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71 Cards in this Set
- Front
- Back
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Edward Jenner, 1798
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- Milkmaids with mild cowpox = resistant to small pox
- inoculating from cowpox pus would protect from small pox |
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Louis Pasteur, 1855
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induction of immunity to cholera (chickens), anthrax (sheep) and Rabies (Human)
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Two types of Immunity
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- Innate (natural)
- Adaptive (acquired) |
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Main Line of Defense for Non-Specific Immune Response
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Neutrophils - Phagocytose invading organisms
Macrophages - Phagocytosis and Intracelular killing. Extracellular killing of infelcted self target cells. Contribute to repair and APC's Natural Killer and Lymphokine Activated Killer cells - kill virus infected and tumour cells (no inflammatory response) Eosinophils - Contains proteins in granules to kill some parasites |
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PAMPs and PRRs
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Components of innate immune response recognise Pathogen Associated Molecular Patterns (PAMP) using Pattern Recognision Receptors (PRR) which instigates a cellular response
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Adaptive Immune System Responds to Non-Self
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Non-Self = forgein antigen whcih is usually a foreign protein
pollen, pollutants, toxins and heavy metals can result in a nuisance immune responses which show no beneficial effect |
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Characteristics of Adaptive Immune System
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Diversity - Can recognise billions of unique structures
Specificity - Specific for an antigen and can discriminate between subtle differences among antigens Memory - Can remember previous antigen contact, can respond quicker 2nd time around forming the basis of Vacinations |
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2 Main Cell Groups of Immunity
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- Lymphocytes (B and T) WBCs of Adaptive Immunity
- APCs, Phagocytic (Macrophages and Dendritic cells) which bellong to Innate Immunity |
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The Lymphoid System
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Lymphocytes arise from stem cels in bone marrow and lymphoid tissues. B cells = Bone marrow, T cells = Thymus Gland. Migrate to Peripheral Lymphoid Tissues such as Lymph Nodes. These sites are activated by antigens where lyphocytes circulate the blood unitl they meet an antigen. Lymph = extracellular fluid from peripheral tissues which carry antigens and re-circulating lymphocytes
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Structure of Lymph Node
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Outer Cortex - B cells orginised into lymphoid follicles
Paracortex - T cells Medullary Cords - Macrophages and plasma cells Medullary Sinus Lymph in via the afferent pathway which carries antigens and lymph out by efferent pathway |
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Clonal Selection
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Many Lypmocytes are made with different specific receptors on. When a new antigen is recognised by a lymphocyte, it gets activated. This lyphocyte clones itself to cells with identicle receptors on which then differenciate into effector and memory cells. cells which can bind to self antigens are deleted early on
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Antibody Respnose (humoral) activating B cells
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B1 Cells (T cell independant) activated by antigen, differentiate into Plasma and memory cells, Plasma cells produce IgM to inactivate antigen
B2 Cells (T cell dependant) activated T cells activate B2 cells via cytokines, differentiate into Plasma and memory cells, Plasma cells produce IgA and IgG to inactivate antigen |
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Cell-Mediated Response
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Antigen Presentation - T cells have T cell Receptors (TCR) which recognises antigens that are rpesented to them by APCs which activates the T cell
T cell Activation - Activated T cells differentiate into T helper cells which produce IL-2 which causes T cells to divide. Cytotoxic T cells - Some acitvated T cells will become cytotoxic T cells which kill infected cells. Others will become memory cytotoxic T cells |
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Albumins and Globulins
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Albumins - Proteins which are soluble in water which precipetate by full saturation with Amonium Sulphate
Globulins - Less soluble proteins which precipetate by half saturation with Amonium Sulphate |
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Immunoglobulin Structure
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Major Immunoglobulin class that is a heterodimer. intrachain disulphide bonds making a loop of 60 AA's to retain 3D quarternary structure. Variable region of both heavy and light = 108 AA's. Light chain constant region = 106 AA's. The heavy chain comprises of 446 AA's. 3 segments on both H and L chain show even more varience (CDR's). CDR H3 contributes to most antigen binding. Mainly H chain CDRs which interact with antigen
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Precipertation of Antigens/Antibodies
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Precipitation occurs when near equivelent antibody and antigen concentration is in a solution forming a large latice
Polyclonal antibody solution + antigens with multiple epitopes forms a latice Monoclonal antiboy solution + antigens with repeating epitope forms a latice |
