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ICB > Antibodies > Flashcards

Antibodies Flashcards

1
Q

How do Antibodies combat infection

A
  • neutralising antibodies block infection via spike protein or toxin binding
  • activate complement causing enhanced opsonisation, inflammation, direct pathogen lysis
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2
Q

Immunoglobulin Molecule Structure

A
  • 4 chain unit heterodimer: 2 identical H chains and 2 identical L chains
  • variable and constant domains (3 C domains in H chain and 1 in the L chain)
  • Ab class defined by heavy chain type (Ig class)
  • variable hinge disulfide joins H chains and the H/L
  • other disulfide bonds for stability
  • mediate antigen binding and various biological activities (complement, cell binding, crossing tissues)
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3
Q

Ab chain types

A
  • H: 5 classes

- L: 2 types encoded by different genetic loci

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4
Q

Complementarity Determining Region

A
  • CDRs are 3 hypervariable regions in the Vl and Vh domains

- determine the shape of binding site and contact the antigen

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5
Q

Immunoglobulin Fold

A
  • light chain contains the Ig fold structure and proximity of CDRs
  • 2 sets of B strands connected via disulphide bonds
  • Vl domain contains CDR loops
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6
Q

Antigen Binding Site

A
  • CDRs of H and L chains combine to create the antigen binding site
  • antigen cleft
  • binding is via noncovalent interaction but high affinity
  • requires close interaction and high degree of complementarity between antigen epitope (specific part of the antigen which the antibody binds to) and the paratope (Ab binding site)
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7
Q

Types of Epitopes

A
  • protein antigens contain multiple epitopes
  • multivalent antigen with different epitopes
  • multivalent antigen with a repeated epitope
  • linear epitope: binds to protein antigen linear sequence
  • discontinuous epitope: binds to discontinuous sequence of the protein
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8
Q

Monoclonal Antibodies

A
  • serum antibodies are polyclonal: antibodies from normal cells have binding affinity/specificity that differs
  • monoclonal antibodies are derived from a single plasma cell
  • monoclonal: single B cell making identical antibodies, very specific
    1. inject antigen with different epitopes into mouse
    2. remove spleen cells
    3. hybridise plasma and myeloma cells to form hybridomas
    4. select hybridomas and separate to ensure all antibodies made are monoclonal : same epitope binding
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9
Q

Monoclonal Antibody Treatment

A
  • monoclonal antibodies can be used to treat disease
  • humanisation of antibodies reduces their perception as foreign
  • chimeric antibodies: variable chains mouse and constant human
  • humanised: CDRs are mouse and rest human
    eg. Adalimumab: human MAb - targets TNF-a for inflammation treatment
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10
Q

Immunoglobulin Classes

A
  • isotypes
  • each antibody isotype has specialised functions (determined by Fc region) which promote diversity of immune responses against pathogens
    1. IgM: membrane bound monomer acts as antigen receptor on naive B cells / secreted IgM is pentameric and first antibody made in primary response
    2. IgG: monomeric produced after IgM in primary response and quickly in secondary response
    3. IgA: monomeric in blood but dimeric and major antibody in mucosal secretions
    4. IgE: monomeric and produced in response to helminth parasites, allergens
    5. IgD: triggers receptor on basophils to eliminate bacteria
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11
Q

B Cell Activation

A
  • B cell receptor is a membrane bound antibody and co-receptor (Iga/IgB)
  • IgM is the antigen receptor on naive B cell
  • binding to ag stimulates receptor clustering and recruitment of tyrosine kinases that phosphorylate residues on ITAMs on Iga/IgB
  • Syk recruited to the doubly phosphorylated ITAMs leads to activation
  • changes in nuclear gene expression
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12
Q

Primary vs Secondary Antibody responses

A
  • in a primary antibody response IgM is made first following by class switching to IgG and other classes with the same antigen specificity
  • IgM is low affinity but IgG and other antibody classes undergo affinity maturation over several weeks to bind the antigen more strongly
  • population of memory B cells with this antigen specificity is generated so that re-exposure to the same antigen stimulates a much quicker and bigger antibody response
  • most memory B cells have IgG as antigen receptor
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13
Q

