effective complement activator?

A

IgG3

35
Q

What has been developed from recombinant antibodies?

A
  • Recombinant antibody has developed – rodent Ab, chimeric Ab, CDR-grafted (humanized) Ab, human Ab, receptor + Fc Ab
  • Murine mAbs – 0% sequences similar to human mAbs
  • Chimeric mAbs – 75% similar to human mAbs
  • CDR-grafted (humanized) mAbs – 90% similar to human mAbs
  • Cept = receptor
  • Direct antibody biological activities
  • Indirect Ab activities

Year 3 - Biotechnology (11 decks)

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