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Evidence for IgG Having Identicle Binding Sites
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The ability for monoclonal antibodies to cross link
The effect of proteolytic degradation of IgG on cross linking both H and L chains involved |
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Effect of Proteolytic Enzymes on Crosslinking
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Papain - produces 2 Fab's (Fragment antigen binding) that cant crosslink and a Fc (Fragment crystalisable) which has no antigent binding site but has biological properties
Pepsin - produces many Fc fragments and 1 F(ab)2 fragment with the ability to crosslink. |
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F(ab)2 advantages
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Smaller than whole IgG's allowing easier penetration of tissue resulting in better antigen recognition in immunohistochemistry
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5 Different Immunoglobulins
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IgG, IgE, IgM, IgD, IgA, based on the different amino acid sequences in the constant region of the heavy chain. they can also be classified by the type of constant light chain they have, either Kappa or Lambda
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IgG
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Most abundant Ig in serum (12mg/ml, 80%)
4 human subclasses of IgG due to slightly different aa sequence in constant heavy region IgG1-4 High affinity for antigen neutralises bacterial toxin, binds to microorganisms, inactivates viruses |
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Features of Complement System
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A collection of serum proteuins (15-20) which can:
Perforate cells Opsonization Recruit/activate cells Participate in regulation of antibody responses Aid in clearance of immune complexes & apoptotic cells |
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Mechanisms of Compliment
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Binds to Ig or to membrane compnents of cells
Proenzymes (activated) cleave other complement proteins Cleavage products activate cells Increase permeability or opzonise of bacteria |
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Classical Pathway of Compliment
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C1 binds to antigen/antibody complex activating it, cleaving C2 and C4 into C2a/C2b and C4a/C4b. C2b and C4b combine to make a protease C3 convertase, cleaving C3 into C3a/C3b
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Alternative Pathway of Compliment
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Endotoxins, polysaccarides and cell wall components react with C3b, which then reacts with proteins factor B factor D and properdin to form the C3 convertase protein
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Inflammation due to Compliment
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C3a is involved in inflammation. C3b can bind to surpahces of microorganisms, phagocytes have binding sites for C3b. addition of properdin to C3 convertase makes C5 convertase which cleaves C5 into C5a and C5b. C5a induces inflammation by acting as a chemoattractant for phagocytes. C5b acts with proteins C6-9 making a membrane attack complex resulting in holes in the cell which lyses it.
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Oxygen Dependant and Independant Phagocytosis
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Phagocytosis - increse in oxygen and glucose consumption (Dependant)
Pre formed substances in granules or lysosome that fuse with phagosome (Independant) |
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IgG Antibody Dependant Cell mediated Cytotoxicity (ADCC)
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Antibody binds to virus infected cell which a NK cell recognises via the Fc region and excretes cytokines which attack and kill the cell.
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Fc Receptors
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Variety of different specific Fc receptors for different classes of Ig.
FcgammaR1 specifically binds to Fc region of IgG Half life ~20 days |
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Maternal Importance of IgG
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Only Ig which can cross the placenta to foetus, important in first few weeks of foetus immunity (transcytosis)
Present in colostrum (Milk from mammary gland) helps further immunity in newborn, travels through intestinal wall into blood stream (Receptor mediated endocytosis |
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IgA
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10%-15% in serum, Predominant in secretory fluids e.g. tears and saliva protecting external surphaces.
exists as a monomer in serum but can exist as dimer, trimer or tetramer. Secratory IgA exists as dimer or tetramer, linked by a J chain which aids in binding to receptor. part of receptor remains bound, thought to protect from bacterial proteases. |
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IgM Features
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First class of Ig to appear on developing B cells (monomeric) and secreted into blood for early stages of primary immune response (pentameric).
Most effective Ig for activating compliment. Efficent agglutinating and cytolytic agent . fights microbial infection in blood |
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IgM Structure
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Has an extra Constant heavy domain and exists in either monomeric or pentameric. held together by J-hcain and disulphide bonds. J-chain allows binding to secretory cells. 10 antigen binding sites but only acces to 5.
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IgD
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0.2% in serum.
