antigens and immunoglobulins

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week 5 clinical immunology

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39 Terms

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immunoglobulin (Ig) structure

  • heavy and light chains contain amino terminal variable (V) region that consists of 100-110 AAs (differs between ABs)

  • constant C regions exhibit limited variation and define the 2 light chain subtypes and the 5 heavy chain subtypes

  • some heavy chains (α, γ, δ) also contain proline-rich hinge region

  • amino terminal portions, corresponding to the V regions, bind to the antigen

  • effector functions are mediated by other domains

  • the μ and ε heavy chains (lack hinge region) contain additional domain in the middle of molecule

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history of Ig structure

  • proteolytic enzymes (proteases) were important tool in early AB structure studies

  • limited digestion:

    • papain cleaves AB molecules into 3 fragments

    • pepsin cuts on the carboxyl terminal side of the disulphide bonds, producing F (ab’)2 fragment, in which 2 antigen-binding arms of the AB must remain linked

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proteolytic cleavage of immunoglobulins

  1. papain cuts the AB molecules on the amino-terminal side of the disulphide bonds that link 2 heavy chains

    link 2 heavy chains, releasing the 2 arms of AB molecule as 2 identical fragments that contain antigen-binding activity

  2. Fab fragments- fragment antigen binding

    Fc fragment-fragment crystallisable

    Fc fragment does not interact with antigen but rather interacts with effector molecules and cells (different between heavy chain isotypes)

  3. pepsin cuts on the carboxyl terminal side of the disulphide bonds

    pepsin cuts the remaining part of the heavy chain into several small fragments

  4. F (ab’) 2 fragment in which the 2 antigen binding arms of the AB molecule remain linked

    F (ab’)2 fragment has the same antigen-binding characteristics as AB but is unable to interact with any effector molecule

  5. pFC’- largest remaining Fc fragment

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production of monoclonal ABs

  • mice spleen cells immunised with antigen A produce/secrete a specific AB

  • spleen cells die after a few days in culture

  • fusing spleen cells with immortal myeloma cells by use of polyethylene glycol (PEG) results in hybrid cell line called hybridoma

  • HGPRT gene contributes by the spleen cell allows hybrid cells to survive in the HAT medium and only hybrid cells can grow continually in culture (selective pressure)

  • unfused myeloma cells and unfused spleen cells die in the HAT medium

  • individual hybridomas are obtained by single-cell diultion and screened for AB production

  • single clones that make AB of desired specificity can be isolated and grown

  • as each hybridoma is descended from a single cell, all cells of a hybridoma cell line make the same AB molecule hence monoclonal AB

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immunoglobulin fold

  • characteristic structural motif of all Ig domains

  • sandwich of β pleated sheets

  • 3 or 4 antiparallel strand of AA strands are connected by loops

  • β strands are characterised by alternating hydrophilic and hydrophobic AAs

  • side chains are perpendicular to the plane of paper

  • hydrophobic interactions stabilise the structure

  • disulphide bond stabilises structure

  • loops are where the complementarity is

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hypervariable regions

  • also called complementarity determining regions (CDRs)

  • 3 hypervariable regions acount for 15-209% of domain

  • 80-85% are less variable, known as framework regions

  • hypervariable regions form the antigen binding site of the AB molecule

  • hypervariable CDRs are located on loops at the end of the Fv regions

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Ig gene superfamily (IgSF)

  • Ig domains are not restricted to Ig genes

  • superfamily of related genes, particularly those encoding proteins crucial to cell-cell interactions and molecular recognition systems (MHC class I and class II molecules)

  • IgSF molecules are found in most cell types and are present across taxonomic boundaries

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difference between antigen and immunogen

  • although a substance that induces a specific immune response is usually called an antigen, it’s more appropriately called an immunogen

  • immunogen is necessarily an antigen but an antigen may not necessarily be an immunogen

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antigen vs immunogen

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immunogens

  • induce AB production because the immune system recognises them as a threat

    • proteins

    • carbs

    • lipids

    • phospholipids

    • nucleic acids

  • only macromolecules can stimulate humoral immune response

  • lipid and nucleic acid must be conjugated to a carrier protein or polysaccharide to be immunogenic

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antigens are not always immunogenic

  • immunogenicity is not always an intrinsic property

    • eg Bovine Serum Albumin

    • BSA is not immunogenic to rabbits

    • BSA is immunogenic to rabbits

  • haptens are another example

  • immunologists tend to use proteins or polysaccharides as immunogens in most experimental studies of humoral immunity

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Hapten

  • a molecules that is incapable, alone, of causing the production of ABs

  • needs to be conjugated to a larger antigenic molecule- carrier

  • hapten carrier effect

  • immune system will then recognise the hapten as part of a larger molecule/structure