IgG subclasses

A
  • differ in number and arrangement of disulphide bonds linking H chains
  • IgG1: major antibody to protein antigens
  • IgG2: less flexible made against carb agents
  • IgG3: good at complement activation
  • IgG4: unique ability to exchange 1H/L chain pair with another IgG4 molecule so funtionally monovalent
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14
Q

IgM

A
  • pentameric
  • J chain required for polymerisation
  • secreted form: 5 x 4 chain units linked by disulfide bonds
  • agglutinate bacteria
  • J chain promotes the polymerisation
  • generally low affinity but has 10 antigen binding sites
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15
Q

IgA

A
  • major Ig class in mucosal secretions
  • mostly dimeric with J chain and secretory component
  • 2 x 4 chain units
  • monomeric in blood plasma but dimeric in mucosal secretions
  • J chain required for polymerisation of 4 chain units
  • has additional secretory component
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16
Q

Secretory IgA

A
  • poly-Ig receptor required for transport across epithelia
  • IgA made by plasma cells in submucosa
  • dimeric IgA with J chain
  • binds to poly-Ig receptor
  • subsequent transcytosis and cleavage of poly-Ig receptor leaves secretory component bound to IgA
  • secretory component protects hinge regions from cleavage
  • IgA bound to mucins at mucosal surface and protects against microbial attachment and colonisation
17
Q

IgE

A
  • bound to mast cell receptors
  • cross linking by allergen causes release of allergic mediators
  • free IgE very labile
  • stabilised by interaction with Fc-eR1 on basophils in blood and mast cells in tissues
  • cross linking causes degranulation of mast cells and basophils
  • mediators released cause allergy symptoms + parasitic expulsion
18
Q

Fc receptors

A
  • different cells have different Fc receptors
  • allow cells to be activated by antibody bound to antigens/pathogens
  • FcyRI is the high affinity receptor for IgG1-IgG3
  • can bind antibody in absence of antigen
  • a chain binds antibody and signalling through y chain
  • allows pathogen trapping by other immune cells to target them
19
Q

Immunoglobulin Diversity

A
  • classic models can’t account for vast diversity of antigen binding specificities
  • Ig heavy and Ig light chains with variable N terminus and constant C terminus
20
Q

Dreyer and Bennett Model

A
  • V region and C region encoded by separate genes which create one protein
  • idea that a small number of genes forms large number of antibodies with different binding specificity
  • goes against 1 gene = 1 polypeptide
  • diversity caused by mutation/rearrangement
21
Q

V region sequence

A
  • DNA for the V region is assembled from 2/3 gene segments
  • recombination of gene segments creates diversity
  • light chain V region encoded by V and J gene segments
  • heavy chain V region encoded by V,D, and J gene segments
  • V region gene segments must first be selected and assembled to produce an exon
22
Q

Recombinatorial Dveristy

A
  • recombination of gene segments generates different V region exons and diversity in antigen binding sites
  • during B cell development, V,D,J segments cut and spliced back by DNA recombination (somatic recombination) to form exon encoding V region of H/L chain
  • single recombination of L chains (VJ): DJ then VDJ
  • recombination is random
  • CDR1 and 2 encoded in V gene segments
  • CDR3 in L chain determined by V/J gene segment junction
  • CDR in H chain determined by D segments
23
Q

Recombination

A

Light chain

  • germline DNA
  • VJ joined rearranged DNA

Heavy chain

  • germline DNA
  • DJ joined DNA
  • VDJ joined DNA
24
Q

Combinatorial Diversity

A
  • many different V region sequences
  • K chain has 175 sequence combinations
  • y chain has 120 combinations
  • antibody binding site is created by combination of H chain and L chain V regions
  • 1.6 million specificities possible
25
Q

Recombination Signal Sequences

A
  • flank each V,D,J gene segment
  • RSS are conserved heptamers and nonamers flanking the gene segments
  • provide recognition sites for enzymes cutting and ligating DNA
  • ensure segments are joined in the right order
  • separated by 23, 12 nucleotides (spacing means V can’t be joined directly to J segments)
26
Q

Junctional Diversity

A
  • created by addition and subtraction of nucleotides at the joints between gene segments
  • dsDNA break precise but joins imprecise
  • variation in hairpin cleavage creating new palindromic nucleotides and variation in addition of N (not germline encoded) nucleotides
  • many rearrangments unproductive on both alleles and B cells die
  • but this junctional diversity at CDR3 can greatly increase the diversity of binding specificity
27
Q