Hinge region is extended More suseptable to proteolytic degradation Found on surphace of B lymphocytes Regulation of lymphocyte activation and suppression possibly in self tollerance stage |
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IgE
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Very low concentration in blood and has extra C region on heavy chain. High affinity for receptors on mast cells which mediates hypersensitivity reactions responsible for symptoms such as hayfever and asthma.
receptors on mast cells bind to IgE complex to realese histamine, resulting in vasopermeability. Also releases factors that attract eosinophils. |
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Contribution of the immunogen
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Foreigness
Size Chemical Composition (more complex, more immunogenic) Physical Form Degradability |
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Haptens
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Small molecules which posses a single epitope, cannot cause an immune response on its own but when bound to a protein it can elicit a response
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Ag - Ab Interactions
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Glutamine residue of lysozyme, protudes between VH and VL domains of antigen binding site. forms hydrogen bonds important for Ag-Ab interaction
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Ag - Ab Reversable Bonding
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Non - covalent bonds
disrupted by high salt, extreme pH many bonds so could be strong hydrophobic bonds drive them together direct contact involves Hydrogen bonds, Van der waals forces and electrostatic. |
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High and Low Affinity Antibodies
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High affinity have High attraction/low repulsion with a good fit (usually more specific)
Low affinity have High repulsion/Low attraction with a poor fit. |
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B cell Development
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B-cell precurser -> Pro B cell -> Pre B cell -> immature B cell -> Mature B cell -> Memory cell or Plasma cell
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Bone Marrow (Antigen Independent Phase)
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Progenitor cell undergo complex rearrangment of the Ig gene to get numerous Igs with different variable regions that are specific to one antigen. apoptosis occurs when bound to self antigen. mature B cell expresses Ab on surface.
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Periphery (Antigen Dependent Phase)
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In absence of Ag 90% of B cells die.
In Pressence of Ag and T helper cells, B cells get activated for further maturation to Plasma cells or Memory cells. Plasma cells have extenceive Rough ER |
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Role of APC's
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Both B cells and DCs present antigens on their surface MHC's
Only DCsare capable of inducing primary immune response in naive T cells Lysosomal proccessing is required from endocytosed material to present on MHC's Peptides displayed on surface of MHC in 30-60 mins following B cell activation |
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Activation of Th1 response
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Three signals required
Ag on MHC Co-stimulatory molecules CD80-86 and CD28 Cytokines IL-12 |
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Toll-Like Receptors
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TLR's involved in microbial molecular pattern detection (Innate immune response) on surface of DC's induce cytokine and chemokine release
B cells were found to respond to specific TLR agonsits aswells as Ag and CD40L on T cells |
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Memory Cell
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Express High affinity antigen Receptors (Ig)
When activated by antigen, memory cells become plasma cells secreting antibodies (secondary immune response) |
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Animal Antibody Production
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Primary Immunisation -> Rest -> Boost 1 -> Rest (Test Bleed) -> Boost 2 -> Rest -> Recover of antiserum (polyclonal) and Splenocytes (Monoclonal).
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Choice of Animal Depends on
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Animal house facillity
Monoclonal or polyclonal Ab wanted Volume needed Amount of immunogen available |
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Immunisation Considerations
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Dose - Ammount used for primary immunisation can affect immunogenicity
Route - The route of antigen administration can also affect the nature of the response (how much for 2nd dose) Adjuvants - Substances that make a hieghtened response to Ag, usualy needed but not for intact cells e.g. bacteria |
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Adjuvants - Mechanism of Action
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Enhance long term release of Ag increasing exposure time of immune system to Ag
Acting as non-specific Mediators of immune cell function by stimulating immune cells Enhance Phagocytosis after binding the Ag as a carrier |
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Types of Adjuvants
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FCA - Mineral Oil Adjuvant containing water-in-oil emulsion and heat killed mycobacteria. Used for primary immunisation
FIA - Same as FCA without mycobacteria. used for booster or as primary immunisation if antigen is highly immunogenic Aluminium Salt Adjuvant - Most frequently used in human vaccination, found in HepA, HepB, Diptheria and tetanus, generally weaker than emulsion s used with highly immunogenics. mild inflammatory reactions |
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Monoclonal Antibody Production
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Immunization of mouse to produce antibodies. Antibody producing cells are isolated from spleen which are fused with cultivated tumour cells using polyethylene glycol to form hybridomas. Hybridomas screened for Ab production which are then cloned and isolated
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Hypoxanthene-Aminopterin-Thymide Selection
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HAT added to cell culture growth medium to select for hybridomas. The enzyme Hypoxanthene:Guanine Phosphoribosyl transferase is required for survival in HAT containing growth medium. HGPRT is expressed in spleen cells thus making hybridomas HGPRT+
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Binding Assays
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Measure binding of Ab-Ag, need a detection method. Can be (Non)competitive and used to detect concentration of either Ab or Ag by labelling. Typical assays include: Western blotting, Immunoflouresence, Enzyme immunoassay, Radio immunoassay.