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factors that affect immunogenicity

  • degree of dissimilarity with self molecules

  • molecular size

  • chemical composition

  • degradability

  • genotype of recipient

  • immune dosage

  • route of administration

  • adjuvants

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adjuvant

  • substances that enhance the immunogenicity of an antigen in a mixture

    • antigen has low immmunogenicity

    • small amounts of antigen are available

    • AB response of mice to immunsisation with BSA can be increased 5 fold or more if BSA is administered with an adjuvant

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the effect of adjuvants

  • prolonged antigen persistence

  • co stimulatory signals are enhanced

  • local inflammation increased

  • non specific proliferation of lymphocytes is stimulated

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epitopes: the basic recognition unit

  • the smallest individually identifiable part of an antigen is known as an epitope

  • the immunologically active regions of an immunogen that interact with membrane receptors on lymphocytes or with secreted ABs

  • protein antigen: epitope is about 5 or 6 AA residues

  • carb antigen: epitope is about 5 or 6 hexose units

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antigens can be classified in order of their origins

  • exogenous antigens

  • endogenous antigens

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exogenous antigens

  • entered the body from the outside (inhalation, ingestion, injection)

  • taken up by endocytosis or phagocytosis

  • processed into fragments in the APCs

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endogenous antigens

  • generated within the cell

    • normal cell metabolism

    • viral or intracellular bacterial infection

    • cancer, tumour antigens

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continuous and discontinuous epitopes

  • an epitope on a protein antigen may involve elements of the primary, secondary tertiary and quaternary structure of the protein

  • continuous/ linear epitopes consist of continuous AA residues on a protein sequence

  • discontinuous/conformational epitopes consist of AA residues that are discontinuous in the protein sequence but are brought together in the 3D structure

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antigen recognition by T cells vs B cells

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paratope

  • 3D structure formed by the complementary binding of the tip part of variable regions (light and heavy chains) of the AB

  • 5-10 AAs that recognise and bind the epitope of an antigen

  • present in the antibody’s Fv region

  • fragment antigen binding (Fab region)

  • antibody-antigen binding is the paratope-epitope interaction

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4 types of noncovalent forces hold together the antigen-antibody complex

  • hydrogen bonds

  • VdWs

  • hydrophobic interactions (account for most of the binding energy)

  • electrostatic interactions occur between charged AA side chains as in salt bridges

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affinity

  • strength of binding of one molecule to another at a single site, such as the binding of a monovalent Fab fragment of AB to a monovalent antigen

  • strong interaction depends on a very close fit between antigen and AB

  • high degree of complementarity between antigen and AB

  • exquisite specificity

  • Ag + Ab ⇌ Ag-Ab

  • Ka = [Ag-Ab] / [Ab] [Ag]

  • Kd = [Ab] [Ag] / [Ab-Ag

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quantifying AB-antigen interactions

  • low affinity Ag-Ab complexes have Ka values between 10^4 and 10^5 L/mol

  • high affinity Ag-Ab complexes have Ka values as high as 10^11 L/mol

  • very stable complexes have very low Kd values

  • low affinity ABs bind antigen weakly and dissociate readily

  • high affinity Ab bind Ag more strongly and remain bound longer

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avidity: strength of Ab-Ag attachment

  • the sum total of the strength of binding of 2 molecules (eg Ab and Ag) or cells to one another at multiple sites

  • distinct from affinity which is the strength of binding of one site on a molecule to its ligand

  • high avidity can compensate for low affinity

  • better measure within biological systems

  • eg IgM can interact at 10 different binding sites

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AB cross-reactivity

  • cross reactivity occurs if 2 different antigens share an identical or very similar epitope (lower affinity than for the original epitope)

  • eg Streptococcus pyogenes expresses cell wall proteins called M antigens

    • ABs against streptococcal cell wall antigens cross react with antigens on heart tissue

    • some ABs cross react with the heart valves others do not

    • an epitope in the heart is structurally similar but not identical to bacterial epitope

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superantigens bind directly to TCRs and MHC molecules

  • distinct class of antigens that stimulate a primary T cell response

  • recognised by T cells without being processed into peptides that are captured by MHC molecules

  • activation of superantigens causes massive production of cytokines by CD4 T cells

  • cytokine in turn cause systemic toxicity and suppression of the adaptive immune response

  • eg: staph enterotoxins, food poisoning

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5 main classes of immunoglobulin isotypes

  • five different heavy-chain constant regions: μ, δ, γ, ε, α

  • each of these 5 different heavy chains is called an isotypes

  • heavy chain determines the class

    • IgM (μ)