Junctional Diversity Process

A
  1. cleavage at heptamer site
  2. RAG complex cleaves heptamer RSSs from teh D and J gene segments to give hairpins
  3. RAG complex opens hairpins by nicking one strand of the DNA: generates palindromic P nucleotides
  4. N nucleotide additions by TdT
  5. pairing of strands
  6. unpaired nucleotides are removed by an exonuclease
  7. gaps are filled by DNA synthesis and ligation to form coding joint
28
Q

Somatic Hypermutation

A
  • targets rearranged gene segments encoding variable region
  • diversity created by gene rearrangement concentrated at CDR3s of Vh and Vl domains
  • point mutations throughout V domain coding sequence
  • Activation Induced Adenosine Deaminase made only by proliferating B cells
  • AID converts cytosine to uracil during transcription creating point mutation
29
Q

Affinity Maturation

A
  • somatic hypermutation allows B cell selection making higher affinity antibodies
  • if mutation is in antigen binding site (CDR) and increases affinity of binding: B cell preferentially selected for maturation to plasma cells
  • somatic mutation increases number of antigen specificities : rapid evolution
30
Q

Heavy Chain Gene

A

-

31
Q

Heavy Chain Gene

A
  • VDJ rearrangement produces functional heavy chain gene (variable region of heavy chain)
  • C regions downstream of rearranged VDJ segments
  • rearranged gene transcribed, RNA spliced, and H chain expressed
  • B cells initially co-express IgM/IgD
  • transcription intiiated at Vh promoter and reads through Cu and Co genes
  • long primary transcript cleaved and polyadenylated at u site or o site then spliced to yield mRNA for u and o heavy chains
32
Q

Allelic Exclusion

A
  • although B cells have 2 copies of heavy chain locus and light chain locus: only 1 H-chain locus and 1 L-chain locus rearranged to produce functional genes
  • this is known as allelic exclusion
  • ensures only 1 H chain and 1 L chain are finally expressed and every B cell expresses identical Ig with same antigen binding site
  • B cell first makes membrane bound IgM/IgD with same antigen specificity
  • needs to associate to Iga/B co-receptor to be transported to plasma membrane
33
Q

Alternative Splicing

A
  • alternative splicing of H chain mRNA generates membrane bound or secreted Ig
  • membrane bound contains mRNA with MC segment
  • secreted contains SC segment
  • naive B cells express membrane bound IgM and IgD
  • upon antigen activation B cells differentiate to plasma cells and produce secreted Abs
34
Q

Secreted vs Membrane bound IgM

A
  • secreted IgM has hydrophilic C terminus encoded at the 3’ end of the Cu4 exon
  • ## membrane bound IgM has a hydrophobic C terminus encoded by 2 separate exons downstream of Cu4
35
Q

Class Switching

A
  • antibody responses involved class switching
  • after antigen stimulation, rearranged VDJ gene segments combine with any C gene to create new antibody class with same binding site
  • expresses Abs with different functional properties but same antigen
  • cytokines regulate switching to specific Ch genes
  • pathogen type leads to cytokine environment leads to immune response + Ab type
36
Q

Steps of Class Switching

A
  • involves recombination of heavy chain constant region genes between specific switch sequences
  • AID targets switch sequences
  • DNA in the switch regions nicked on both strands
  • looping out and switch region recombination to produce different class
  • AID converts cytosine to uracil: uracil removed and endonucleases create nicks in both strands of DNA at this site: recombination between VDJ and downstream C gene
37
Q

Summary of changes in Ig genes during B cell maturation

A
  1. V region assembly from gene fragment: somatic recombination of genomic DNA
  2. generation of junctional diversity: imprecision in joining rearranged DNA segments adding P and N nucleotides
  3. assembly of transcriptional controlling elements: promoter and enhancer brought close by V region assembly
  4. transcription activated with coexpression of surface IgM and IgD
  5. synthesis changes to secreted antibody
  6. somatic hypermutation: point mutation
  7. isotype switch: somatic recombination
38
Q

B cell development

A
  • initial development in bone marrow (antigen independent)
  • mature B cells bearing membrane bound IgM and D released
  • B cells activated in peripheral lymphoid tissues
  • differentiate into plasma cells secreted different classes of antibodies
  • memory B cells generated

ICB (15 decks)

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