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Western Blotting
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Uses Ab's to detect proteins after seperation from electrophoresis.
Protein samples loaded into gel well to separate, then transfered from gel to membrane(PVDF, Nitrocellulose) with top and bottom filtter paper stack. Block membrane then add primary Ab, then add 2ndary Ab to detect binding. |
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Immunohistochemistry (IHC) and Immunofluorescense (IFA)
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Detect Expression and Localisation of a protein in cells. IHC use specific staining to view location under light microscope (Enzyme Conjugated Antibody; HRP or AP)
IFA, simmilar to IHC but uses fluorescently tagged Ab's which are detected when binding occurs. Direct = Primary Ab is tagged, Indirect = Ab is specific for Ag, not tagged |
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Competitive Immunoassays
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Detect both large and small molecules (Antigens, Haptens)
Can involve a single mAb which recognises a single epitope called one site assays. can be used to measure Ab or Ag(Common) e.g. RIA |
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RIA
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Add Ag, wash, Add Ab, wash, Add radiolabelled ligand, wash, count
Competition between labelled and unlabelled Ags for limiting amount of Ab Labeled bound to wells and counted, known ammounts of unlabbeled used to make displacement curve |
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Non-Competitive Assays
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One-site, Two-site or many sites
Reagents are in excess Quick Reaction May not be as sensitive as RIA |
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Two Site Non-Competitive Immunoassay
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only suitable for large molecules as requires more than one epitope. Two antibodies aer involved, Capture Ab which is immobilised on plastic to bind to epitope 1 on Ag, Detection Ab which is labeled and binds to captured Ag epitope 2, bound label proportional to Ag conc. Highly specific and sensitive
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ELISA
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Uses HRP enzyme label. Direct = one-site, non competitive immunoassay, Indirect = Sandwich ELISA
Direct Elisa - |
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Direct ELISA and Sandwich ELISA
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Direct - Add Ag then block exposed sites, introduce first Ab, Introduce enzyme labelled second Ab, add enzyme substrate and measure product development
Sandwich - Add Ab to plastic and introduce Ag. Ass 2nd Ab with enzyme attached which binds to different epitope on Ag. Add anxyme substrate and measure product. |
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Immunoprecipitation
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Add Ab to protein of interest, Add proteins A/G linked beads which bind to Fc region of Ab, Add protein sample buffer and boil to remove immunoprecipitates from beads, Load samples to SDS-PADE gel and detect bands on western blot.
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Agglutination Reactions
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IgM
Simple to perform, inexpensive, can detect small Ag amounts Heamagglutination used in blood typing - RBCs, ABO Ag's Serial dilution of serum, Aglutination only occurs if sufficient Ab's are present to crosslink. |
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Source Materials for Antibody Purificationd and their Main Contaminants
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Antiserum - Other Ab's, Serum proteins, Lipids etc (variable yield of specific Ab)
Hybridoma Cell Culture Medium - Serum proteins and lipids (yields 0.01-0.5mg Ab/ml) Ascites Fluid - Body fluids, Lipids and Cell debris (1-15mg Ab/ml) |
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Sample Preperation for Purification of Ab's
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Removal of Gross impurities and some specific impurities
Salt Precipitation - Ammonium sulphate selectively precipitates IgG, Caprylic acid selectively precipitates serum proteins, salt removal needed before further steps Clarification - Precipitation of lipids using dextran sulphate pollyvinyl pyrollidone Centrifugation or Filtration - nitrocellulose membrane filters to remove aggregates Buffer Exchange - This involves desalting by gel filtration chromatography |
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Main Purification
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Affinity Chromatography - exploits binding of Ab to a ligand covalently bound to an insoluble support matrix
Ligand types: Fc specific Protein A (Human, Rabbit or mouse IgG specific) or protien G (Rat, Goat, Cow and Guinea Pig IgG Specific), Immunospecific, purified Ag or Ab raised against the relevant antibody class and species |
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Further Purification
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Seperation by size e.g. IgM from IgG on sephadex g-200
Hydrophobic interaction chromatography - based on differences by hydrophoicity at high salt concentration, elutes at low salt conc |
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Biotin as an Ab Label
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Covalently binds with Fc region of Ab
20-30 biotin/Fc domain label attached to secondary antigen, allows greater signal amplification in 3 steps |
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Gold as an Ab Label
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Gold - Ab + Colloidal Gold + PEG, centrifuge conjugates on density gradient, application = immunogold electron microscopy
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