    • IgG (γ)

    • IgA (α)

    • IgD (δ)

    • IgE (ε)

  • Fc structure is common to all specificities of AB within an isotype (although there are allotypes)

  • different classes activate distinct effector mechanisms in response to an antigen, triggering different elements of the innate immune system

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immunoglobulin M (IgM)

  • IgM is the first AB secreted by the adaptive immune system

  • monomeric IgM is a heterotetramer of approx 180kDa

  • secreted form of IgM exists predominantly in a pentametric configuration with a molecular weight greater than 900 kDa

  • participates in neutralisation and clearance of pathogens

  • powerful activator of complement

  • helps activate inflammation, opsonisation and destruction of pathogens

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immunoglobulin D (Igd)

  • IgD is co expressed with IgM on B cells due to differential RNA splicing

  • conjugation of IgD with antigen can activate, delete or anergise B cells

  • IgD plasma cells are found in the respiratory mucosa

  • IgD can bind to basophils and mast cells and activate these cells to produce antimicrovial factors to participate in respiratory immune defence in humans

  • also stimulates basophils to release B cell homeostatic factors

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immunoglobulin A (IgA)

  • crucial role in the immune function of mucous membranes

  • up to 15% of total immunoglobulins produced throughout the body

  • IgA exists in 2 subclasses (IgA1 and IgA2)

  • can be produced as a monomeric or dimeric form

  • the IgA dimeric form is the most prevalent also called secretory IgA (slgA)

    • IgA1 is mostly found in serum and made by bone marrow B cells

    • IgA2 is mostly found in mucosal secretions, colostrum and milk and is made by B cells located in the mucosa

  • slgA provides a line of defence against bacteria such as salmonella, cholera, gonorrhoea and viruses like polio, flu and reovirus

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secretory IgA (slgA) and transcytosis

  1. IgA adsorbs on the layer of mucus covering the epithelium

    in addition to aggregating commensal bacteria, it can neutralise pathogens and toxins preventing their access to tissues and inhibiting their functions

  2. pathogens and toxins internalised by the epithelial cell can meet and be neutralised by IgA in endosomes

  3. toxins/pathogens encounter pathogen specific IgA in lamina propria, resulting complexes are exported across the epithelial cell as the dimeric IgA is secreted

  4. antigen bound to SlgA in the lumen can bind via carb residues on the Fc portion of IgA to dectin-1 on M cells in Peyer’s patches and be transported to underlying dendritic cells

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immunoglobulin E (IgE)

  • only found in mammals

  • synthesised by plasma cells

  • utilised during immune defense against certain protozoan parasites such as plasmodium falciparum

  • IgE has an essential role in type I hypersensitivity- associated with allergy

  • Ig E also plays a pivotal role in responses to allergens

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immunoglobulin G: most common type of Ig

  • approx 75% of serum ABs in humans

  • provides long term protection

  • important role in the elimination of microbial pathogens (agglutination, opsonisation, neutralisation, activation of the classical complement pathway, AB-dependent cellular cytotoxicity and intracellular AB mediated proteolysis- direct marked virions to the proteasome in the cytosol)

  • associated with type II and type III hypersensitivity reactions

  • 4 IgG subclasses (IgG1, 2, 3 and 4) in humans that differ in their constant region which binds to IgG-Fc receptors (FcγR) and C1q

  • different subclasses have different effector functions

  • IgG1 and IgG3 ABs are generally induced in reesponse to protein antigens whereas IgG2 and IgG4 are associated with polysaccharide antigens

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Fc receptors for IgG

  • family of Fc receptors for IgG (FcγRs) is broadly expressed by cells of haematopoietic origin

  • can be acitvating or inhibiting

  • FcγRI is an activating receptor with high affinity for IgG and is expressed on monocytic DCs and on monocytes/macrophages

  • FcγRIIb is an inhibitory receptor expressed on B cells, DCs and basophils

  • Fc receptor binding to ABs stimulates phagocytic or cytotoxic cells to destroy microbes or infected cells by ADCP or ADCC

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ABs can protect against infectious agents by 5 mechanisms:

  1. agglutination

  2. neutralisation

  3. opsonisation

  4. activation of complement

  5. AB dependent cell mediated cytotoxicity

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factors affecting AB functional response

  • activation threshold at AB doses that vary per effector mechanism

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why ABs need an Fc region

  • Fc region of AB interacts with cell surface receptors called Fc receptors and some proteins of the complement system

  • this property allows ABs to activate the immune system

<ul><li><p>Fc region of AB interacts with cell surface receptors called Fc receptors and some proteins of the complement system</p></li><li><p>this property allows ABs to activate the immune system</p></li></ul><p></p>